Biomedical subjects

Toshinori Hayashi

Publications and source records attributed to Toshinori Hayashi.

10 recordsLinked to original sources

PubMed2007

Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti.

Previous studies have demonstrated the importance of FGF signaling at several stages in the development of the cochlea. At early stages of embryogenesis, Fgfr1, Fgfr2, and several FGFs are critical for both the induction of the otic vesicle and the initial development of the sensory epithelium. At late stages of cochlear development, Fgfr3 is necessary for the development of the tunnel of Corti. To determine the stage of development when Fgfr3 is required, we examined the expression of Fgfr3 and Fgf8 at various developmental stages. We also re-examined the Fgfr3 -/- mouse with additional markers for developing supporting cells. We confirmed the previous analysis of the Fgfr3 -/- mice, indicating that there are deficiencies in support cell differentiation. Specifically, we find that the inner pillar cell never develops, while the outer pillar cell is stalled in its differentiation. In addition, we found an extra row of outer hair cells, and accompanying Deiters' cells, in the apical two thirds of the organ of Corti in the Fgfr3 mutant. Thus, in addition to controlling the fate decision between pillar cells and Deiters' cells, we find that Fgfr3 also regulates the width of the sensory epithelium.

Animals↗

PubMed2006

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye.

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.

Animals↗

PubMed2006

Expression of Prox1 during mouse cochlear development.

We carried out an analysis of the expression of Prox1, a homeo-domain transcription factor, during mouse inner ear development with particular emphasis on the auditory system. Prox1 is expressed in the otocyst beginning at embryonic day (E)11, in the developing vestibular sensory patches. Expression is down regulated in maturing (myosin VIIA immunoreactive) vestibular hair cells and subsequently in the underlying support cell layer by E16.5. In the auditory sensory epithelium, Prox1 is initially expressed at embryonic day 14.5 in a narrow stripe of cells at the base of the cochlea. This stripe encompasses the full thickness of the sensory epithelium, including developing hair cells and support cells. Over the next several days the stripe of expression extends to the apex, and as the sensory epithelium differentiates Prox1 becomes restricted to a subset of support cells. Double labeling for Prox1 and cell-type-specific markers revealed that the outer hair cells transiently express Prox1. After E18, Prox1 protein is no longer detectable in hair cells, but it continues to be expressed in support cells for the rest of embryogenesis and into the second postnatal week. During this time, Prox1 is not expressed in all support cell types in the organ of Corti, but is restricted to developing Deiters' and pillar cells. The expression is maintained in these cells into the second week of postnatal life, at which time Prox1 is dynamically down regulated. These studies form a baseline from which we can analyze the role of Prox1 in vertebrate sensory development.

Animals↗

PubMed2005

Rapid detection of Mycobacterium tuberculosis in respiratory samples by transcription-reverse transcription concerted reaction with an automated system.

The aim of this study was to evaluate the performance of the transcription-reverse transcription concerted (TRC) method for the detection of Mycobacterium tuberculosis complex (MTC) 16S rRNA in clinical respiratory samples for the diagnosis of pulmonary tuberculosis. TRC is a novel method that enables the rapid and the completely homogeneous real-time monitoring of isothermal sequence RNA amplification without any postamplification procedure. The detection limit of the TRC method for MTC was one organism per 100 mul of sputum. The specificity of the method was confirmed by the absence of positive signals for sputum containing 10(6) M. avium or M. kansasii organisms per 100 microl. A total of 201 respiratory samples from patients diagnosed with or suspected of having tuberculosis were tested. Of the 72 MTC culture-positive samples, the TRC method was positive for 52 (sensitivity, 72.2%), whereas the Roche COBAS AMPLICOR PCR was positive for 58 (sensitivity, 80.6%). Both the TRC method and the COBAS AMPLICOR PCR showed no positive identification for any of the 129 culture-negative samples. The percent agreement between the two methods was 95% (191 of 201 samples). The high sensitivity and specificity together with shorter detection time (within 1 h) of the TRC method allow it to be proposed as a useful method for the rapid detection of MTC in respiratory samples.

Bacteriological Techniques↗

PubMed2004

FGF2 triggers iris-derived lens regeneration in newt eye.

Lens regeneration in newts occurs exclusively from the dorsal aspect of the iris pigment epithelium. Although the phenomenon has been a paradigm of experimental tissue regeneration, little is understood about how it is initiated and restricted to the dorsal iris. Here we show among various growth factors injected in an intact eye, a single injection of FGF2 specifically caused morphological changes of the iris characteristic of lens regeneration, induced expression of transcription factor genes Pax6, Sox2 and MafB, as well as endogenous Fgf2 in both dorsal and ventral halves, and provoked second lens development only from the dorsal iris. FGF2 protein accumulated in the iris tissue after the lens was removed, and injection of a soluble form of FGF receptor titrating FGF2 inhibited all reactions observed after the lens removal or after administration of FGF2. These results indicate that FGF2 and/or related molecules trigger lens regeneration from the dorsal iris in the newt. The observations also indicate that the absence of lens regeneration from the ventral iris is due to a block in a later phase of lens developmental pathway.

Animals↗

PubMed2004

Rapid detection of tdh and trh mRNAs of Vibrio parahaemolyticus by the transcription-reverse transcription concerted (TRC) method.

We developed a novel method named the transcription-reverse transcription concerted (TRC) method and an instrument that allowed rapid and completely homogeneous real-time monitoring of RNA isothermal sequence amplification without any post-amplification analysis in our previous study [Ishiguro et al., Anal. Biochem., 314, 77-86 (2003)]. In this study, we newly established rapid and sensitive TRC systems for the detection of the mRNAs transcribed from two major virulence genes of Vibrio parahaemolyticus: the tdh gene encoding the thermostable direct hemolysin (tdh) and the trh gene encoding the thermostable direct hemolysin-related hemolysin. Examination of the standard RNAs prepared in vitro showed that a positive result, increase in the fluorescence intensity to the cut-off value within 25 min, was obtained for as few as 100 copies of RNA. The TRC method specific to the trh mRNA detected the mRNAs transcribed from the trh1 and trh2 genes, two representative trh variants sharing 84% sequence identity. The detection time gave a linear relationship to the logarithm of starting RNA copies ranging from 10(3) to 10(7) copies, showing that quantitative analysis is possible. The detection time for 10(3) copies of the standard RNAs ranged from 11 to 15 min. Examination of the total RNAs extracted from the standard strains of V. parahaemolyticus demonstrated that the new TRC systems are sufficiently sensitive to detect as few as 100 CFUs of the strains carrying the target genes. Total RNA preparations extracted from 24 strains of V. parahaemolyticus, 52 strains belonging to 31 other species of the genus Vibrio and 11 strains belonging to 8 species of non-Vibrio genera were examined. The results of the detection of tdh- and trh-specific mRNAs by the two TRC systems and those of the respective genes by the DNA colony hybridization method agreed. We conclude that the new TRC systems are rapid, highly sensitive, easy to manipulate, and are suitable for routine examination of virulent strains of V. parahaemolyticus in microbiological laboratories.

Journal Article↗

PubMed2004

Rapid and specific detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by transcription-reverse transcription concerted reaction with an automated system.

Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 10(3) to 10(7) copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups.

Automation↗

PubMed2004

Rapid genetic diagnosis with the transcription-reverse transcription concerted reaction system for cancer micrometastasis.

BACKGROUND: Detection of cancer micrometastases is required for improvement of cancer therapy. The aim of this study was to establish a rapid and practical genetic assay to detect micrometastasis in gastric cancer and to assess its clinical significance with respect to prognosis. METHODS: A novel RNA amplification system with transcription-reverse transcription concerted reaction (TRC) was introduced for quantitative detection of cancer-specific carcinoembryonic antigen messenger RNA. The sensitivity and quantitative aspects of the assay were assessed with the full-length carcinoembryonic antigen messenger RNA, a gastric cancer cell line (MKN-45), and metastatic lymph nodes obtained from patients with gastric cancer. Peritoneal lavage fluid specimens that were collected from gastric cancer surgery were subjected to the assay, and the clinical significance of the results was examined for prediction of recurrence and survival. RESULTS: The quantification, sensitivity, and reproducibility of the assay with the TRC reaction were equal to those of quantitative reverse transcriptase-polymerase chain reaction with LightCycler. The most important advantages of the assay were its simplicity and rapidity. Molecular diagnosis of peritoneal lavage fluid by the TRC reaction significantly correlated with depth of invasion, peritoneal metastasis, clinical stage, overall survival, and peritoneal recurrence-free survival. CONCLUSIONS: Molecular diagnosis of peritoneal lavage fluid with the TRC reaction could be a useful prognostic indicator for peritoneal recurrence and survival. Because the TRC reaction is more rapid and simpler than reverse transcriptase-polymerase chain reaction as a format for detecting RNA sequences, it may enhance the genetic diagnosis of cancer micrometastasis and may improve cancer therapy.

Biomarkers, Tumor↗

PubMed2003

Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel.

We developed a completely homogeneous and isothermal method of detecting RNA sequences and demonstrated ultrarapid and direct quantification of pathogenic gene expression with high sensitivity. The assay is based on performing isothermal RNA sequence amplification in the presence of our novel DNA probe, an intercalation activating fluorescence DNA probe, and measuring the fluorescence intensity of the reaction mixture. When detecting mecA gene expression of methicillin-resistant Staphylococcus aureus, we quantified starting copies ranging from 10 to 10(7) copies within 10min. The primer sequences were designed to bind to secondary structure-free sites of the target RNA, which enabled a totally isothermal protocol to quantify mRNA specifically in a sample of existing genomic DNA. When we applied this to quantifying the expression of marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG strain, the results correlated well with the viability of each bacterium. We also demonstrated monitoring Pab gene expression of M. bovis BCG during cultivation with antibiotics. The present method can potentially realize rapid antimicrobial susceptibility testing of slowly growing organisms, such as tuberculosis.

Base Sequence↗

PubMed2002

Regulated lens regeneration from isolated pigmented epithelial cells of newt iris in culture in response to FGF2/4.

When a lens is removed from the newt eye, a new lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish lens regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a lens with the synthesis of lens-specific crystallins, recapitulating the normal lens regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro lens regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in lens forming ability between them.

Animals↗