Sequestration of RBM10 in Nuclear Bodies: Targeting Sequences and Biological Significance.

RBM10 is an RNA-binding protein that regulates alternative splicing (AS). It localizes to the extra-nucleolar nucleoplasm and S1-1 nuclear bodies (NBs) in the nucleus. We investigated the biological significance of this localization in relation to its molecular function. Our analyses, employing deletion mutants, revealed that RBM10 possesses two S1-1 NB-targeting sequences (NBTSs), one in the KEKE motif region and another in the C2H2 Zn finger (ZnF). These NBTSs act synergistically to localize RBM10 to S1-1 NBs. The C2H2 ZnF not only acts as an NBTS, but is also essential for AS regulation by RBM10. Moreover, RBM10 does not participate in S1-1 NB formation, and without alterations of RBM10 protein levels, its NB-localization changes, increasing as cellular transcriptional activity declines, and vice versa. These results indicate that RBM10 is a transient component of S1-1 NBs and is sequestered in NBs via its NBTSs when cellular transcription decreases. We propose that the C2H2 ZnF exerts its NB-targeting activity when RBM10 is unbound by pre-mRNAs, and that NB-localization of RBM10 is a mechanism to control its AS activity in the nucleus.

Improvement of malaria diagnostic system based on acridine orange staining.

BACKGROUND: Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. METHODS: The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. RESULTS: Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. CONCLUSIONS: Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Selections, frameshift mutations, and copy number variation detected on the surf 4.1 gene in the western Kenyan Plasmodium falciparum population.

BACKGROUND: Plasmodium falciparum SURFIN4.1 is a putative ligand expressed on the merozoite and likely on the infected red blood cell, whose gene was suggested to be under directional selection in the eastern Kenyan population, but under balancing selection in the Thai population. To understand this difference, surf 4.1 sequences of western Kenyan P. falciparum isolates were analysed. Frameshift mutations and copy number variation (CNV) were also examined for the parasites from western Kenya and Thailand. RESULTS: Positively significant departures from neutral expectations were detected on the surf 4.1 region encoding C-terminus of the variable region 2 (Var2) by 3 population-based tests in the western Kenyan population as similar in the Thai population, which was not covered by the previous analysis for eastern Kenyan population. Significant excess of non-synonymous substitutions per nonsynonymous site over synonymous substitutions per synonymous site was also detected in the Var2 region. Negatively significant departures from neutral expectations was detected on the region encoding Var1 C-terminus consistent to the previous observation in the eastern Kenyan population. Parasites possessing a frameshift mutation resulting a product without intracellular Trp-rich (WR) domains were 22/23 in western Kenya and 22/36 in Thailand. More than one copy of surf 4.1 gene was detected in western Kenya (4/24), but no CNV was found in Thailand (0/36). CONCLUSIONS: The authors infer that the high polymorphism of SURFIN4.1 Var2 C-terminus in both Kenyan and Thai populations were shaped-up by diversifying selection and maintained by balancing selection. These phenomena were most likely driven by immunological pressure. Whereas the SURFIN4.1 Var1 C-terminus is suggested to be under directional selection consistent to the previous report for the eastern Kenyan population. Most western Kenyan isolates possess a frameshift mutation that would limit the expression of SURFIN4.1 on the merozoite, but only 60% of Thai isolates possess this frameshift, which would affect the level and type of the selection pressure against this protein as seen in the two extremities of Tajima's D values for Var1 C-terminus between Kenyan and Thai populations. CNV observed in Kenyan isolates may be a consequence of this frameshift mutation to increase benefits on the merozoite surface.

Characteristic age distribution of Plasmodium vivax infections after malaria elimination on Aneityum Island, Vanuatu.

Resurgence is a major concern after malaria elimination. After the initiation of the elimination program on Aneityum Island in 1991, microscopy showed that Plasmodium falciparum disappeared immediately, whereas P. vivax disappeared from 1996 onward, until P. vivax cases were reported in January 2002. By conducting malariometric surveys of the entire population of Aneityum, we investigated the age distribution of individuals with parasites during this epidemic in the context of antimalarial antibody levels and parasite antigen diversity. In July 2002, P. vivax infections were detected by microscopy in 22/759 individuals: 20/298 born after the beginning of the elimination program in 1991, 2/126 born between 1982 and 1991, and none of 335 born before 1982. PCR increased the number of infections detected to 77, distributed among all age groups. Prevalences were 12.1%, 16.7%, and 6.0%, respectively (P < 0.001). In November, a similar age pattern was found, but with fewer infections: 6/746 and 39/741 individuals were found to be infected by microscopy and PCR, respectively. The frequencies of antibody responses to P. vivax were significantly higher in individuals born before 1991 than in younger age groups and were similar to those on Malakula Island, an area of endemicity. Remarkably low antigen diversity (h, 0.15) of P. vivax infections was observed on Aneityum compared with the other islands (h, 0.89 to 1.0). A P. vivax resurgence was observed among children and teenagers on Aneityum, an age distribution similar to those before elimination and on islands where P. vivax is endemic, suggesting that in the absence of significant exposure, immunity may persist, limiting infection levels in adults. The limited parasite gene pool on islands may contribute to this protection.

S1-1/RBM10: multiplicity and cooperativity of nuclear localisation domains.

BACKGROUND INFORMATION: S1-1, also called RBM10, is an RNA-binding protein of 852 residues. An alteration of its activity causes TARP syndrome, a severe X-linked disorder with pre- or post-natal lethality in affected males. Its molecular function, although still largely unknown, has been suggested to be transcription and alternative splicing. In fact, S1-1 localises in the nucleus in tissue cells and cultured cells. RESULTS: By deletion and substitution mutagenesis, a classical 17-amino-acid (aa) nuclear localisation sequence (NLS1) was identified at aa 743-759 in the C-terminal region of S1-1. NLS1 was bipartite, with its N-terminal basic cluster weakly contributing to the NLS activity. S1-1 contained two additional NLSs. One was in the aa 60-136 RNA recognition motif region (NLS2), and the other was a novel NLS motif sequence in the aa 481-540 octamer-repeat (OCRE) region (NLS3). The OCRE is a domain known to be critical in splicing regulation, as shown with RBM5, a close homologue of RBM10 [Bonnal et al. (2008) Mol. Cell 32, 81-95]. The NLS activities were verified by expressing each DNA sequence linked to EGFP or a FLAG tag. These multiple NLSs acted cooperatively, and S1-1 became completely cytoplasmic after the concomitant removal of all NLS domains. In some cell types, however, S1-1 was partly cytoplasmic, suggesting that cellular localisation of S1-1 is subjected to regulation. CONCLUSIONS: The present results indicate that S1-1 contains multiple NLSs that act cooperatively. Among them, the OCRE is a hitherto unreported NLS. The nuclear localisation of S1-1 appears to be regulated under certain circumstances. We discuss these NLSs in relation to the biochemical processes they are involved in.

Enhanced radiosensitization by liposome-encapsulated pimonidazole for anticancer effects on human melanoma cells.

The nitroimidazole-related hypoxic radiosensitizer, pimonidazole (Pmz) was encapsulated in liposome composed of dipalmitoylphosphatidylcholine, cholesterol and dipalmitoylphosphatidylglycerol (molar ratio = 1:1:0.2; diameter = 112.9 nm), and the radiosensitization was evaluated in human melanoma cells HMV-II. Cell proliferation was examined by WST-8 assay after X-ray irradiation in the presence of liposomal Pmz or free-Pmz under hypoxic conditions. On 7th day after X-ray irradiation of 5 Gy, cell proliferation decreased more markedly in the administration of liposomal Pmz than free-Pmz at equivalent Pmz doses. Chromatin fragmentation or nuclear condensation was observed in liposomal Pmz-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with Pmz amounts of 250-2000 microM contained in liposomal Pmz. Intracellular uptake was more abundant for liposomal Pmz for 60-240 min than for free-Pmz. Thus liposomal Pmz has a potential to overcome radiation resistance in hypoxia, owing to enhanced intracellular uptake by melanoma cells.

S1-1 nuclear domains: characterization and dynamics as a function of transcriptional activity.

BACKGROUND INFORMATION: The RNA-binding protein S1-1, also called RBM10 (RNA-binding motif 10), is a paralogue of putative tumour suppressor RBM5 and has been correlated with cancer proliferation and apoptosis. In the present study, we have investigated the cell biology of S1-1. RESULTS: In the extranucleolar nucleoplasm, S1-1 occurred in hundreds of punctate and irregular domains. Some 10-40 of these domains were larger than 0.5 mum and prominent for S1-1 immunostaining. These domains (S1-1 nuclear bodies) were commonly present in tissue cells and in cultured cells. When cellular transcription was globally reduced by heat shock, serum starvation, culture at high cell densities or inhibition with RNA polymerase II inhibitors, small S1-1 domains (S1-1 granules), with weak immunostaining signals, reduced in number, whereas S1-1 nuclear bodies became prominent and increased in size. These altered S1-1 domains were returned to initial states when the cells were placed under normal conditions. Similar to paraspeckles, S1-1 nuclear bodies occurred closely adjacent to nuclear speckles or IGCs (interchromatin granule clusters), as determined by immunoelectron microscopy. However, the S1-1 nuclear bodies did not correspond to paraspeckles or IGAZs (interchromatin-granule-associated zones), but coincided with TIDRs (transcription-inactivation-dependent RNA domains), which we had characterized previously at the RNA level. The enlarged S1-1 nuclear bodies/TIDRs accumulated the S1-1 protein and microinjected primary and spliced mRNAs, presumably for later elevation of gene expression. In addition, electron microscopy revealed that S1-1 was also present on perichromatin fibrils, suggesting the structure of S1-1 granules seen at higher resolution. CONCLUSIONS: S1-1 constitutes hundreds of nuclear domains, which dynamically change their structures in a reversible manner. Upon globally reducing RNA polymerase II transcription, S1-1 nuclear bodies enlarge and decrease in number. They are novel domains different from paraspeckles or IGAZs, despite their similar occurrence adjacent to nuclear speckles. We discuss S1-1 granules in terms of their association with gene expression. In addition, this is the first report of a TIDR-localized protein.

Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs.

Development and application of a rapid and sensitive genotyping method for pharmacogene variants using the single-stranded tag hybridization chromatographic printed-array strip (STH-PAS).

Genetic polymorphisms contribute to inter-individual variability in the metabolism of multiple clinical drugs, including warfarin, thiopurines, primaquine, and aminoglycosides. A rapid and sensitive clinical assessment of various genome biomarkers is, therefore, required to predict the individual responsiveness of each patient to these drugs. In this study, we developed a novel genotyping method for the detection of nine pharmacogene variants that are important in the prediction of drug efficiency and toxicity. This genotyping method uses competitive allele-specific PCR and a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) that can unambiguously determine the presence or absence of the gene variant by displaying visible blue lines on the chromatographic printed-array strip. Notably, the results of our STH-PAS method were in 100% agreement with those obtained using standard Sanger sequencing and KASP assay genotyping methods for CYP4F2 gene deletion. Moreover, the results were obtained within 90 min, including the PCR amplification and signal detection processes. The sensitive and rapid nature of this novel method make it ideal for clinical genetic testing to predict drug efficacy and toxicity, and in doing so will aid in the development of individualized medicine and better patient care.

High and Heterogeneous Prevalence of Asymptomatic and Sub-microscopic Malaria Infections on Islands in Lake Victoria, Kenya.

Kenya is intensifying its national efforts in malaria control to achieve malaria elimination. Detailed characterization of malaria infection among populations living in the areas where the disease is endemic in Kenya is a crucial priority, especially for planning and evaluating future malaria elimination strategy. This study aimed to investigate the distribution and extent of malaria infection on islands in Lake Victoria of Kenya to aid in designing new interventions for malaria elimination. Five cross-sectional surveys were conducted between January 2012 and August 2014 on four islands (Mfangano, Takawiri, Kibuogi and Ngodhe) in Lake Victoria and a coastal mainland (Ungoye). Malaria prevalence varied significantly among settings: highest in Ungoye, followed by the large island of Mfangano and lowest in the three remaining small islands. Of the 3867 malaria infections detected by PCR, 91.8% were asymptomatic, 50.3% were sub-microscopic, of which 94% were also asymptomatic. We observed geographical differences and age dependency in both proportion of sub-microscopic infections and asymptomatic parasite carriage. Our findings highlighted the local heterogeneity in malaria prevalence on islands and a coastal area in Lake Victoria, and provided support for the inclusion of mass drug administration as a component of the intervention package to eliminate malaria on islands.

Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene.

Detection of sub-microscopic parasitemia is crucial for all malaria elimination programs. PCR-based methods have proven to be sensitive, but two rounds of amplification (nested PCR) are often needed to detect the presence of Plasmodium DNA. To simplify the detection process, we designed a nested PCR method whereby only the primary PCR is required for the detection of the four major human Plasmodium species. Primers designed for the detection of the fifth species, Plasmodium knowlesi, were not included in this study due to the absence of appropriate field samples. Compared to the standard 18S rDNA PCR method, our cytochrome c oxidase III (cox3) method detected 10-50% more cases while maintaining high sensitivities (1.00) for all four Plasmodium species in our samples from Vanuatu (n=77) and Kenya (n=76). Improvement in detection efficiency was more substantial for samples with sub-microscopic parasitemia (54%) than those with observable parasitemia (10-16%). Our method will contribute to improved malaria surveillance in low endemicity settings.

Alterations and diversity in the cytoplasmic tail of the fusion protein of subacute sclerosing panencephalitis virus strains isolated in Osaka, Japan.

We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan, from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain, five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed.

Relative frequencies of polymorphisms of variation in Block 2 repeats and 5' recombinant types of Plasmodium falciparum msp1 alleles.

The mechanisms producing the genetic polymorphism at Plasmodium falciparum merozoite surface antigen-1 locus (pfmsp1) include the insertion and deletion of the different type of dimorphic Block 2 9-nucleotide repeat units as well as the intragenic recombination. To study relative occurrence frequencies of these two distinct mechanisms, we have developed a sensitive PCR strategy to identify both 5' recombinant types and the number of Block 2 repeats from the same sample. This method can specifically detect the target 5' recombinant type (Blocks 2-6) at the sensitivity of 1-4 copies of the pfmsp1. Applying the new method to field isolates from the Solomon Islands enabled us to identify six different 5' recombinant types and variation in Block 2 repeat number in three of them, thus distinguishing 10 different alleles. Distribution of these alleles in local three villages in the study area suggests that frequencies of variation in the number of Block 2 9-bp repeats and recombination events within Blocks 2-6 are mutually independent and the frequency of repeat variation is relatively high as compared to that of recombination events at the pfmsp1 locus in P. falciparum populations from the Solomon Islands.

Unusual plasmodium malariae-like parasites in southeast Asia.

During malaria surveys in Myanmar, 2 peculiar forms of Plasmodium malariae-like parasites were found. The morphologies of their early trophozoite stages were distinct from that of the typical P. malariae, resembling instead that of Plasmodium vivax, var. minuta, reported by Emin, and Plasmodium tenue, reported by Stephens, both in 1914. Two polymerase chain reaction (PCR)-based diagnoses, which target the same regions in the small subunit ribosomal RNA (SSUrRNA) genes, indicated that these parasites were new variant forms of P. malariae and that they could be separated into 2 genetic types that correlated with the 2 morphological types. Sequence analysis of the SSUrRNA and the circumsporozoite protein genes revealed that they were distinct both from each other and from other known P. malariae isolates and that the P. tenue-like type was closer to a monkey quartan malaria parasite, Plasmodium brasilianum. These results illustrate that the microscopic appearance of human P. malariae parasites may be more varied than previously assumed and suggest the value of molecular tools in the evaluation of malaria morphological variants.

Enhanced radiosensitization by the cationic liposome-encapsulated thymidine analogue BrdU through the increased intracellular BrdU-uptake on human melanoma as compared to anionic or nonionic liposomal or free BrdU.

The synthetic thymidine analogue, 5-bromo-2'-deoxy-uridine (BrdU) was encapsulated in cationic liposome composed of dipalmitoylphosphatidylcholine, cholesterol and stearylamine (molar ratio = 1/1/0.2; diameter = 120 nm), and the radiosensitization of cationic liposomal BrdU was assessed on human melanoma cells HMV-II, with comparing to anionic or nonionic liposomal BrdU and free-BrdU. HMV-II cells were pretreated by cationic liposomal BrdU or free-BrdU and then exposed to X-ray, followed by WST-8 assay to examine cell proliferation. The radiation-induced change of nuclei was defined with Hoechst33342 staining. The rates of thymidine replacement by BrdU and DNA double-strand breaks on HMV-II cells were determined with an anti-BrdU antibody and anti-53BP1 antibody, respectively. On 7th day after X-ray irradiation at 3 Gy, cell proliferation was suppressed more markedly in the administration of cationic liposomal BrdU than free-BrdU at equivalent BrdU doses. Giant polykaryocytes were observed in cationic liposomal BrdU-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with BrdU doses of 0.1-0.8 µM in the order: cationic liposomal BrdU > anionic liposomal BrdU > nonionic liposomal BrdU (see symbol) free-BrdU. Similarly, the cationic liposomal BrdU augmented the rate of thymidine-moiety replacement by BrdU and DNA double-strand breaks more appreciably than free-BrdU. Therefore, the cationic liposome-encapsulation of BrdU would be one of favorable drug deliveries for facilitating the X-ray therapy against cancer.

RBM10 regulates alternative splicing.

RBM10, originally called S1-1, is a nuclear RNA-binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis-related genes, Fas and cFLIP. In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl-x genes. RBM10 promotes exon skipping of Fas pre-mRNA as well as selection of an internal 5'-splice site in Bcl-x pre-mRNA. We propose a consensus RBM10-binding sequence at 5'-splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.

Preservation of wild isolates of human malaria parasites in wet ice and adaptation efficacy to in vitro culture.

Wild isolates of malaria parasites were preserved in wet ice for 2-12 days and cultivated by a candle jar method. In four isolates of Plasmodium falciparum collected from Myanmar and preserved for 12 days, all failed to grow. In 31 isolates preserved for 5-10 days, nine were transformed to young gametocytes, but 22 isolates grew well. From Ranong, Thailand, nine isolates preserved for 7 days were examined, and six grew well. On the other hand, all of the 59 isolates collected from eastern Indonesian islands failed to establish as culture-adapted isolates, even most of them were preserved only for 2-3 days: 10 isolates stopped to grow, and 49 isolates were transformed to sexual stages by Day 10. These results indicated that a great difference in adaptation to in vitro culture may exist between wild isolates distributed in continental Southeast Asia and in eastern Indonesia and that gametocytogenesis might be easily switched on in Indonesian isolates. In wild isolates of P. vivax, P. malariae and P. ovale preserved for 2-9 days, ring forms or young trophozoites survived, but adaptation to in vitro culture failed. These results indicate that wild isolates can be preserved in wet ice for 9-10 days.

Antitumor effects of nano-bubble hydrogen-dissolved water are enhanced by coexistent platinum colloid and the combined hyperthermia with apoptosis-like cell death.

In order to erase reactive oxygen species (ROS) related with the proliferation of tumor cells by reducing activity of hydrogen, we developed functional water containing nano-bubbles (diameters: <900 nm for 71%/population) hydrogen of 1.1-1.5 ppm (the theoretical maximum: 1.6 ppm) with a reducing ability (an oxidation-reduction potential -650 mV, normal water: +100-200 mV) using a microporous-filter hydrogen-jetting device. We showed that hydrogen water erased ROS indispensable for tumor cell growth by ESR/spin trap, the redox indicator CDCFH-DA assay, and was cytotoxic to Ehrlich ascites tumor cells as assessed by WST-8 assay, crystal violet dye stain and scanning electron microscopy, after 24-h or 48-h incubation sequent to warming at 37°C or 42°C. Hydrogen water supplemented with platinum colloid (0.3 ppm Pt in 4% polyvinylpyrrolidone) had more antitumor activity than hydrogen water alone, mineral water alone (15.6%), hydrogen water plus mineral water, or platinum colloid alone as observed by decreased cell numbers, cell shrinkage and pycnosis (nuclear condensation)/karyorrhexis (nuclear fragmentation) indicative of apoptosis, together with cell deformation and disappearance of microvilli on the membrane surface. These antitumor effects were promoted by combination with hyperthermia at 42°C. Thus, the nano-bubble hydrogen water with platinum colloid is potent as an anti-tumor agent.

In vitro effect of dehydroepiandrosterone sulfate on steroid receptors, aromatase, cyclooxygenase-2 expression, and steroid hormone production in preovulatory human granulosa cells.

OBJECTIVE: To examine the effect of a low concentration of DHEAS on the expression of the androgen receptor, estrogen receptor alpha and beta, progesterone receptor, aromatase, 3-beta-hydroxysteroid dehydrogenase, and cyclooxygenase-2 in human preovulatory granulosa cells, and to measure their production of steroid hormones (estrone, estradiol, progesterone, androstenedione, and testosterone). DESIGN: Analysis of cultured primary human preovulatory granulosa cells by real-time reverse-transcriptase polymerase chain reaction and assays of hormone production. SETTING: Osaka City University Postgraduate School of Medicine. INTERVENTION(S): Preovulatory granulosa cells were collected from follicular fluid obtained from patients undergoing transvaginal oocyte retrieval with ultrasound guidance. The cells were cultured in the absence or presence of a low concentration of DHEAS. Real-time reverse-transcriptase polymerase chain reaction was performed to quantify the RNA expression of the investigated genes, and steroid hormone (estrone, estradiol, progesterone, androstenedione, and testosterone) levels were measured in the culture medium. MAIN OUTCOME MEASURE(S): Changes in [1] the levels of mRNAs encoding androgen receptor, estrogen receptor alpha, estrogen receptor beta, progesterone receptor, aromatase, 3-beta-hydroxysteroid dehydrogenase, and cyclooxygenase-2; and [2] the levels of steroid hormones (estrone, estradiol, progesterone, androstenedione, and testosterone) in the culture medium. RESULT(S): Treatment of granulosa cells with 20 ng/mL DHEAS increased the expression of androgen receptor, aromatase, 3-beta-hydroxysteroid dehydrogenase, and cyclooxygenase-2, reduced the expression of estrogen receptor beta, and increased estrone and estradiol levels, but had no effect on progesterone, androstenedione, or testosterone levels. CONCLUSION(S): DHEAS may be an essential trigger of ovulation.

Molecular analysis of the alpha-N-acetylglucosaminidase gene in seven Japanese patients from six unrelated families with mucopolysaccharidosis IIIB (Sanfilippo type B), including two novel mutations.

Molecular analysis of the alpha-N-acetylglucosaminidase gene in seven Japanese patients with Sanfilippo syndrome type B from six unrelated families was carried out, and six disease-causing mutations were found. The parents of Patient 2 had a consanguinous marriage, but other families did not have any record of consanguinity. Two families were from Okinawa Island, where more patients with Sanfilippo syndrome were found than in other areas in Japan. Patients 1 and 6 showed the most severe phenotype with rapid progression. Patients 2, 5, and 7 were moderate. Patients 3 and 4 (sib cases) showed an attenuated form compared with other patients. Patients 1, 2, and 6 were homozygous for R482W, R565W, and R565P, respectively. Patients 3 and 4 were compound heterozygous for F314L and R565P. Patient 5 had delTG2171-2172 in exon 6 in one allele, and the other allele was unknown. Patient 7 was compound heterozygous for V241M and R482W. The family of Patients 3 and 4 and that of Patient 6 are unrelated, although both families are from Okinawa Island, and the patients have the same mutation, R565P; thus, R565P might be a common mutation in the Okinawa district. F314L and V241M are novel mutations.