Biomedical subjects

T Harigaya

Publications and source records attributed to T Harigaya.

16 recordsLinked to original sources

PubMed1999

Linear SRY transcript in equine testis.

Employing a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques, the complete coding sequence of cDNA for the equine SRY gene was determined. We also attempted to make clear whether the equine SRY gene transcript is expressed in the adult testis, and whether the type of transcript is expressed as linear or circular RNA. As a result, in total a 1420 bp cDNA sequence was determined. Accomplishment of 3' RACE infers that equine SRY gene was expressed as a linear RNA transcript in testicular tissue just after puberty, in contrast to the situation in mice.

Amino Acid Sequence↗

PubMed1997

Expression of prolactin gene in mouse placenta during late pregnancy: detection of mRNA and its translation product.

To examine the existence of PRL messenger ribonucleic acid (mRNA) in the mouse placenta during late pregnancy, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis were carried out followed by nucleotide sequence analysis of cDNA. Total RNA extracted from each tissue was reverse-transcribed, followed by PCR with two oligonucleotide primers specific or a part of mouse PRL (mPRL) cDNA. An amplified RT-PCR product of predicted size was detected in all samples from the placenta of days 16 and 18 pregnant mice. This product was specifically hybridized with a probe overlapping an entire sequence of mPRL cDNA in Southern blot analysis. Nucleotide sequence analysis also provided evidence that the amplified cDNA had a nucleotide sequence completely identical to the mPRL cDNA sequence reported previously. Furthermore, mPRL with a slightly bigger molecular weight than that of pituitary PRL was detected in the placenta of days 12, 14, 16 and 18 pregnancy by immunoblot analysis. These results suggest that PRL mRNA and its translation product are synthesized in mouse placenta during late pregnancy.

Animals↗

PubMed1996

Variable expression of human transgenes in SHN mice.

The mouse mammary tumour virus/human transforming growth factor alpha (MMTV/hTGF alpha) gene or the mouse whey acidic protein/human growth hormone (mWAP/hGH) gene was introduced to a high mammary tumour strain of SHN virgin females by mating with males bearing each gene. We maintained transgenic mice by backcrossing males with the hTGF alpha transgene or high serum hGH levels (> 50 ng/ml) to SHN virgins in the subsequent generations. Expression of the transgenes was examined at each generation. At the first and the second generations of backcrossing (N1 and N2), females showed hTGF alpha mRNA in the mammary glands associated with a marked outgrowth of the glands. Both females and males had high hGH levels in the circulation, but these characteristics disappeared completely after the third generation (N3). All findings indicate that SHN mice are specific in the resistance to the expression of some human transgenes.

Animals↗

PubMed1996

The mRNA expression of neurocan, a brain-specific chondroitin sulfate proteoglycan, in neoplastic mammary glands in mice.

In the experiment of mouse transforming growth factor alpha (TGF alpha) gene expression in mammary tumors, various sizes of amplified products by reverse transcriptase-polymerase chain reaction (RT-PCR) using mouse TGF alpha primers were detected in addition to a predicted size in four strains of mice. During the further analysis of these RT-PCR products in mouse mammary tumors, the transcript of neurocan gene was detected in the mammary tumor from SHN mice by the cloning and nucleotide sequence analysis after RT-PCR reaction using mouse TGF alpha primers. The 5'-nucleotide sequence of sequential 246bp in the amplified cDNA of 527bp was completely identical to a middle part of mouse neurocan cDNA sequence, one of the chondroitin-sulfate proteoglycan expressed in the nervous tissue.

Animals↗

PubMed1994

Cause of failure of lactation in mouse mammary tumor virus/human transforming growth factor alpha transgenic mice.

Transgenic female mice bearing human transforming growth factor-alpha (TGF alpha) cDNA under the control of the mouse mammary tumor virus enhancer/promoter became pregnant but failed lactation. TGF alpha mRNA was detected in the mammary glands of these mice by the reverse transcriptase-polymerase chain reaction. By the use of collagenase-dissociated mammary epithelial cells, the binding of prolactin to its receptor was determined before and after parturition. At the end of pregnancy, the binding in TGF alpha transgenic (TGF alpha [+]) mice was small and its amount was comparable to that in the TGF alpha negative (TGF alpha [-]) mice. On the day of parturition, prolactin binding in TGF alpha (+) mice increased approximately 1.9-fold (insignificant), while that in TGF alpha (-) mice elevated over 5.3-fold (P < 0.01). The binding sites per cell were also higher in TGF alpha (-)mice. Radioimmunoassay of prolactin suggested that in TGF alpha (+) mice the low level of prolactin binding after parturition was not due to masking effect of serum prolactin. Among six TGF alpha (+) mice assayed, one mother with the highest prolactin binding activity (3.7-fold increase) initiated lactation, but the others did not. As there was little difference between groups in the growth and synthesis in the mammary glands, it was concluded that the failure of lactation in TGF alpha (+) mice is principally due to the lack of elevation of mammary prolactin receptor after parturition. At present, the role of TGF alpha in this process is obscure; however, TGF alpha was revealed not to interfere with the binding of prolactin to the receptor.

Animals↗

PubMed1994

Different gene expression of mouse transforming growth factor alpha between pregnant mammary glands and mammary tumors in C3H/He mice.

Mouse transforming growth factor alpha (TGF alpha) gene expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) method in pregnant mammary glands and mammary tumors in C3H/He mice. Both normal pregnant and tumorous mammary tissues expressed mRNA for mouse TGF alpha. When primers from rat TGF alpha cDNA sequence as well as those in the previous report [12] were used, the PCR amplified products were the same sizes in both normal pregnant mammary gland and tumor. However, there were obviously different PCR products between pregnant mammary gland and tumor in case of using primers according to the mouse TGF alpha cDNA sequence. In tumors, various sizes of PCR products were detected in addition of a predicted size. This is unlikely the experimental artifact, but the polymorphic TGF alpha gene expression is induced in mammary tumors.

Animals↗

PubMed1992

Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity.

Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5' non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5'-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16 kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.

Amino Acid Sequence↗

PubMed1988

Hepatic placental lactogen receptors during pregnancy in the mouse.

The binding sites for mouse placental lactogen-II (mPL-II) in virgin and pregnant mouse hepatic membranes were analyzed by Scatchard analysis and affinity cross-linking. Competitive binding studies showed that mPL-II and mouse PRL (mPRL) bound to the same receptors in all liver membrane preparations, although the affinity of mPRL binding was lower than that of mPL-II binding. Two classes of receptors for mPL-II, high and low affinity, were found by Scatchard analysis. The concentration of both types of sites in liver membranes increased during pregnancy. In contrast, the affinity of both sites for mPL-II was highest in virgin female mice and declined during pregnancy. Cross-linking of [125I]iodo-mPL-II to maternal liver membranes resulted in the specific labeling of one major protein species of 67,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Under nonreducing conditions, two bands of approximately 63,000 and 60,000 daltons were apparent. Subtraction of the mol wt of mPL-II (22,000, reduced; 20,000, nonreduced) from the mol wt of the cross-linked complex indicated that the mol wt of the receptor was 45,000 under reducing conditions and 43,000 and 40,000 under nonreducing conditions. These observations suggest that mPL-II receptors may be present in mouse liver membranes in at least two forms.

Animals↗

PubMed1988

Immunohistochemical identification of prolactin-producing cells in pituitary isografts in CBA mice.

Prolactin producing cells were investigated in pituitary isografts placed from female donor to female host CBA mice and also in the in situ pituitary gland of the graft-bearing host mice. Prolactin-producing cells were identified immunohistochemically using anti-mouse prolactin serum. Light microscopically, the cells with immunoreactive prolactin granules are present throughout the entire area of pituitary isografts, but less frequently in the in situ pituitary gland of the host mice. Electron microscopically, prolactin-producing cells may be classified into 3 types mainly based upon immunoreactive prolactin granules. Prolactin-producing cells in the pituitary grafts are classified as modified type III cells containing numerous immunoreactive prolactin granules resembling those found in the type III cells well-developed endoplasmic reticulum and enlarged Golgi apparatus. In contrast, following 5 d of the treatment with 2-bromo-alpha-ergocriptine, both type III and modified type III cells were decreased in number and type II cells were increased, and the cytoplasmic organella in these prolactin-producing cells appeared to be poorly developed.

Animals↗

PubMed1986

Cloning and sequence analysis of cDNA for mouse prolactin.

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.

Amino Acid Sequence↗

PubMed1985

Sexual dimorphism in amino acid compositions of mouse prolactin.

Sexual dimorphism was found in amino acid compositions and immunogenecity of variant types of prolactin (PRL) purified from the pituitary gland of normal adult C57BL mice by a high performance liquid chromatography. From the pituitary gland of female mice, three female variant types of PRL were isolated, whereas from the pituitary gland of male mice, two male variant types of PRL (M1-PRL and M3-PRL) and a female variant type of PRL (M2-PRL) were obtained. The amino acid composition of M3-PRL was different from any of female variant types which were very similar to each other and contained less varine, isoleucine, leucine, tyrosine and lysine but more alanine than the latter. Immunoreactivity of any of female variant types of PRL against anti-PRL serum was 100%, whereas that of M1-PRL was as much as 85% and that of M3-PRL was nearly undetectable.

Amino Acids↗

PubMed1985

Effects of hyperprolactinaemia on reproduction in male mice.

Hyperprolactinaemia induced by pituitary isografts in male host mice was confirmed by radioimmunoassay, but plasma testosterone levels determined by radioimmunoassay in these mice showed no changes. Immunoenzyme electron microscopic observations revealed large spherical-shaped immunoreactive prolactin granules in pituitary grafts in male hosts, regardless of the sex of the donor mice, indicating the disappearance of sexual dimorphism in prolactin-producing cells in hyperprolactinaemic mice. In hyperprolactinaemic host mice the male accessory sex glands, particularly the seminal vesicle and the ventral prostate, exhibited considerable proliferation and significant increase in weight. These phenomena do not seem to be mediated by the increased action of testosterone. Such biological effects in host mice were much greater when the donor was female rather than male, and were more noticeable in C57BL mice than in C3H mice.

Animals↗

PubMed1982

Influence of glucocorticoids on mammary prolactin receptors in pregnant mice after ovariectomy.

Regulation of mammary prolactin receptors by steroid hormones was investigated in ovariectomized mid-pregnant mice. Ovariectomy increased the number of mammary prolactin receptors per cell with no effect or a slight decrease in dissociation constant (Kd). The simultaneous removal of adrenals prevented this increase in numbers. A single injection of glucocorticoid (corticosterone or cortisol) in ovariectomized-adrenalectomized mice restored the number of prolactin receptors in mammary glands to the same level as that in ovariectomized controls without changing the Kd. Aldosterone, deoxycorticosterone and oestradiol did not affect the number of mammary prolactin receptors after ovariectomy-adrenalectomy. Serum concentration of prolactin was not influenced by the hormone manipulation except with injections of oestradiol or cortisol and apparently did not correlate with the number of prolactin receptors. These results indicated that glucocorticoids are required for the increase in the number of mammary prolactin receptors induced by ovariectomy in mid-pregnant mice.

Adrenalectomy↗

PubMed1980

Specific binding of prolactin to the mammary gland and lactose synthesis in pregnant rats after removal of ovaries or ovaries and adrenals.

Ovariectomy performed at mid pregnancy increased the specific binding of 125I-prolactin to the mammary gland (expressed as cpm/mg DNA) in rats 40 hr after the operation. When adrenals were removed simultaneously at the time of ovariectomy, no increase in the specific binding was observed. An appreciable amount of lactose was found in all mammary glands of ovariectomized-adrenalectomized animals, though the content was less than half of that of the ovariectomized animals. The plasma level of prolactin in ovariectomized and ovariectomized-adrenalectomized rats was slightly higher than that in sham operated control rats, though the differences were not statistically significant. These results seem to indicate that the adrenal gland is necessary for the induction of the site of specific binding of prolactin in the mammary gland of the rat and that an increase in the circulating levels of prolactin does not seem to play an essential role in the initiation of milk synthesis in this species.

Adrenal Glands↗

PubMed1978

Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.

Animals↗

PubMedDate not supplied

Normal and neoplastic mammary gland growth in MMTV/TGF alpha transgenic mice.

Biochemical and Dynamic change of mammary glands in different reproductive states were studied in comparison with histological structures in female and male transgenic mice bearing human transforming growth factor alpha (TGF alpha) cDNA under the control of the mouse mammary tumour virus enhancer/promoter. Female and male F1 mice between SHN female and transgenic male mice were divided into TGF alpha (+) and TGF alpha (-) groups according to the presence of TGF alpha gene at approximately 50 days of age. While there was little difference in mammary gland contents of DNA and RNA in females at 2 months of age, both nucleic acid contents were elevated markedly in TGF alpha (+) female mice with large variations at 4 months. These extremely high DNA and RNA contents in the TGF alpha (+) group declined to the level of the TGF alpha (-) group in the middle of pregnancy and at the end of pregnancy, respectively. Thymidine kinase (TK) activity in the mammary glands as an index of DNA synthesis was significantly higher in TGF alpha (+) mice than in TGF alpha (-) mice at both 2 and 4 months of age and the high TK in TGF alpha (+) mice also declined to the level of TGF alpha (-) mice with pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗