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Biomedical subjects

Y Hanaoka

Publications and source records attributed to Y Hanaoka.

At least 19 recordsLinked to original sources
PubMed2001

Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)

Animals↗
PubMed2001

Advantages of the digital X-ray system in dental identification of persons with reference to two murder cases.

Direct digital X-ray technology was applied to dental identification of victims in two murder cases using the Compuray system. In both cases the digital radiography proved to be simple to use, quick and effective, allowing superimposition, enlargement and transportability to a mortuary. These are the first reported uses of the technology in Japan and further development promises the transmitability of data and images electronically to remote locations, further enhancing its usefulness. Comparing the skull with the dental ante-mortem X-ray films and records of a specific person who was reported "missing", we found many identical points between the two, especially in regard to the X-ray findings with the Compuray. In both cases we obtained a large number of X-ray images in a remarkably short time and this was very useful for identification by means of the teeth.

Adolescent↗
PubMed2000

An autopsy case of Klinefelter's syndrome suspected and its DNA analysis.

UNLABELLED: We experienced an autopsy case, small testes and tall stature, which suggested Klinefelter's syndrome. DNA analysis was performed to confirm the genetic abnormality. CASE HISTORY: A 28-year-old man who was single and lived with his parents. He suddenly lost his consciousness in a sitting room and died. Autopsy findings: He was 176 cm in height and 57 kg in weight. The post-mortem hypostasis was red-purple on his back, and rigor mortis was strong in each joint of the whole body. The heart weighted 340 g, in which dark red fluidal blood (300 ml) without coagulation was contained. The testes were smaller than normal adult male (left and right testes with epididymides weighted 8.1 g and 6.0 g, respectively). As a results of pathological examination, clumped Leydig cells, sclerotic and hyalined tubules were observed. Some germ cells with spermatozoid were also present. DNA Analysis: Generally, Klinefelter's syndrome is determined by karyotype analysis and/or the detection of sex chromatin. However, in this case, karyotype analysis and the detection of sex chromatin could not be demonstrated, because the blood which was collected in the autopsy became too old. Therefore, we tried sex determination and STR analysis (HPRT, HUMARA and DXS 1470) using DNA extracted from stored blood materials. Consequently, in the sex determination, no different situation was found in the X- and Y-specific bands from normal male's and as results of STR analyses, we could not corroborate the Klinefelter's syndrome.

Adult↗
PubMed2000

Molecular cloning of cDNA encoding Xenopus laevis deoxyribonuclease I.

A 1200-bp cDNA encoding Xenopus laevis deoxyribonuclease I (X. laevis DNase I) was constructed from the total RNA of a X. laevis pancreas using a rapid amplification of cDNA ends method. When the cDNA was transiently transfected into COS-7 cells, the recombinant polypeptide exhibited similar enzymological properties to those of the native pancreatic DNase I. The recombinant enzyme was considerably more labile than most other vertebrate DNase I enzymes. The X. laevis DNase I polypeptide was larger than any other known vertebrate DNase I, containing a unique Cys-rich stretch of 68 or 70 amino acid residues at the carboxyl terminus, and it had less well conserved binding sites for the Ca2+, G-actin and DNA, and two DNase I signature motifs. These alterations might account for its heat instability.

Amino Acid Sequence↗
PubMed2000

Cloning and expression of the gene encoding thermostable poly(3-hydroxybutyrate) depolymerase.

The gene encoding extracellular poly(3-hydroxybutyrate) depolymerase from a thermophilic poly(3-hydroxybutyrate)-degrading bacterium, strain HS, was cloned and intracellularly expressed in Escherichia coli. The gene was found to consist of 1485-bp nucleotide sequence coding for a 22-amino-acid signal peptide and a 473-amino-acid mature protein. Phylogenetic analysis and domain structure showed that the enzyme was clustered with type II PHB depolymerases. The gene was expressed in E. coli under the control of the tac promoter. A 46-kDa protein was detected in the cell extract by SDS-PAGE. The N-terminal sequence of the protein agreed with that of the original enzyme. The crude enzyme was able to degrade PHB particles at 70 degrees C.

Journal Article↗
PubMed1999

Xenopus laevis pancreatic DNase I: purification and immunological characterization.

Deoxyribonuclease I (DNase I) was purified from Xenopus laevis pancreas to apparent electrophoretic homogeneity using a series of column chromatographies. The purified enzyme showed a molecular mass of about 36 kDa and maximum activity at pH 7.0-8.0, required divalent cations, Ca2+ and Mg2+, for its activity, and was inhibited by EDTA, EGTA and an antibody specific to the enzyme, but not by G-actin. The N-terminal amino acid sequence of the enzyme up to the 37th residue shared 38-44% homology with that of mammalian DNases I derived from bovine, ovine, porcine, rat, mouse, rabbit and human. A systematic survey of DNase I activity distribution in 20 different kinds of frog tissues showed that the pancreas and rectum produced higher amounts than other tissues. This is the first report concerning the purification and chemical and immunological characterization of frog pancreatic DNase I.

Amino Acid Sequence↗
PubMed1998

Gonadotropins and reproductive function in the anuran amphibian, Rana esculenta.

In this study, the measurement both of peripheral gonadotropins (FSH and LH) and of sex steroids in male and female wild anuran, Rana esculenta, was performed during the annual reproductive cycle; moreover, the role of gonadotropins in the vitellogenic process and in ovarian steroidogenesis was investigated through in vitro experiments. LH plasma changes in males showed high values during autumn-winter months and during the mating period, when high androgen levels were found. Conversely, for the first time in male vertebrates, a clear correspondence between plasma FSH and estradiol-17beta (E2) was shown. In females, FSH peak values were found at the beginning of the mating period in parallel with those of plasma vitellogenin (VTG) and E2; in contrast, high LH levels went together with ovarian weight (gonadosomatic index), which is considered a good marker for the plasma sequestration of VTG by growing oocytes. The in vivo results are corroborated by in vitro studies showing the direct effects of both FSH and LH in inducing hepatic VTG synthesis and release in the culture media. Lastly, although it is not yet known whether or not FSH and LH have separate functions in amphibians, it was clearly shown that they induce ovarian steroid production. These results are discussed in terms of the high seasonality previously demonstrated in this wild frog.

Androgens↗
PubMed1998

D4S43 locus DNA typing in the Japanese population and application to teeth with degraded DNA.

VNTR polymorphism at the D4S43 locus was investigated, and allele frequencies in the Japanese population sample were determined by the polymerase chain reaction. Eleven different alleles and 16 genotypes were observed in 131 unrelated Japanese. The most common allele was one repeat unit (60.3%), the heterozygosity index of the present study was 58.7%, and the polymorphic information content was calculated at 0.55. In addition, we found four new size variations in the one-repeat-unit allele, which was the most common allele among the Japanese population. These variations were believed to be different from variations by repeats of the basic 14-bp unit. The one-repeat-unit allele at the D4S43 locus, which comes in four sizes, was detected in all of the samples, including degraded DNA samples obtained from hard tooth tissue. Therefore, this new variation is useful in personal identification by DNA analysis using forensic scientific degraded DNA samples.

Alleles↗
PubMed1997

Hyperpolarization-activated Cl- current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes.

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1-10 microM) activated an inward rectifier current at membrane potentials more negative than -60 mV without causing any significant change in currents at potentials more positive than -60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at -22.5 mV, close to the theoretical value of Cl- equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl-]o. This current was blocked by Cl- channel blocker SITS and Ba2+. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl- current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.

Animals↗
PubMed1996

A catechol derivative (4-methylcatechol) accelerates the recovery from experimental acrylamide-induced neuropathy.

Acrylamide (ACR) monomer produces neuropathy of the dying-back type and 4-methylcatechol (4-MC) is a potent stimulator of endogenous nerve growth factor synthesis. In the present study, we investigated the efficacy of 4-MC in promoting recovery from experimental ACR neuropathy in rats. Twenty-two Sprague-Dawley rats were made neuropathic by ACR injections. They showed hindlimb paralysis, increment of landing foot spread distance and a statistically significant reduction in motor nerve conduction velocity. After the ACR neuropathy had been established, 12 of the rats were administered 4-MC for 2 weeks, and the other 10 were injected with phosphate-buffered saline alone. 4-MC-administered ACR neuropathy rats showed improvement, i.e., a decrease in landing foot spread distance, increase in motor nerve conduction velocity and increase in nerve growth factor content in the sciatic nerves in comparison with the corresponding values for ACR neuropathy rats given phosphate-buffered saline alone. A decreased number of large myelinated fiber with a reciprocal increase in small myelinated fiber number also was seen in the ACR neuropathy rats; however, this change was ameliorated in part by the administration of 4-MC. Therefore, these findings suggest that 4-MC can accelerate the recovery process clinically, electrophysiologically, biochemically and neuropathologically.

Acrylamides↗
PubMed1996

Sex determination from blood and teeth by PCR amplification of the alphoid satellite family.

Sex determination from blood and teeth by PCR amplification of the alphoid satellite family according to the method of Witt and Erickson was investigated. Amplification of the X-(131 bp) and Y-(172 bp) specific sequences in males and that of the X-specific sequence in females was satisfactory in almost all samples. However, of the 22 samples (blood: 5, dental pulp: 12, tooth hard tissue: 5), a false positive fragment was detected at 172 bp among the female samples (blood: 17, dental pulp: 30, tooth hard tissue: 23) as a result of silver staining. These bands were not amplified by the dual PCR method and were different from the Y-specific band. Subsequent restriction enzyme digestion (DdeI, HinfI, and NciI) of these bands resulted in different patterns from the typical Y-specific band. Furthermore, in five male samples of hard tooth tissue specimens (n = 14), a Y-specific band was detected as a weak band, and in two of these five samples, this weak specific band was not amplified by dual PCR in spite of the male origin of the samples. These results showed that this method is useful for sex determination, but that dual PCR and restriction analysis should be used together in practical applications.

Base Sequence↗
PubMed1995

Development and application of a homologous radioimmunoassay for Xenopus prolactin.

A specific and sensitive homologous radioimmunoassay (RIA) for Xenopus prolactin (xPRL) was developed. PRL isolated from X. laevis pituitary glands was used for generating antiserum in a rabbit, for radioligand and for the standard. Pituitary homogenates and plasma from adult Xenopus produced displacement curves parallel to the xPRL standard. Plasma from hypophysectomized Xenopus showed negligible cross-reactivity. Purified PRLs from other amphibian species such as the bullfrog (Rana catesbeiana) and toad (Bufo japonicus) gave inhibition curves which did not parallel the standard. Ovine PRL, mouse PRL, newt (Cynops pyrrhogaster) PRL, bullfrog GH, and bullfrog LH showed no inhibition of binding even at relatively high doses in this RIA. The sensitivity of the RIA was 0.122 +/- 0.005 ng xPRL/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 2.46 and 6.65%, respectively. Histological studies of Xenopus adenohypophyses revealed that the cells which reacted immunologically with the antiserum against xPRL corresponded to those positively stained with antiserum against bullfrog PRL. A preliminary application of this homologous RIA for xPRL was performed by evaluating plasma and pituitary PRL levels in subadult and adult Xenopus of both sexes.

Animals↗
PubMed1995

Comparison of cardiovascular responses to airway management: fiberoptic intubation using a new adapter, laryngeal mask insertion, or conventional laryngoscopic intubation.

STUDY OBJECTIVES: To evaluate the circulatory effects of fiberoptic intubation with a newly developed mask adapter and to compare these effects with those obtained with a conventional laryngoscope and laryngeal mask airway with general anesthesia. DESIGN: Randomized study. SETTING: Operating room at a university hospital. PATIENTS: 30 ASA physical status I adult patients scheduled for minor elective surgery. INTERVENTIONS: A Macintosh laryngoscope was used for intubation in Group L (n = 10), a fiberoptic bronchoscope with newly developed mask adapter was used in Group FI (n = 10), and a laryngeal mask airway was used in Group LMA (n = 10). Anesthesia was induced with thiamylal, fentanyl, and vecuronium. Patients were manually ventilated for 4 minutes with sevoflurane. Then their tracheas were intubated with a laryngoscope or fiberoptic bronchoscope, or a laryngeal mask airway was inserted. MEASUREMENTS AND MAIN RESULTS: Blood pressure (BP) and heart rate (HR) were measured continuously. After intubation, the magnitude of change of both BP and HR was greatest in Group L, next highest in Group FI, and lowest in Group LMA. CONCLUSION: Fiberoptic intubation with general anesthesia using the newly developed mask adapter offers an advantage over the standard laryngoscope for patients in whom pressor response of intubation is potentially hazardous.

Adult↗
PubMed1995

[Fundamental and practical study for DNA analysis using tooth as a source of DNA].

Degree of degradation and the yield of DNA extracted from dental pulp tissues were examined on the tooth samples (n = 50) stored at room temperature and the method of DNA extraction from tooth hard tissues was also investigated. The DNA samples obtained were also applied to forensic odontological material examination including DNA fingerprinting using a probe Myo and VNTR (variable number of tandem repeat) analysis in D4S43 locus by PCR. The amount of DNA obtained from the dental pulp tissue of a single tooth varied approximately from 3 to 40 micrograms. In most cases, high molecular weight DNA was still present in samples stored at room temperature for at least 336 days. When the dental pulp tissue samples were less than 5 mg in weight, the amount DNA extracted was usually less than 10 micrograms, however when the samples were more than 5 mg in weight, the amount of DNA extracted was more than 10 micrograms. No correlation was observed between the storage period of the tooth samples and the DNA extraction ratio (the amount of extracted DNA weight, micrograms/pulp weight, mg). The efficiency of DNA extraction from tooth hard tissues was investigated under different conditions using 0.005 M and 0.5 M EDTA solutions for decalcification. DNA was efficiently extracted from the tooth samples which were decalcified for one week without changing the 0.5 M EDTA solution or by changing the solution once within a week. Rapid decalcification using formic acid buffer was not suitable for DNA extraction from tooth hard tissues. Southern blot hybridization of DNA samples extracted from pulp tissues using Myo probe gave multiple bands. Finger print patterns obtained from DNA recovered from dental pulp and tooth hard tissues samples were identical, however, the number of hybridizing bands obtained from tooth hard tissues was less than that obtained from blood and dental pulp tissues. The D4S43 typing using DNA recovered from blood stains, dental pulp tissues and tooth hard tissues of the same individuals was in agreement with each other and the 184bp fragment was efficiently amplified in all the samples tested. The DNA obtained from dental pulp tissues usually contains high molecular weight DNA and was suitable for multilocus probe and PCR analysis. However, the DNA obtained from tooth hard tissues was suitable only for PCR analysis.

Adolescent↗