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T Higashiyama

Publications and source records attributed to T Higashiyama.

At least 37 records · Page 2Linked to original sources
PubMed1999

Measurement of the expression of oncofetal fibronectin mRNA in thyroid carcinomas by competitive reverse transcription-polymerase chain reaction.

Abundant expression of oncofetal fibronectin mRNA has been observed in thyroid papillary and anaplastic carcinomas. In this study, we measured relative expression levels of oncofetal fibronectin mRNA in thyroid cancer tissues by competitive reverse transcription polymerase chain reaction (RT-PCR) using thyroglobulin mRNA as an internal control. By this method, all papillary and anaplastic carcinomas and 3 of 6 follicular carcinomas were distinguished from benign tissues, such as normal thyroid tissues, follicular adenomas, and adenomatous goiters. Furthermore, 2 anaplastic carcinomas were clearly distinguished from differentiated carcinomas. These results suggest the possibility of establishing a more accurate preoperative or postoperative diagnosis of papillary and anaplastic carcinomas by measuring the relative expression level of oncofetal fibronectin to thyroglobulin in thyroid tumors.

Adenoma↗
PubMed1999

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia.

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.

Base Sequence↗
PubMed1999

Preoperative diagnosis of medullary thyroid carcinoma by RT-PCR using RNA extracted from leftover cells within a needle used for fine needle aspiration biopsy.

Fine needle aspiration Biopsy (FNAB) is commonly used to diagnose thyroid tumors. In some clinical situations, however, accurate diagnosis requires a more objective method than cytological examination alone. Medullary thyroid carcinomas (MTC) derive from C cells in the thyroid and express some specific messenger RNAs (mRNA), such as those transcribed from the RET proto-oncogene, the calcitonin gene, and the gene for carcinoembryonic antigen (CEA), which usually do not exist in normal thyroid follicular cells or thyroid tumors of follicular epithelial descent. Recently, we established a new method for the molecular diagnosis of thyroid tumors without additional invasion to the patient by extracting RNA for RT-PCR from the leftover cells inside the needles used for fine needle aspiration biopsy (Aspiration Biopsy-Reverse Transcription-Polymerase Chain Reaction, ABRP). By applying the ABRP method to the detection of RET, calcitonin, and CEA mRNAs, an accurate molecular-based diagnosis for MTC maybe established as an adjunct to cytological diagnosis. In this study, 35 aspirates were obtained at the time of surgery from thyroid tumors, including 11 MTCs. The expression of these mRNAs in the leftover cells inside the needles used for the aspiration was then examined. Transcripts from all three genes were detected in the samples from all 11 MTCs, but none of these mRNAs were detected in the other tumors or normal thyroid tissues. Furthermore, MTC was preoperatively diagnosed in three patients by ABRP detection of these mRNAs, and these diagnoses were confirmed by subsequent cytological and histopathological analyses. Thus RT-PCR detection of RET, calcitonin, and CEA mRNAs in FNABs may be an efficient molecular adjunct for diagnosing MTC.

Adult↗
PubMed1999

Screening of specific changes in mRNAs in thyroid tumors by sequence specific differential display: decreased expression of c-fos mRNA in papillary carcinoma.

To examine the specific changes in gene expression in thyroid carcinomas, we performed sequence specific-differential display (SS-DD) analysis by using a specific degenerate primer for the superfamily of the small G protein. After subcloning and sequencing analysis, one of the genes that showed decreased expression in thyroid papillary carcinomas was revealed to be the proto-oncogene c-fos. Expression of c-fos mRNA in benign and malignant tissues was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. The expression of c-fos was detected in all twelve normal thyroid tissues, whereas it decreased in thirteen of fifteen papillary carcinomas. These results indicate that the increased expression of c-fos mRNA is not necessarily to be an oncogenic feature of thyroid tumors. Further, its constant expression in normal thyroid tissues may play a role in the maintenance of thyroid function.

Adenoma↗
PubMed1998

Accurate and objective preoperative diagnosis of thyroid papillary carcinomas by reverse transcription-PCR detection of oncofetal fibronectin messenger RNA in fine-needle aspiration biopsies.

Recently, the restricted expression of oncofetal fibronectin mRNA was reported in thyroid papillary and anaplastic carcinomas. In this study, by extracting RNA from the leftover cells inside the needles used for fine-needle aspiration biopsy, we establish a new method for gene diagnosis of these carcinomas without further invasiveness to the patient (aspiration biopsy-reverse transcription-PCR, ABRP). RNA was extracted from 177 fine-needle aspiration biopsies of thyroid nodules that were suspicious for malignancy, and then the gene diagnoses made by reverse transciption-PCR detection of oncofetal fibronectin mRNA were compared with cytological diagnoses. Thirty-five (94.6%) of 37 samples that were diagnosed as papillary or anaplastic carcinomas by cytological examination showed a positive result by gene diagnosis, whereas only 4 (3.7%) of 109 samples that were cytologically diagnosed negative for both carcinomas showed a positive result. Among all of the cases, 50 patients underwent surgery, and a histological diagnosis was consequently made. The sensitivity and specificity of this method were 96.9 and 100%, respectively. A combined examination using both genetic and cytological approaches may contribute to a more precise preoperative diagnosis of papillary and anaplastic carcinomas.

Adolescent↗
PubMed1998

The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells.

In the isogamous green alga Chlamydomonas reinhardtii, the chloroplast genome is transmitted from the mt+ parent, while the mitochondrial genes are believed to be inherited from the mt- parent. Chloroplast nucleoids have been visualized by DAPI (4,6-diamidino-2-phenylindole) staining, and the preferential digestion of the mt- chloroplast nucleoids has been observed in young zygotes. However, the mitochondrial nucleoids have never been visualized, and their behavior is only deduced from genetic and biochemical studies. We discovered that the mitochondrial and chloroplast genomes can be visualized simultaneously in living cells, using the fluorescent dye SYBR Green I. The ability to visualize the mitochondrial and chloroplast genome in vivo permits the direct observation of the number, distribution and behavior of the chloroplast and mitochondrial nucleoids in young zygotes. Using this method, the biparental transmission of the mitochondrial genome was revealed.

Animals↗
PubMed1998

Guidance in vitro of the pollen tube to the naked embryo sac of torenia fournieri

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.

Journal Article↗
PubMed1997

Zepp, a LINE-like retrotransposon accumulated in the Chlorella telomeric region.

Six copies of insertion elements accumulate in the subtelomeric region immediately proximal to the telomeric repeats on Chlorella chromosome I. The elements, designated Zepps, bear the characteristic features of non-viral (LINE-like) retrotransposons, including a poly(A) tail, 5'-truncations, a retroviral reverse transcriptase-like ORF and flanking target duplications. Detailed sequence analysis of the Chlorella subtelomeric region revealed a novel mechanism of Zepp transposition; successive insertions of each Zepp element into another Zepp as a target, leaving a tandem array of their 3'-regions with poly(A) tracts facing toward the centromere. Only the most distal Zepp copy was inverted to connect its poly(A) tail with the telomeric repeats. A similar Zepp cluster but without the telomeric repeats was also found at the terminus of another Chlorella chromosome. These structures contrast with that proposed for the addition of HeT-A and TART elements to Drosophila telomeres. Expression of Zepp elements is induced by heat shock treatment. Possible roles of the subtelomeric retrotransposons in formation and maintenance of telomeres are discussed.

Amino Acid Sequence↗
PubMed1997

DNA staining for fluorescence and laser confocal microscopy.

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

Animals↗
PubMed1997

Diverse function of aromatase and the N-terminal sequence deleted form.

The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

Aromatase↗
PubMed1996

Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450.

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.

Animals↗
PubMed1996

Diverse functions of aromatase: O-deethylation of 7-ethoxycoumarin.

In studying the diverse functions of aromatase we found that purified and reconstituted aromatase also catalyzes O-deethylation of 7-ethoxycoumarin. Aromatase cytochrome P450 was purified from human term placentas by monoclonal antiaromatase P450 antibody-Sepharose 4B column chromatography. Kinetic analysis of the O-deethylation of 7-ethoxycoumarin by reconstituted aromatase showed Km of 200 microM, Vmax of 12.5 nmol.min-1.mg-1, and turnover rate of 1.06 min-1. 7-Ethoxycoumarin competitively inhibited androstenedione aromatization, the Ki was 180 microM. Fadrozole (CGS16949A), a specific competitive aromatase inhibitor, and MAb3-2C2, an antiaromatase P450 monoclonal antibody, inhibited both aromatase and 7-ethoxycoumarin O-deethylase activities dose responsively. The IC50 of Fadrozole was 33 nM for aromatase and 67 nM for 7-ethoxycoumarin O-deethylase. The IC50 of MAb3-2C2 was 1.1 micrograms IgG for aromatase and 4.0 micrograms IgG for 7-ethoxycoumarin O-deethylase. These results indicate that the two enzyme activities are catalyzed by the same active site of the cytochrome P450. Contrary to the previous postulate on the mechanism-based inactivation of microsomal aromatase by 4-androstene-3,6,17-trione, we found that with purified aromatase, both the initial 19-hydroxylase and the after lyase reactions are simultaneously inactivated by the steroid suicide inhibitor.

Androstenes↗
PubMed1995

Structural study of the glycosylated and unglycosylated forms of a genetic variant of human serum albumin (63 Asp-->Asn).

A genetic variant of human serum albumin (alloalbumin) exhibited atypical electrophoretic mobility and chromatographic behavior apparently because of the effect of a point substitution on the molecular conformation. Three forms of albumin were isolated by DEAE HPLC chromatography: normal albumin, and two variant forms V1 and V2. The point substitution (Asp-63-->Asn) generated a canonical tripeptide acceptor sequence for glycosylation with an N-linked oligosaccharide (Asn-Lys-Ser). Neuraminidase digestion followed by electrophoresis showed that the V2 variant form was glycosylated and the V1 form was not. Time-of-flight mass spectrometry yielded a molecular weight of about 2000 for the carbohydrate. Structural analysis of the carbohydrate was done by chromatographic comparison of the pyridylaminated derivatives with standards and was confirmed by proton NMR of the three pronase glycopeptides and of the pyridylaminated oligosaccharide. The oligosaccharide had a complex biantennary structure with two sialic acid residues. In normal albumin Asp-63 is exposed and is adjacent to the first disulfide bond, Cys-62-->Cys-53. The apparent effect on molecular conformation resulting in incomplete glycosylation and atypical electrophoretic mobility suggests that glycosylation may interfere with disulfide bond formation at this site.

Amino Acid Sequence↗
PubMed1995

Molecular organization of Chlorella vulgaris chromosome I: presence of telomeric repeats that are conserved in higher plants.

The unicellular green alga Chlorella vulgaris (strain C-169) has a small genome (38.8 Mb) consisting of 16 chromosomes, which can be easily separated by CHEF gel electrophoresis. We have isolated and characterized the smallest chromosome (chromosome I, 980 kb) to elucidate the fundamental molecular organization of a plant-type chromosome. Restriction mapping and sequence analyses revealed that the telomeres of this chromosome consist of 5'-TTTAGGG repeats running from the centromere towards the termini; this sequence is identical to those reported for several higher plants. This sequence is reiterated approximately 70 times at both termini, although individual clones exhibited microheterogeneity in both sequence and copy number of the repeats. Subtelomeric sequences proximal to the termini were totally different from each other: on the left arm, unique sequence elements (14-20 bp) which were specific to chromosome I, form a repeat array of 1.7 kb, whereas a 1.0 kb sequence on the right arm contained a poly(A)-associated element immediately next to the telomeric repeats. This element is repeated several times on chromosome I and many times on all the other chromosomes of this organism.

Base Sequence↗
PubMed1995

Telomere and LINE-like elements at the termini of the Chlorella chromosome I.

The telomeres of Chlorella chromosomes consisted of 5'-TTTAGGG repeats, which are exactly the same as those of higher plants. This sequence was reiterated approximately 70 times at both termini of chromosome I. Subtelomeric sequences next to the telomeres were totally different between the right and left arms. On the left-side subtelomeric region, polyA associated LINE (long interspersed element)-like elements were found tandemly repeated just next to the telomeric repeats. It is very interesting to compare this unique structure with telomeres of Drosophila, where a transposable element play a major role in forming telomerase-generated repeats. We propose a mechanism of transposon-mediated healing of a broken chromosomal end that would operate in the unicellular green alga Chlorella.

Animals↗
PubMed1995

Molecular characterization of Chlorella chromosomes: screening of bent DNAs.

As a model for plant-type chromosomes, we have been characterizing molecular organization of Chlorella vulgaris (C-169) chromosome I. To locate structurally important chromosome regions, bent DNA fragments of this chromosome were systematically obtained and analyzed. By two-dimensional polyacrylamide gel electrophoresis, 150 clones containing bent DNA fragments were screened from C. vulgaris genomic libraries. These clones were classified into 3 groups by Southern hybridization. A group that hybridized with all Chlorella chromosomes contained fragments carrying a retroposon-like element. A clone (p2DS-66A) derived from chromosome VI strongly hybridized with chromosome I as well as the others. The corresponding chromosome I sequences (pI-21 and pI-31) were analyzed in detail.

Base Sequence↗
PubMed1993

Characterization of the terminal inverted repeats and their neighboring tandem repeats in the Chlorella CVK1 virus genome.

A unique group of large icosahedral viruses that infect a unicellular green alga (Chlorella sp. NC64A) were isolated from freshwater sources in Japan. These viruses contain a linear double-stranded DNA (dsDNA) genome with hairpin ends. A physical map was constructed for the genomic DNA of CVK1 (Chlorella virus isolated in Kyoto, no. 1) by pulsed-field gel electrophoresis of restriction fragments. The nucleotide sequences around both termini of the CVK1 DNA revealed the presence of inverted terminal repeats (ITR) of approximately 1.0 kb. Adjacent to the ITR, unique sequence elements of 10 to 20 bp were directly repeated 20 to 30 times in tandem array. Several copies of these repeat elements were deleted in virus mutants that were occasionally generated from Chlorella cells that were in a putative CVK1 carrier state. These repeats might represent a hot spot of rearrangement in the CVK1 genome.

Base Sequence↗