Development of microsatellite markers for the Japanese endemic conifer Thuja standishii and transfer to other East Asian species.

OBJECTIVE: Design polymorphic microsatellite loci that will be useful for studies of the genetic diversity, gene-flow and reproduction in the Japanese endemic conifer Thuja standishii and test the transferability of these loci to the two other East Asian species, T. sutchuenensis and T. koraiensis. RESULTS: Fifteen loci were developed which displayed 3 to 21 alleles per locus (average = 9.2) among 97 samples from three populations of T. standishii. Observed heterozygosity for all samples varied between 0.33 and 0.75 (average = 0.54) while expected heterozygosity values were higher with an average over the 15 loci of 0.62 (0.37-0.91). Low multi-locus probability of identity values (< 0.00002) indicate that these markers will be effective for identifying individuals derived from clonal reproduction. All 15 loci amplified in 13 samples of T. sutchuenensis, the sister species of T. standishii, with 1 to 11 alleles per locus (average = 4.33) while 13 loci amplified in four samples of the more distantly related T. koraiensis with 1 to 5 alleles per locus (average = 2.15).

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) µg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Classification of Cryptococcus neoformans and yeast-like fungus isolates from pigeon droppings by colony phenotyping and ITS genotyping and their seasonal variations in Korea.

Cryptococcus neoformans (C neoformans) is a frequent cause of invasive fungal disease in immunocompromised human hosts. Ninety-eight samples of pigeon droppings were collected from the pigeon shelters in Seoul, and cultured on birdseed agar (BSA) and Sabouraud dextrose agar (SDA). One hundred yeast-like colonies were selected and identified via phenotype characteristics, such as colony morphology and biochemical characteristics. This was then followed with genotyping via sequencing of the internal transcribed spacer (ITS) region. The colonies were classified into four kinds of colony color types: brown type (BrT), beige type (BeT), pink type (PT), and white type (WT). Numbers of isolated BrT, BeT, PT, and WT colonies were 22 (22%), 30 (30%), 19 (19%), and 39 (39%), respectively. All BrT colonies were identified as C neoformans. BeT were identified as 19 isolates of Cryptococcus laurentii, 10 isolates of Malassezia furfur, and 1 isolate of Cryptococcus uniguttulatus. PT was divided into two colony color types: light-PT (l-PT) and deep-PT (d-PT). Eighteen of l-PT and one of d-PT were identified as Rhodotorula glutinis and Rhodotorula mucilaginosa, respectively. WT were identified as 34 isolates of Cryptococcus guilliermondii, 3 isolates of Cryptococcus zeylanoides, 1 isolate of Cryptococcus sake, and 1 isolate of Stephanoascus ciferrii. Most strains were classified identically with the use of either phenotype or genotyping techniques, but C uniguttulatus and C sake classified by phenotyping were Pseudozyma aphidis and Cryptococcus famata by genotyping. This rapid screening technique of pathogenic yeast-like fungi by only colony characteristics is also expected to be very useful for primary yeast screening. Additionally, we investigated the seasonal variations of C neoformans and other yeast-like fungi from 379 pigeon-dropping samples that were collected from February 2011 to March 2011. We isolated 685 yeast-like fungi from the samples. Almost all C neoformans and yeast-like fungi were isolated in the fall (298 strains, 43.5%) and spring (244 strains, 35.6%). A few yeast-like fungi were isolated in winter (98 strains, 14.3%) and summer (45 strains, 6%). These results would be used as an important indicator related to epidemiology and prevention of pathogenic yeast-like fungi infections transmitted through pigeon droppings.

Ependymal cyst in the cerebrum of an African green monkey (Chlorocebus aethiops).

A focal lesion was detected by magnetic resonance imaging in the right caudal occipital lobe of the cerebrum in an African green monkey (Chlorocebus aethiops). Neurological signs were not observed in this animal. At necropsy examination, an 8mm wedge-shaped intracranial cavity was found, which apparently did not communicate with the ventricles. Microscopically, the inner surface of the cavity was lined by ciliated cuboidal epithelium with positive immunoreactivity for S100 protein, glial fibrillary acidic protein and cytokeratin. Based on the gross, microscopical and immunohistochemical findings the lesion was classified as an ependymal cyst. To the best of our knowledge, this is the first report of an ependymal cyst in an African green monkey.

High-speed characteristics of vertical cavity surface emitting lasers and resonant-cavity-enhanced photodetectors based on intracavity-contacted structure.

We fabricated vertical cavity surface emitting lasers (VCSELs) and resonant-cavity-enhanced photodetectors (RCE-PDs) with GaAs/AlGaAs distributed Bragg reflectors (DBRs), operating at lambda approximately 980 nm, based on an intracavity-contacted structure. The top-DBR mesa diameter of the VCSELs was optimized to 18 microm in terms of slope efficiency, differential series resistance, and 3 dB bandwidth. For VCSELs with an oxide aperture of 4.5 microm and a top-DBR mesa diameter of 18 microm, the threshold current was about 1.2 mA, exhibiting maximum output power of approximately 3.49 mW (at 20 degrees C) with good uniformity. The effect of the overetching in the outermost layer of RCE-PDs on the device performance was also investigated. For RCE-PDs based on the VCSEL structure, a peak responsivity of 0.44 A/W (at lambda approximately 979.7 nm) with a spectral width of approximately 3 nm and a dark current of 68 pA under a bias voltage of -5 V at 20 degrees C was obtained. The maximum 3 dB bandwidths of approximately 11.5 GHz with a modulation current efficiency factor of 5.6 GHz/mA(1/2) at -7 mA and 9 GHz at -7 V were achieved for VCSELs and RCE-PDs, respectively.

A possibility of detection of the non-charge based analytes using ultra-thin body field-effect transistors.

Ultra-thin body of p-type field-effect transistors were developed as transducer for biosensors. Changes of conductance resulted from the changes of the surface potentials of ultra-thin body field-effect transistors (UTB-FETs) due to surface chemical modifications were demonstrated. The channel surface of UTB-FETs were modified with N-[3-(trimethoxysilyl)propyl]ethylenediamine (AEAPTMS) and then gold nanoparticles (AuNPs) to immobilize the bio-component, the genetically engineered Delta(5)-3-ketosteroid isomerase (Art_KSI) or the Art_KSI conjugated with charged reporter (Art_KSI_mA51). The binding of charge-based molecules or nanoparticles has been demonstrated to strongly affect the conductivity of UTB-FETs; the increase or decrease of the conductance depends on the polarity of the immobilized molecules or nanoparticles. A new protocol involving the detection of a non-charged analyte relied on the competitive binding of analyte (19-norandrostendione) and a charged reporter (mA51) with KSI. When exposed to a 19-norandrostendione solution (10 microM), the conductance of Art_KSI_mA51-modified UTB-FET increased by 265 nS ( approximately 12%). On the other hand, conductance of Art_KSI-modified UTB-FET showed no distinct change under the same detection conditions.

Detection of the virulent Marek's disease virus genome from feather tips of wild geese in Japan and the Far East region of Russia.

Marek's disease (MD) virus (MDV) is known to cause malignant lymphomas in chickens. In 2001, we first reported an MD case in a white-fronted goose (Anser albifrons) in Japan. Therefore, the prevalence of MDV in the wild geese was surveyed by nested PCR using feather-tip samples in Japan and the Far East region of Russia, breeding habitats of geese migrating to Japan. MDV was detected in about 30% of analyzed white-fronted geese. Furthermore, by nucleotide sequence analysis, we confirmed that this MDV shows high homology to very virulent MDV, suggesting that highly virulent MDV is widespread in white-fronted geese migrating between Japan and Far East region of Russia.

Performance estimation of a Venturi scrubber using a computational model for capturing dust particles with liquid spray.

A Venturi scrubber has dispersed three-phase flow of gas, dust, and liquid. Atomization of a liquid jet and interaction between the phases has a large effect on the performance of Venturi scrubbers. In this study, a computational model for the interactive three-phase flow in a Venturi scrubber has been developed to estimate pressure drop and collection efficiency. The Eulerian-Lagrangian method is used to solve the model numerically. Gas flow is solved using the Eulerian approach by using the Navier-Stokes equations, and the motion of dust and liquid droplets, described by the Basset-Boussinesq-Oseen (B-B-O) equation, is solved using the Lagrangian approach. This model includes interaction between gas and droplets, atomization of a liquid jet, droplet deformation, breakup and collision of droplets, and capture of dust by droplets. A circular Pease-Anthony Venturi scrubber was simulated numerically with this new model. The numerical results were compared with earlier experimental data for pressure drop and collection efficiency, and gave good agreements.

Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis.

In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 x 10(2) plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T(m)s): 89.23 +/- 0.27 degrees C for velogenic strains, 90.17 +/- 0.35 degrees C for pigeon mesogenic strains, 91.25 +/- 0.14 degrees C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.

Molecular characterization of the nucleocapsid protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid identifying method.

Nucleocapsid protein (NP) gene of Newcastle disease virus (NDV) strains mainly isolated in Japan from 1930 to 2001 was genetically characterized. By deduced amino acid sequence comparison, the N-terminal region (from 1 to 401 residues) of the NP protein was found to be highly conserved, while the C-terminal region was highly variable among the NDV isolates. A phylogenetic tree construct based on the nucleotide sequence of the complete NP gene revealed that the old (prior to 1970s) and the new (after 1980s) isolates could be classified into two major different groups, i.e., a group comprising virulent strains, and another group composed of avirulent strains. By restriction enzyme analysis using Pst I, none of the virulent strains were cleaved, while avirulent strains were cleaved. The results may be useful for simple primary screening test for differentiating NDV isolates.

Identification of novel compositions of ferromagnetic shape-memory alloys using composition spreads.

Exploration of new ferroic (ferroelectric, ferromagnetic or ferroelastic) materials continues to be a central theme in condensed matter physics and to drive advances in key areas of technology. Here, using thin-film composition spreads, we have mapped the functional phase diagram of the Ni-Mn-Ga system whose Heusler composition Ni(2)MnGa is a well known ferromagnetic shape-memory alloy. A characterization technique that allows detection of martensitic transitions by visual inspection was combined with quantitative magnetization mapping using scanning SQUID (superconducting quantum interference device) microscopy. We find that a large, previously unexplored region outside the Heusler composition contains reversible martensites that are also ferromagnetic. A clear relationship between magnetization and the martensitic transition temperature is observed, revealing a strong thermodynamical coupling between magnetism and martensitic instability across a large fraction of the phase diagram.

Metabolic adaptations in skeletal muscle overexpressing GLUT4: effects on muscle and physical activity.

To understand the long-term metabolic and functional consequences of increased GLUT4 content, intracellular substrate utilization was investigated in isolated muscles of transgenic mice overexpressing GLUT4 selectively in fast-twitch skeletal muscles. Rates of glycolysis, glycogen synthesis, glucose oxidation, and free fatty acid (FFA) oxidation as well as glycogen content were assessed in isolated EDL (fast-twitch) and soleus (slow-twitch) muscles from female and male MLC-GLUT4 transgenic and control mice. In male MLC-GLUT4 EDL, increased glucose influx predominantly led to increased glycolysis. In contrast, in female MLC-GLUT4 EDL increased glycogen synthesis was observed. In both sexes, GLUT4 overexpression resulted in decreased exogenous FFA oxidation rates. The decreased rate of FFA oxidation in male MLC-GLUT4 EDL was associated with increased lipid content in liver, but not in muscle or at the whole body level. To determine how changes in substrate metabolism and insulin action may influence energy balance in an environment that encouraged physical activity, we measured voluntary training activity, body weight, and food consumption of MLC-GLUT4 and control mice in cages equipped with training wheels. We observed a small decrease in body weight of MLC-GLUT4 mice that was paradoxically accompanied by a 45% increase in food consumption. The results were explained by a marked fourfold increase in voluntary wheel exercise. The changes in substrate metabolism and physical activity in MLC-GLUT4 mice were not associated with dramatic changes in skeletal muscle morphology. Collectively, results of this study demonstrate the feasibility of altering muscle substrate utilization by overexpression of GLUT4. The results also suggest that as a potential treatment for type II diabetes mellitus, increased skeletal muscle GLUT4 expression may provide benefits in addition to improvement of insulin action.

The growth suppressor PML represses transcription by functionally and physically interacting with histone deacetylases.

The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARalpha encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

Comparison of outcome in acute myelogenous leukemia patients with translocation (8;21) found by standard cytogenetic analysis and patients with AML1/ETO fusion transcript found only by PCR testing.

Patients with normal-karyotype acute myelogenous leukemia (NKAML) may have undetected genetic abnormalities that could affect prognosis. Screening for known AML-specific genetic abnormalities using the reverse transcription polymerase chain reaction (RT-PCR) may help in arriving at a more definitive prognosis. To test this hypothesis, 104 patients without translocation (8;21) and inversion(16), as shown by standard cytogenetic (SC) analysis, were screened for these two genetic abnormalities using RT-PCR. Western blot analysis for the AML1/ETO fusion protein and fluorescent in situ hybridization (FISH) analysis for t(8;21) were performed in patients for whom we had samples. The characteristics and outcome after high-dose cytarabine containing treatments in five patients with t(8;21) shown by RT-PCR alone were then compared to 21 patients with t(8;21) detected using SC analysis. Eight of the 104 patients had masked t(8;21) and none had masked inv(16), as shown by RT-PCR. Five of 54 patients with NKAML had a detectable AML1/ETO fusion RNA transcript. Western blot analysis showed the AML1/ETO fusion protein in four of the seven patients for whom we had samples among the eight with masked t(8;21) shown by RT-PCR. All patients with t(8;21) shown by RT-PCR had negative FISH results. Ninety percent (n=19) of the patients with t(8;21) shown by SC analysis and 40% (n= 2) of the patients with t(8;21) shown by RT-PCR alone achieved a complete remission (P value 0.03). These data suggest that the outcome of NKAML patients with t(8;21) shown by RT-PCR is not equivalent to patients with t(8;21) by SC studies.

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.).

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998.

A large scale outbreak of hand-foot-and-mouth disease (HFMD) occurred in Taiwan in 1998, in which more than 80 children died of shock syndrome with pulmonary edema/hemorrhage. Enterovirus 71 was implicated as the cause of this outbreak. In order to understand the virological basis responsible for mortality on this scale, nucleotide sequences of VP1 that is important for serotypic specificity, and the 5'-non-coding region (5'-NCR) that is important for replication efficiency, were analyzed comparatively. Phylogenetic analysis of both VP1 and 5'-NCR of nine EV71 isolates derived from specimens of fatal patients and seven isolates derived from uncomplicated HFMD patients showed that all but one isolate fell into genotype B. The one distinct isolate from a case of uncomplicated HFMD belonged to genotype C that was clustered along with one isolate from Taiwan in 1986. Complete sequence analysis of two selected isolates, one from the spinal cord of a fatal case and one from the vesicle fluid of a patient with mild HFMD, confirmed a high degree (97-100%) of identity in nucleotide sequence throughout the entire genome, except focal regions of 3C and 3'-NCR where the nucleotide homology was 90-91%. The identity of the deduced amino acid sequence in the 3C region that encodes viral proteinase dropped further to 86%, a result of missense mutations at the first nucleotide position of many codons.

Overexpression of thyroid hormone receptor beta1 is associated with thyrotropin receptor gene expression and proliferation in a human thyroid carcinoma cell line.

To correlate the differentiation phenotype of two human thyroid cancer cell lines with their expression of various molecular markers, we analyzed the mRNA levels of four thyroid-specific genes, including thyrotropin receptor (TSHR), thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), and paired-box containing transcription factor-8 (PAX-8) genes. The results showed a differentiation-status-related pattern in which a well-differentiated cell line (WRO) expressed all the four genes, in contrast to an anaplastic cell line (ARO) that expressed TTF-1 and reduced levels of TSHR, but no Tg or PAX-8 genes. Furthermore, to verify the finding of concomitant loss of beta subtype thyroid hormone receptor (TRbeta) and TSHR gene expression in neoplastic thyroid tumors (Bronnegard et al. 1994), we examined the expression levels of TRbeta1 gene in these cell lines. Whereas the WRO cells produced an abundant amount of TRbeta1 protein detectable by immunoprecipitation, the ARO cells produced none. This new observation prompted us to investigate whether overexpression of TRbeta1 protein in ARO cells might produce changes in the differentiation phenotypes. We found that the level of expression of the TSHR gene and the proliferative index of ARO cells were significantly upregulated in the cells stably transfected with wild-type TRbeta1. These findings suggest that TRbeta1 protein overexpression can affect the differentiation phenotypes and induce more efficient cell proliferation of the anaplastic ARO cells.

Comparison of a new screw-tipped intraosseous needle versus a standard bone marrow aspiration needle for infusion.

The purpose of this study is to compare the speed and ease of establishing intraosseous infusion using a standard bone marrow needle (SBMN; $8) and a new screw-tipped intraosseous needle (Sur-Fast; $42). The study is an experimental design. A total of 42 medical students, without prior IO experience, were recruited as study subjects. Subjects were randomized to perform the IO procedures in one of two models: (1) turkey femur or (2) pork ribs. Each subject performed an initial trial using both IO needles without practice (inexperienced) and a second trial using both IO needles after practice (experienced attempt), such that in total, each subject completed four attempts (two with each needle type). IO placement times were measured, and placement difficulty scores were measured using a 10 cm visual analog scale (VAS). The averaged elapsed time to successful IO completion was significantly shorter for the SBMN in the initial "inexperienced" attempt (33 versus 54 seconds, P = .019), but there was no significant difference in the postpractice "experienced" attempt. VAS difficulty scores were lower (easier) for the SBMN for both inexperienced and experienced trials. Success rates were significantly higher for the Sur-Fast needle during the experienced attempt (95% versus 79%, P < .05), but there was no significant difference in success rates during the inexperienced attempt. The Sur-Fast screw-tipped intraosseous needle does not show superiority over the SBMN in this intraosseous model, therefore its higher cost is difficult to justify based on this study.