Biomedical subjects

T Tsubota

Publications and source records attributed to T Tsubota.

At least 37 records · Page 2Linked to original sources

PubMed1998

Serum progesterone, oestradiol, luteinizing hormone and prolactin profiles in the female black bear (Ursus americanus).

Identifying steroid and pituitary hormone profiles in the female black bear (Ursus americanus) throughout pregnancy may provide a greater understanding of the reproductive cycle and indicate which hormones are required for implantation. Our objective was to assess endocrine activity in black bears oestrus onset, at oestrus, during pregnancy and after parturition. Serum samples were obtained from 12 captive, 16 uncollared and five radiocollared free-ranging female black bears from March through the end of December and assayed for serum progesterone, oestradiol, luteinizing hormone (LH) and prolactin (PRL). In captive bears, progesterone concentrations were low at days 0-10 after oestrus and increased significantly days 25-35 and 45-52 after oestrus. Oestradiol concentrations were high at oestrus (day 0) and days 4-10 after oestrus and then decreased days 25-35 and 45-52 after oestrus. LH concentrations were not significantly different throughout the sampling period. Changes in PRL concentrations pattern were similar to those of oestradiol, with elevated levels at oestrus and days 4-10 after oestrus, followed by a significant decrease 45-52 days after oestrus. In non-collared free-ranging bears, progesterone concentrations increased gradually after mating with a further significant increase in November-December. Oestradiol concentrations were highest in March (before mating) and in June (during mating) followed by a significant decrease in July (early delay period) and November-December (peri-implantation period). LH concentrations were low until November-December and then increased significantly. PRL concentrations were low in March (before mating), increased significantly during the mating season in June, decreased slightly in July, and were low in November-December (peri-implantation period). In radiocollared free-ranging bears, serum progesterone concentrations were elevated in pregnant bears in December and extremely low in lactating and non-lactating bears in March. Oestradiol levels were slightly higher in pregnant bears in December than in non-lactating or lactating bears in March. PRL concentrations were considerably higher in lactating bears in March than in pregnant bears in December. Our results suggest that: (1) serum progesterone concentrations are low, but detectable during the early delay implantation period and greatly elevated during the peri-implantation period; (2) serum oestradiol concentrations are elevated at oestrus and decline during the delay period; (3) LH may be involved in luteal activation; and (4) the decline of serum PRL concentrations during short days may be necessary for implantation to occur.

Animals↗

PubMed1998

Immunohistochemical demonstration of cytoskeletal proteins in the testis of the Japanese black bear, Ursus thibetanus japonicus.

The seasonal changes of the cytoskeletal protein expressions were immunohistochemically investigated in the testes of Japanese black bear, Ursus thibetanus japonicus. A strong immunoreaction for alpha-smooth muscle actin is restricted to the vascular smooth muscle cells and the peritubular cells which surround the seminiferous tubules by several layers throughout the year. Weak immunoreactions for B4 antigen and desmin were observed in the vascular smooth muscle cells and in a part of peritubular cells throughout the year. A strong immunoreaction for vimentin was also detected in the fibroblasts and Leydig cells, in addition to the vascular smooth muscle and epithelial cells and the peritubular cells throughout the year. A strong alpha-tubulin immunoreaction was detected in the elongating spermatids during the acrosome phase of spermiogenesis in May and June. The cytoplasm of several Sertoli cells was faintly immunoreacted for vimentin in the basal and lateral region, while an intense alpha-tubulin reaction was seen in the entire cytoplasm in May, April and June. In November, January and March, the immunoreactions for vimentin and alpha-tubulin strongly accumulate in a perinuclear region of Sertoli cells when developmental spermatids are not seen in the seminiferous tubules. These accumulations in the immunoreactions for vimentin and alpha-tubulin seem to be caused by the reduction in size of Sertoli cells cytoplasm with season. However, the seasonal changes of distributions in the cytoskeletal proteins are obscure in the bear testes. These results suggest that the contents of cytoskeletal proteins may not change in relation to the morphological differences with season in the testes of the seasonal breeders.

Actins↗

PubMed1998

Establishment and characterization of "biphenotypic" acute leukemia cell lines with a variant Ph translocation t(9;22;10) (q34;q11;q22).

We established two novel PH-positive acute leukemia cell lines with "biphenotypic" feature, NALM-27 and NALM-28, with t(9;22;10)(q34;q11;q22) from a patient with biphenotypic acute leukemia(BAL). The breakpoint cluster region (bcr) of the BCR gene was found to be rearranged in these cells by Southern blot analysis using a major 3'-side bcr probe. Polymerase chain reaction (PCR) results showed differences in the pattern of expression of the abl-bcr fusion gene in comparison with the diagnosis. In the case of variant Ph translocations, reports have appeared concerning mainly chronic myelogenous leukemia (CML), but there have been few concerning acute leukemia with lymphoid feature. This study thus identifies nonrandomly involved chromosome sites which can then be targeted for detailed molecular analysis to obtain an understanding of abl-bcr fusion in the cells with lymphoid feature. In addition, chromosome band 10q22, involved in this translocation, is the site for several neoplasia. Furthermore, this site is non-randomly involved in the formation of variant Ph translocations in acute lymphoblastic leukemia (ALL). This is the first report on the t(9;22;10)(q34;q11;q22) rearrangement in NALM-27 and NALM-28 cell lines which should prove useful for understanding the translocation of molecular breaks within the bcr of the complex translocation site.

Acute Disease↗

PubMed1997

Seasonal changes in the immunolocalization of steroidogenic enzymes in the testes of the Japanese black bear (Ursus thibetanus japonicus).

Seasonal changes in sites of immunostaining of steroidogenic enzymes were examined in testes of the Japanese black bear, Ursus thibetanus japonicus. In addition, serum concentrations of testosterone and estradiol-17beta were investigated by radioimmunoassay, and the seasonal changes were compared with the results of immunostaining. On the basis of morphological observations of spermatogenic activity, the reproductive cycle was divided into five periods: an active period in May and June; a degenerative period in November; a resting period in January; an early-resumptive period in March; and a late-resumptive period in April. Serum concentrations of testosterone differed with season accompanied by differences in spermatogenic activity, with baseline levels in November and January, increasing levels in March and April, and high levels in May, and April and June of the next year. Immunoreactivities specific for cholesterol side-chain cleaving cytochrome P450, 17alpha-hydroxylase cytochrome P450 and 30-hydroxysteroid dehydrogenase (3beta HSD) were observed in Leydig cells throughout the year. Only the percentages of Leydig cells immunopositive for 3beta HSD exhibited seasonal differences that correlated with serum concentrations of testosterone. Aromatase cytochrome P450 (P450arom) was immunolocalized in Leydig and Sertoli cells throughout the year, in spermatids in May, and April and June of the next year and in myoid cells in January and March. The percentages of Leydig cells immunopositive for this enzyme increased in May, and January, March and June of the next year. On the other hand, no pattern of seasonal change in serum estradiol-17beta concentration was observed. These results suggest that 3beta HSD is a key enzyme in the regulation of the testosterone production in Leydig cells. Furthermore, estrogen derived from Leydig and myoid cells seems to play a role in the regulation of Leydig cells by negative feedback as a paracrine and/or autocrine mediator.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear Ursus americanus.

American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March, 15 June, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggests that aromatase and oestrogen may play a role in re-initiating spermatogenesis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Seasonal changes in subcellular structures of Leydig and Sertoli cells in the Japanese black bear, Ursus thibetanus japonicus.

The fine structure of Leydig and Sertoli cells in Japanese black bears, Ursus thibetanus japonicus, was studied at different stages of reproductive activity in different seasons. Structures of Leydig cells in the testes that were spermatogenically active (May and June), inactive (November and January), and resumptive (March and April) were found to be different. The nucleus and smooth endoplasmic reticulum in Leydig cells had regressed when observed in November and January, and began to develop in March and April. Lipid droplets were numerous in Leydig cells in November and January, but few in March and April. These seasonal variations in the subcellular structures of Leydig cells corresponded to those in the steroidogenic activity of the cells. In Sertoli cells, changes in mitochondria and lipid inclusions with the seasons were detected, but the nuclei and other organelles, including the rough endoplasmic reticulum, did not change throughout the year. Lipid inclusions of both electron-dense and electron-lucent types in Sertoli cells were numerous in November and few in January. This change was reversely proportional to the number of degenerating cell masses in the luminal space, indicating that most of these lipid inclusions might be the end products of germ cells phagocytosed by the Sertoli cells. The results suggest that the seasonal changes in the morphology of Leydig cells are closely associated with their spermatogenic activity

Animals↗

PubMed1996

Structurally altered Evi-1 protein generated in the 3q21q26 syndrome.

Overexpression of the Evi-1 gene appears to be a consistent feature of the 3q21q26 syndrome, an association of myeloid leukemias/myelodysplastic syndrome with a specific chromosomal aberration involving both 3q21 and 3q26, such as t(3;3)(q21;q26) or inv(3)(q21q26). The rearrangement in 3q26 has been reported to occur near the Evi-1 locus, implicating that it is the critical gene deregulated in the 3q21q26 syndrome. Here we present a structural abnormality of Evi-1 protein in a case with the 3q21q26 syndrome. In this case carrying typical inv(3)(q21q26), the 3q26 breakpoint is located within an intron of the Evi-1 gene, and resulted in overexpression of normally unexpressed, an aberrant form of Evi-1 protein, in which the C-terminal 44 amino acids of wild-type Evi-1 protein were truncated and replaced by five amino acids. The truncated Evi-1 protein is shown to increase AP1 activity when expressed in NIH3T3 cells as its wild-type counterpart. We also show that the origin of this peculiar type of rearrangement of the Evi-1 gene is not an artifact during establishment of the cell line, but is the event that occurred in the primary leukemic cells. Our results strongly support that the primary target for the 3q21q26 syndrome is the Evi-1 gene, and provide the first evidence that the structurally altered Evi-1 gene may be involved in the 3q21q26 syndrome.

3T3 Cells↗

PubMed1996

Spermatogenic cycle in the testis of the Japanese black bear (Selenarctos thibetanus japonicus).

Spermatogenic cycle in the testis of the japanese black bear (Selenarctos thibetanus japonicus) was studied by light and transmission electron microscopy. By light microscopy, spermatids were allocated into eleven steps based on morphological changes in the nucleus and the acrosome of spermatids. Cellular associations of the seminiferous epithelium were allocated into eight stages based on the changes in the nucleus and acrosome of spermatids, appearance of meiotic figures and time of spermiation. Cross-sections of the seminiferous tubule seldom contained more than one type of stage. Spermatids at steps 1-2 had the well-developed Golgi complex. The crescent-shaped Golgi complex was accompanied by the acrosome extending over the nucleus at steps 3-5. At step 6, spermatids faced the base, and the outer membrane of the acrosome converged upon the plasma membrane of spermatids. The acrosome projected into the cytoplasm of Sertoli cells at step 9. At step 11, most of the cytoplasm was phagocytosed by Sertoli cells, and spermatids were released in the lumen to become spermatozoa.

Acrosome↗

PubMed1996

Intraovarian immunolocalization of steroidogenic enzymes in a Hokkaido brown bear, Ursus arctos yesoensis during the mating season.

Immunolocalization for four steroidogenic enzymes was performed on an ovary taken from a Hokkaido brown bear during the mating season. This specimen is considered to be in the follicular phase because of the presence of large follicles. In large follicles, cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were immunolocalized in theca interna cells and granulosa cells. 17 alpha-hydroxylase/C17-C20 lyase cytochrome P450 (P450c 17) was immunolocalized in theca interna cells but not in granulosa cells. Aromatase cytochrome P450 (P450arom) was immunolocalized only in granulosa cells. In medium follicles, however, P450scc and 3 beta HSD were immunolocalized only in theca interna cells, and the immunoreactivity of P450arom was detected in neither theca interna cells nor granulosa cells. Immunoreactivities of P450scc, 3 beta HSD and P450c 17 but not P450arom were detected in interstitial cells. This study suggests that estrogen biosynthesis takes place through interrelation between theca cells and granulosa cells and is explained by the so-called two-cell mechanism. Furthermore, the granulosa cells in large follicles have the capability for pregnenolone and progesterone biosynthesis, and the interstitial cell in the bear ovary is also a steroidogenic site.

3-Hydroxysteroid Dehydrogenases↗

PubMed1996

Four subclones with distinct immunoglobulin light chain phenotypes. (kappa+lambda+, kappa+, lambda+ and kappa-lambda-) from acute leukemia.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-9, was established from the peripheral blood specimen of a immunoglobin (lg) phenotype, the established BALM-9 cell line expressed both kappa and lambda light (L) chains simultaneously in a range of 30-80%. Two-color flow cytometric analysis demonstrated that there was a distinct population of kappa lambda double positive cells as well as kappa single, lambda single, and double negative populations. Therefore, subclones were obtained from each population by limiting dilution and were designated BALM-9KL (kappa+lambda+), BALM-9K (kappa+lambda-), BALM 9N (kappa-lambda-). Western blotting confirmed the results of the immunofluorescence test at the protein level. In BALM-9N, L chains were absent even in the cytoplasm as demonstrated by Western blotting. Evidence that the subclones have the same ancestry was provided both by cytogenetic analysis and by Southern blotting, which revealed the 14q32 chromosomal rearrangement as a common abnormality and the same IgH gene arrangement among the subclones. The existence of a kappa lambda positive B cell population suggests a transient stage of normal B cell maturation. These subclones might represent such a stage and thus provide a useful means of analyzing the mechanism of this double light chain expression.

Antigens, CD↗

PubMed1996

Abnormal expression of Evi-1 gene in human leukemias.

The human Evi-1 gene located on chromosome 3q26, encodes a zinc finger protein that functions as a transcription factor. It was frequently overexpressed in leukemias having 3q26 abnormalities such as t(3;3)(q21;q26) and inv(3)(q21 q26), and subjected to structural alteration in t(3;21)(q26;q22). In addition, recent studies indicated that several cases of leukemias without 3q26 abnormalities also expressed Evi-1 gene. In this study we present another case of structural alteration of Evi-1 gene in a case of inv(3)(q21 q26), in which Evi-1 was truncated and a shorter form of Evi-1 protein was expressed upon rearrangement of the gene. We also studied expression of the Evi-1 gene in a variety of leukemias by northern blot analysis. Evi-1 was overexpressed not only in leukemias with 3q26 abnormalities, but, in those without 3q26 abnormalities, especially in blast crisis of CML. Our result also supports an idea that Evi-1 is a relevant oncogene whose overexpression or structural changes might play a crucial role in development of human leukemias.

Adult↗

PubMed1995

Validity of retrograde corpora lutea of pregnancy as an index of past gestation in Sika deer, Cervus nippon.

Corpora lutea of the sika deer (Cervus nippon, Cervidae) were histologically examined in order to elucidate their retrograding processes after parturition. They showed a remarkable regression within one month as luteal cells decreased in number and, by nine months after parturition, turned into masses of blood vessels containing many arterioles with a diameter of 30 microns or less. In retrograde corpora lutea (RCL), older than 1.5 years after parturition, arterioles of these dimensions decreased markedly, while those measuring 50 microns or more became dominant. These findings indicate that an examination of RCL can distinguish females less than nine months after parturition from those more than 1.5 years after parturition. Based on the findings of RCL from the ovaries of a 10.5-year-old female, it is suggested that RCL remain in the ovaries for at least 8.5 years after parturition. It was, however, difficult to distinguish RCL of pregnancy from retrograde accessory corpora lutea (RACL). In order to arrive at a complete count of RCL, the ovaries should be examined at thicknesses of 0.5 mm. To check the presence/absence of RCL of pregnancy within nine months or less after parturition, sectioning at thicknesses of 2 mm is sufficient.

Animals↗

PubMed1995

Annual changes in serum concentrations of prolactin in captive male black bears (Ursus americanus).

Prolactin may be involved in the regulation of reproduction in black bears (Ursus americanus) as it is a mediator of photoperiodic changes in a number of species. The objectives of this study were to validate a radioimmunoassay to measure prolactin in bear serum and to describe seasonal changes in serum prolactin concentrations in captive male bears. Serum samples were obtained nine times during a year from three captive male black bears that were denning between November and March and active during the other months. The heterologous prolactin radioimmunoassay, using pig 125I-labelled prolactin and goat anti-pig prolactin as a primary antibody, was validated. Injection of thyrotrophin-releasing hormone into the three male bears in June resulted in a rapid increase in serum concentrations of prolactin (t = 0, 11.4-14.8 ng ml-1; t = 15-30 min, 18.4-28.7 ng ml-1). The sensitivity of the assay was 0.08 ng per tube. Intra- and interassay coefficients of variation were 5.5% (n = 6) and 5.7% (n = 6), respectively. Serum concentrations of prolactin changed seasonally, with the lowest concentrations in December (mean +/- SD = 1.1 +/- 0.1 ng ml-1); this was followed by a gradual increase between January (2.6 +/- 0.6 ng ml-1) and April (6.4 +/- 1.2 ng ml-1) and the highest concentrations in May (17.6 +/- 4.7 ng ml-1), preceding peak testosterone concentrations in June. The observation that prolactin secretion increased with increasing daylength suggests that photoperiod may be an external regulator. The presence of high concentrations of prolactin before peak testosterone concentrations suggests that prolactin may play a role in regulating seasonal changes in the testes.

Animals↗

PubMed1994

Immunolocalization of steroidogenic enzymes P450scc, 3 beta HSD, P450c17 and P450arom in the corpus luteum of the Hokkaido brown bear (Ursus arctos yesoensis) in relation to delayed implantation.

The objective was the immunocytochemical localization of steroidogenic enzymes in the corpus luteum of Hokkaido brown bears during the period of delayed implantation. Cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) were localized as biosynthetic sites of pregnenolone, progesterone, androgens, and oestrogens, respectively. Ovaries containing corpora lutea were obtained from three mature bears during the expected delayed implantation period and ovarian sections were immunostained by the avidin-biotin-peroxidase complex method using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. P450scc and 3 beta HSD were localized in all luteal cells, whereas P450c17 (0.4-5.1% of 1000 cells) and P450arom (7.1-11.2% of 1000 cells) were localized in only a few luteal cells. These data suggest that luteal cells contain steroidogenic enzymes required for progesterone synthesis but also have a minimum capability for synthesizing androgen and oestrogen during the delayed implantation period in Hokkaido brown bears.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Establishment and characterization of new pre-B cell leukemia cell line NALM-26.

A new pre-B cell leukemia cell line, NALM-26, was established from the peripheral blood of a 24-year-old male patient with acute pre-B cell leukemia. NALM-26 is unique in its expression of T cell-associated CD5 and myeloid cell-associated CD13 antigens. Interleukin-7 (IL-7) receptor (CDw127) was detected by flow cytometric analysis. After PMA treatment, NALM-26 was induced to express CD20, CD25 and CD28, and to increase its expression of both CD5 and CD13. The expression of CDw127 was down-modulated.

Adult↗

PubMed1994

Establishment of multiple leukemia cell lines with diverse myeloid and/or megakaryoblastoid characteristics from a single Ph1 positive chronic myelogenous leukemia blood sample.

Seven cell lines, MOLM-6, -7, -8, -9, -10, -11 and -12, were established from a single blood sample from a patient with chronic myelogenous leukemia (CML) in blastic phase having the Ph1 chromosome abnormality. Based on immunophenotyping, two of these seven cell lines, MOLM-7 and -11, represented the megakaryoblastoid lineage, and the other five cell lines represented two different maturation stages of the myeloid lineage.

Adult↗

PubMed1993

Determination of aspoxicillin in broncho-alveolar lavage fluid by high-performance liquid chromatography with photolysis and electrochemical detection.

A determination method for the penicillin antibiotic aspoxicillin in broncho-alveolar lavage fluid has been developed, involving high-performance liquid chromatography and post-column photolysis. The method enabled the determination of aspoxicillin at concentrations below 100 ng/ml, with a linear calibration curve at concentrations of 1-1000 ng/ml for 100 microliters of lavage fluid. The detection limit was 1 ng/ml at a signal-to-noise ratio of 3:1.

Amoxicillin↗

PubMed1993

Immunolocalization of steroidogenic enzymes, P450scc, 3 beta-HSD, P450c17, and P450arom in the Hokkaido brown bear (Ursus arctos yesoensis) testis.

Hokkaido brown bears (Ursus arctos yesoensis) are seasonal breeders and the profile of their serum testosterone concentrations undergoes annual changes. However, precise sites of steroidogenesis in the bear testis have not been identified. Therefore, our objective was to localize steroidogenic enzymes by immunocytochemistry using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. The steroidogenic enzymes localized were cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) as biosynthetic sites of pregnenolone, progesterone or androstenedione, androgens, and estrogens, respectively. Testes were collected from three adult bears prior to the mating season (April, Hokkaido, Japan) and prepared for immunostaining with primary antibodies, followed by an avidin-biotin-peroxidase method. P450scc was localized in Leydig cells and spermatids. 3 beta-HSD was found only in Leydig cells. P450c17 was identified in Leydig cells and spermatids. Finally, P450arom was found in Leydig cells and in spermatids that stained very intensely. Therefore, Leydig cells appear to be a site of progestin (both delta 4 and delta 5 C21 steroids), androgen, and estrogen production, whereas spermatids appear to be a site of pregnenolone, androgen, and estrogen production. Immunolocalization of steroidogenic enzymes suggests that steroidogenesis may occur not only in Leydig cells, but also in spermatids of the Hokkaido brown bear testis prior to the mating season. We also suggest that Leydig cells and spermatids are the predominant sites of androgen and estrogen synthesis, respectively, in the Hokkaido brown bear testis.

3-Hydroxysteroid Dehydrogenases↗