Kazumi Umeki

Regional localization of the gene for thyroid peroxidase to human chromosome 2p25 and mouse chromosome 12C

Thyroid peroxidase (TPO) plays a central role in thyroid gland function. The enzyme catalyzes two important reactions of thyroid hormone synthesis, i.e., the iodination of tyrosine residues in thyroglobulin and phenoxy-ester formation between pairs of iodinated tyrosines to generate the thyroid hormones, thyroxine and triiodothyronine. Previously, we isolated the cDNAs encoding human and mouse TPOs and assigned the human TPO gene to the short arm of chromosome 2 by somatic cell hybrid mapping. By a similar analysis of DNA from somatic cell hybrids, the human TPO gene was mapped to 2pter-p12. The mouse TPO gene was localized to chromosome 12 using a rat TPO cDNA as a probe to hybridize with mouse-hamster somatic cell hybrids. In this study, we used fluorescence in situ hybridization (FISH) to confirm the localization of human and mouse TPO genes to human chromosome 2 and mouse chromosome 12 and to assign them regionally to 2p25 and 12C, respectively. 7 refs., 1 fig.

Partial iodide organification defect caused by a novel mutation of the thyroid peroxidase gene in three siblings

background Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808–13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their fathers DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well‐conserved amino acids in the peroxidase superfamily, Gly533Cys‐ and Asp574/Leu575del‐TPOs were thought to be affected structurally or functionally. In expression studies using CHO‐Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild‐type TPO and had enzyme activity, although Gly533Cys‐TPO was slightly lower in efficiency of expression and more degenerative than wild‐type TPO.

IMMUNOHISTOCHEMICAL LOSS OF THYROID PEROXIDASE IN PAPILLARY THYROID CARCINOMA: STRONG SUPPRESSION OF PEROXIDASE GENE EXPRESSION

It is believed that qualitative changes in thyroid peroxidase (TPO) cause decreased enzyme activity in differentiated thyroid carcinoma. To re‐evaluate TPO expression in thyroid cancer, TPO mRNA expression was compared with TPO protein expression in 38 samples of thyroid tissue obtained from patients with various thyroid diseases. In Northern blot studies, while TPO mRNA was highly expressed in tissues from all 18 benign lesions, it was strongly suppressed in 14 tumours, including 12 out of 12 papillary carcinomas, one of seven follicular carcinomas, and one medullary carcinoma. TPO mRNA was not detected in six carcinomas, of which four were papillary, one follicular, and one medullary, by the usual Northern blot method. The 14 cases with strong underexpression of TPO mRNA were very weakly stained with anti‐TPO monoclonal antibody 38E, whereas all 18 benign tissues were strongly stained. Moreover, a comparative study of TPO expression by Northern blot and immunohistochemical analysis revealed a positive correlation between TPO mRNA expression and the staining intensity of TPO protein. These results suggest that strong suppression of TPO mRNA transcription causes low TPO activity in papillary carcinoma; immunohistochemical loss of TPO may be a useful diagnostic marker. TPO mRNA expression in differentiated thyroid carcinomas did not always correlate with the mRNA expression of thyroglobulin, thyroid stimulating hormone receptor, and thyroid transcription factor 1.

Stable high level expression of human thyroid peroxidase in cultured Chinese hamster ovary cells

An expression plasmid containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs was transfected into chinese hamster ovary cells. The stably transformed cells constitutively expressed immunoreactive thyroid peroxidase on the cell surface. These cells were further used to establish a subline producing a large amount of thyroid peroxidase by selecting clones resistant to methotrexate. The molecular weight of the expressed thyroid peroxidase was the same as purified human thyroid peroxidase. This expressed protein had peroxidase activity when determined by guaiacol oxidation. Furthermore, the expressed thyroid peroxidase was immunoreactive to sera of patients with autoimmune thyroid disease in which autoantibodies to thyroid peroxidase appeared.

Iodide organification defects resulting from cosegregation of mutated and null thyroid peroxidase alleles.

This report describes an intriguing combination of the thyroid peroxidase (TPO) alleles resulting in an iodide organification defect. Sequence analysis of the patients TPO gene showed the presence of T-deletion in exon 14 of the TPO gene (T2512del). From the sequencing pattern, this new mutation of the TPO gene was thought to be homozygous. mRNA transfection studies in which mutated mRNA was transfected to CHO-K(1) cells by electroporation showed that the cells transfected with mutated mRNA expressed smaller TPO molecules than those of cells transfected with wild-type mRNA and that they had TPO activity. However, the smaller TPO molecules could not translocate onto the cell surface. To investigate T2512del in the parents, their genomic DNAs were sequenced. Results showed that the mother had T2512del but the father did not. However, when seven polymorphic positions reported earlier were analyzed, the mother showed two kinds of nucleotides at four positions but the patient and father showed only one nucleotide at all seven positions. We suspected a deletion of the TPO gene (2p25) in one of two second chromosomes, and analyzed the patients chromosomes by FISH using TPO cDNA and N-myc genomic DNA as probes. N-myc genomic DNA exhibited two signals and TPO cDNA only one signal, although the G-band showed no morphological abnormalities. T2512-deleted and 2p25-deleted null alleles cosegregated from her parents, resulting in iodide organification defect in the patient.

Engraftment of peripheral blood mononuclear cells from human T-lymphotropic virus Type 1 carriers in NOD/SCID/γcnull (NOG) mice

The transmission of human T‐lymphotropic virus Type 1 (HTLV‐1) occurs mainly via breast‐feeding, sexual intercourse and blood transfusions. After transmission, the HTLV‐1 infection is predominantly maintained by cell‐to‐cell infection and clonal expansion; however, the details have not yet been clarified. To investigate how HTLV‐1 infected cells act in an environment without an effective immune reaction, peripheral blood mononuclear cells (PBMCs) from asymptomatic HTLV‐1 carriers were inoculated into nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull (NOG) mice, which have immunological dysfunctions of T‐ and B‐lymphocytes and NK cells. Human mononuclear cells including both CD4+ and CD8+ T cells were found to have infiltrated into various organs, including the liver, kidney, spleen and lung, when the mice were sacrificed 1 month after inoculation. The copy numbers of HTLV‐1 provirus detected in the tissue‐infiltrating human cells were much higher than those in the original PBMCs from the carriers. The expression of HTLV‐1 mRNA was demonstrated in the tissue‐infiltrating cells by reverse transcriptase‐polymerase chain reaction. Inverse‐long polymerase chain reaction showed that the pattern of HTLV‐1 proviral integration was different from that of the original carrier and that it varied among NOG mice inoculated with PBMCs from the same carrier. These results suggest the selective proliferation of particular clones of HTLV‐1 infected cells in NOG mice. Alternatively, transmission and new integration of HTLV‐1 from infected cells to noninfected cells might have occurred in an environment without an effective immune reaction. The NOG mouse is considered a good animal model for the patho‐physiological study of HTLV‐1 infection with immunodeficiency.

Rapid Identification and Subtyping of Helicobacter cinaedi Strains by Intact-Cell Mass Spectrometry Profiling with the Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Helicobacter cinaedi infection is recognized as an increasingly important emerging disease in humans. Although H. cinaedi-like strains have been isolated from a variety of animals, it is difficult to identify particular isolates due to their unusual phenotypic profiles and the limited number of biochemical tests for detecting helicobacters. Moreover, analyses of the 16S rRNA gene sequences are also limited due to the high levels of similarity among closely related helicobacters. This study was conducted to evaluate intact-cell mass spectrometry (ICMS) profiling using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a tool for the identification of H. cinaedi. A total of 68 strains of H. cinaedi isolated from humans, dogs, a cat, and hamsters were examined in addition to other Helicobacter species. The major ICMS profiles of H. cinaedi were identical and differed from those of Helicobacter bilis, which show >98% sequence similarity at the 16S rRNA sequence level. A phyloproteomic analysis of the H. cinaedi strains examined in this work revealed that human isolates formed a single cluster that was distinct from that of the animal isolates, with the exception of two strains from dogs. These phyloproteomic results agreed with those of the phylogenetic analysis based on the nucleotide sequences of the hsp60 gene. Because they formed a distinct cluster in both analyses, our data suggest that animal strains may not be a major source of infection in humans. In conclusion, the ICMS profiles obtained using a MALDI-TOF MS approach may be useful for the identification and subtyping of H. cinaedi.

Downregulation of CDKN1A in Adult T-Cell Leukemia/Lymphoma despite Overexpression of CDKN1A in Human T-Lymphotropic Virus 1-Infected Cell Lines

ABSTRACT Human T-lymphotropic virus 1 (HTLV-1) causes an aggressive malignancy of T lymphocytes called adult T-cell leukemia/lymphoma (ATLL), and expression of HTLV-1 Tax influences cell survival, proliferation, and genomic stability in the infected T lymphocytes. Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21waf1/Cip1) is upregulated by Tax, without perturbation of cell cycle control. During an analysis of the gene expression profiles of ATLL cells, we found very low expression of CDKN1A in ATLL-derived cell lines and ATLL cells from patient samples, and epigenetic abnormalities including promoter methylation are one of the mechanisms for the low CDKN1A expression in ATLL cells. Three HTLV-1-infected cell lines showed high levels of expression of both CDKN1A and Tax, but expression of CDKN1A was detected in only two of six ATLL-derived cell lines. In both the HTLV-1-infected and ATLL cell lines, we found that activated Akt phosphorylates CDKN1A at threonine 145 (T145), leading to cytoplasmic localization of CDKNIA. In HTLV-1-infected cell lines, cytoplasmic CDKN1A did not inhibit the cell cycle after UV irradiation; however, following treatment with LY294002, a PI3K inhibitor, CDKN1A was dephosphorylated and relocalized to the nucleus, resulting in suppression of the cell cycle. In the ATLL cell lines, treatment with LY294002 did not inhibit the cell cycle but induced apoptosis with the cytoplasmic localization. Therefore, the low CDKN1A expression in ATLL cells may be a key player in ATLL leukemogenesis, and the abnormal genomic methylation may influence the expression of not only HTLV-1 Tax but also CDKN1A during long-term development of ATLL from the HTLV-1-infected T lymphocytes.

Proviral loads and clonal expansion of HTLV-1-infected cells following vertical transmission: a 10-year follow-up of children in Jamaica.

Objective: Few studies have specifically examined proviral load (PVL) and clonal evolution of human T-lymphotropic virus type 1 (HTLV-1)-infected cells in vertically infected children. Methods: Sequential samples (from ages 1 to 16 years) from 3 HTLV-1-infected children (cases A, B and C) in the Jamaica Mother Infant Cohort Study were analyzed for their PVL and clonal expansion of HTLV-1-infected cells in peripheral blood mononuclear cells (PBMCs) by inverse-long PCR. Results: The baseline PVL (per 100,000 PBMCs) of case A was 260 (at 1 year of age) and of case B it was 1,867 (at 3 years of age), and they remained constant for more than 10 years. Stochastic patterns of clonal expansion of HTLV-1-infected cells were predominately detected. In contrast, case C, who had lymphadenopathy, seborrheic dermatitis and hyperreflexia, showed an increase in PVL from 2,819 at 1.9 years to 13,358 at 13 years of age, and expansion of 2 dominant clones. Conclusion: The clonal expansion of HTLV-1-infected cells is induced in early childhood after infection acquired from their mothers. Youths with high PVL and any signs and symptoms associated with HTLV-1 infection should be closely monitored.

A monoclonal antibody to rat liver arylsulfatase C and its application in immunohistochemistry

We purified arylsulfatase C from rat liver microsomes and prepared a monoclonal antibody (P42C2) to the purified enzyme. By SDS-PAGE and immunoblotting analysis using P42C2, the molecular weight of the purified enzyme and of the enzyme in liver and kidney microsomes were estimated at 62,000 daltons. P42C2 caused little inhibition of arylsulfatase C activity, and was bound only slightly to liver microsomes. Localization of arylsulfatase C was studied at the light and electron microscopic level by the indirect immunoperoxidase method using P42C2. In rat liver, arylsulfatase C was detected mainly in the hepatocytes, and less frequently in endothelial cells, Kupffers cells, and Itos cells. In rat kidney, strong staining was observed in the straight portions of the proximal tubules. The podocytes, interstitial cells, endothelial cells, and epithelial cells of Henles thin limbs were stained faintly. By electron microscopy, arylsulfatase C was found localized on the membranes of the endoplasmic reticulum and nuclear envelopes in these cells. These immunohistochemical findings agree with the localization demonstrated by an enzyme-histochemical method which we had previously developed.