Biomedical subjects

J Enami

Publications and source records attributed to J Enami.

At least 19 recordsLinked to original sources

PubMed2001

Establishment of heterotropic liver tissue mass with direct link to the host liver following implantation of hepatocytes transfected with vascular endothelial growth factor gene in mice.

One of the major goals of tissue engineering is to establish an integrated organ in vivo. We have previously shown that transfection of vascular endothelial growth factor (VEGF) gene into hepatocytes promotes tissue formation by engrafted cells. Here we show that tissue growth was significantly enhanced by co-transplantation of hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF alpha) gene transfected hepatocytes with VEGF-gene transfected cells, but tissue islands were scattered nonspecifically in the abdomen of mice. The result brought us forward to the next step to establish an integrated mass and structural formation of liver tissue. We entrapped VEGF gene transfected hepatocytes in a nylon mesh bag and intraperitoneally engrafted close to the liver. Three weeks later, the bag was covered by a thick network of blood vessels, compared to the control. Histological examination showed that the blood vessels penetrated the parenchyma of the engrafted bag and formed a well-developed vessel network in the region. The use of hepatocytes from lacZ transgenic mice and PCR analysis demonstrated survival and albumin production by hepatocytes in the engrafted bag. Our model can potentially be developed into a heterotropic artificial liver with direct access to the host blood circulation.

Actins↗

PubMed1999

Hepatocyte growth factor supports androgen stimulation of growth of mouse ventral prostate epithelial cells in collagen gel matrix culture.

To assess the role of hepatocyte growth factor (HGF) and androgen in growth of prostate epithelial cells, we isolated mouse ventral prostate epithelial cells and cultured them in a three-dimensional type I collagen gel matrix under serum-free conditions. Although the prostate epithelial cells tended to die in the insulin-supplemented basal medium, 5alpha-dihydrotestosterone (DHT) prevented the cell death, and HGF slightly stimulated the growth. By contrast, coexistence of DHT and HGF greatly augmented the growth and branching morphogenesis of the epithelial cells. Some of the outgrowths formed under these conditions showed enlarged structures resembling the prostate ducts or alveoli. Examination of the stromal cell-conditioned medium revealed that a growth-stimulating activity is present in the conditioned medium. A major portion of this activity was abolished by anti-HGF IgG. These observations suggest that HGF is produced by the stromal cells of the prostate gland and supports the androgen stimulation of growth of the epithelial cells.

Animals↗

PubMed1999

Effects of 1alpha,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel.

We examined the effects of 1alpha,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10(-11)-10(-9) M) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham's F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10(-8)-10(-7) M) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25-DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10(-11)-10(-9) M) and inhibiting it above 10(-8) M. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25-DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25-DHCC does not substitute for glucocorticoids in the differentiation of the cells.

Animals↗

PubMed1999

Mammary fibroblast-derived hepatocyte growth factor and mammogenic hormones stimulate the growth of mouse mammary epithelial cells in primary culture.

We recently isolated a mammary growth factor from mouse mammary fibroblast-conditioned medium and identified it as a homologue of human HGF (hepatocyte growth factor) (Sasaki et al. Biochem Biophys Res Commun 199: 772-779, 1994). To elucidate the role of mammary fibroblast-derived HGF in mammary epithelial cell growth in vivo, we used recombinant mouse HGF and examined its effect on the growth of primary cultures of mouse mammary epithelial cells in this study. HGF stimulated the growth of mammary epithelial cells not only in monolayer culture but also in three-dimensional collagen gel matrix culture. In contrast, mammogenic hormones, i.e., prolactin and progesterone, stimulated the growth of mammary epithelial cells only when the cells were cultured in a collagen gel matrix, suggesting that the three-dimensional culture conditions are necessary to observe the growth-stimulatory effect of mammogenic hormones seen in vivo. Under these three-dimensional culture conditions, an additive effect of HGF and mammogenic hormones was also seen. Northern blot analysis revealed that HGF mRNA was expressed in the mammary gland as well as in cultured mammary fibroblasts but not in cultured mammary epithelial cells, indicating that HGF is produced preferentially by the stromal fibroblasts of the mammary gland. These observations thus suggest a role for HGF as a mammary stromal fibroblast-derived growth factor which, in cooperation with mammogenic hormones, stimulates the growth of mammary epithelial cells in vivo.

Animals↗

PubMed1998

Mammary fibroblast-derived hepatocyte growth factor stimulates growth and morphogenesis of mouse mammary tumor cells in primary culture.

We have recently isolated a mammary growth factor from the conditioned medium of mouse mammary stromal fibroblasts and identified it as a mouse homologue of human HGF (hepatocyte growth factor). To elucidate the role of HGF in mouse mammary tumorigenesis, we produced recombinant mouse HGF and examined its effects on primary cultures of mouse mammary tumor cells in this study. HGF at concentrations above 20 ng/ml maximally stimulated the growth of mammary tumor cells in primary monolayer culture. HGF also stimulated the three-dimensional growth and branching morphogenesis of mammary tumor cells cultured inside collagen gels. A comparison of the growth-stimulating activity of HGF with that of EGF (epidermal growth factor) and KGF (keratinocyte growth factor) revealed that HGF is the most potent growth factor among the three. Immunological studies using an antibody against mouse HGF demonstrated that 74% of the growth-stimulating activity present in the mammary fibroblast-conditioned medium was abolished by the antibody, indicating that HGF is the major growth factor produced by the fibroblasts. These observations thus suggest a role for HGF as a mammary stromal fibroblast-derived factor which stimulates growth and morphogenesis of adjacent mammary tumor cells in vivo.

Animals↗

PubMed1997

Corticosterone is required for the prolactin receptor gene expression in the late pregnant mouse mammary gland.

In order to clarify the prolactin receptor (PRL-R) gene expression at lactogenesis, the levels of the long and short forms of PRL-R mRNA were determined by the competitive RT-PCR in the pregnant, lactating and ovariectomized midpregnant mouse mammary gland. Plasma concentrations of corticosterone and progesterone were determined by RIA. The long form of PRL-R mRNA level was low until 10:00 on day 18, increased 3.3-fold at 22:00 on day 18 of pregnancy and further increased to 4.6-fold at 10:00 on day 0 of lactation. The short form of PRL-R mRNA level remained unchanged during this time period. The corticosterone:progesterone ratio increased 15.5-fold during the last 1.5 days of pregnancy. Corticosterone increased the long form of PRL-R mRNA level when the tissues on day 17 were cultured. On day 12 of pregnancy and following ovariectomy, corticosterone was exceedingly high from 2 h to 8 h and the corticosterone:progesterone ratio changed prior to the increase in the long form of PRL-R mRNA level. We conclude that corticosterone increases the PRL-R gene expression in the mammary gland before the onset of parturition.

Animals↗

PubMed1997

Removal of milk by suckling acutely increases the prolactin receptor gene expression in the lactating mouse mammary gland.

To determine the effect of suckling on the prolactin receptor (PRL-R) gene expression, we measured the quantity of PRL-R mRNA in the lactating mouse mammary gland. When the pups were separated from their mother on day 5 of lactation, the long form of PRL-R (PRL-R[L]) mRNA disappeared with a half-life of 12.5 h for the first 9 h and 3.0 h for the following 9-15 h. By supplying pups to mice which had been weaned for 24 h, PRL-R(L) mRNA increased 2.5-fold during the next 6 h-period. The increase in PRL-R(L) mRNA was found in the mammary glands from which the pups removed milk. The number of mammary PRL-R protein decreased or increased following weaning or following the removal of milk by suckling, respectively. From these observations. it was concluded that the removal of milk acutely increases the level of PRL-R(L) mRNA during lactation.

Animals↗

PubMed1997

D-amino-acid oxidase is not present in the mouse liver.

Since there are conflict reports on the presence of D-amino-acid oxidase in the mouse liver, this problem was examined. D-Amino-acid oxidase activity was not detected in the homogenates of the mouse liver, lung, or heart, whereas it was detected in the homogenates of the mouse kidney and brain. Western blotting showed that a protein which reacted with the antiserum against pig D-amino-acid oxidase was present in the homogenates of the mouse kidney and brain but not in those of the liver or heart. Northern hybridization using a D-amino-acid oxidase cDNA probe detected a hybridizing signal in poly(A)+ RNAs extracted from the mouse kidney and brain but not in those from the liver, heart, or lung. Reverse transcription-polymerase chain reaction using three primer pairs always amplified D-amino-acid oxidase cDNA fragments of expected sizes in the mouse kidney and brain but very rarely did so in the liver, heart, or lung. The results indicate that D-amino-acid oxidase is not present in the mouse liver in a measurable amount.

Animals↗

PubMed1997

The regulation of the prolactin receptor gene expression in the mammary gland of early pregnant mouse.

The purpose of this research was to examine the hormonal regulation of the PRL receptor (PRL-R) gene expression in the mammary gland of early pregnant mouse. Following reverse-transcription, the quantity of PRL-R mRNA was determined by the competitive polymerase chain reaction. The level of long form PRL-R (PRL-RL) mRNA changed cyclically with the highest at estrus and the lowest at diestrus II. PRL-RL mRNA was maintained at high levels for the first 3 days of pregnancy but declined to lower levels on day 5. Mice ovariectomized on day 2 of pregnancy maintained the same level of PRL-RL mRNA during the 24 h-period. The level of PRL-R mRNA increased more than 2- and 2.7-fold with 17 beta-estradiol and PRL, respectively, and the progesterone concentration decreased its levels to 71% of the vehicle-injected control. The increasing action with 17 beta-estradiol and PRL was suppressed by administration of progesterone. Mice ovariectomized on day 3 had a 1.8-fold higher level than that of the sham-operated control. The short form of PRL-R remained at low levels throughout the experiments. The results suggested that the expression of the PRL-R gene was suppressed when serum progesterone increased during early pregnancy.

Animals↗

PubMed1996

Structure and expression of a murine homologue of sky receptor tyrosine kinase gene.

To identify new receptor tyrosine kinases (RTKs), we screened cDNAs from mouse mammary tumor cells and mouse brain. A homology search of the complete cDNA sequences obtained showed that one cDNA was a murine homologue of recently reported human sky [Ohashi, K. et al. (1994) Oncogene 9, 699-705]. Another cDNA obtained was also related to sky but had a 5' upstream sequence similar to brt [Fujimoto, J. and Yamamoto, T. (1994) Oncogene 9, 693-698]. Analysis of the 5' region of the sky genomic DNA revealed that brt-type and sky-type sequences are encoded by the sky gene in different exons. The upstream region of the sky-type coding exon is highly GC-rich and contains potential recognition sites for the Sp1 trans-acting factor, but lacks TATA and CAAT boxes, features commonly found in promoters of other RTKs. To examine whether this upstream region functions as a promoter, we fused it with chloramphenicol acetyltransferase (CAT) gene and transfected the construct into COS-7 cells. The results of the CAT assay showed that the sky upstream region retains a significant promoter activity. Furthermore, primer extension analysis revealed that the transcription starts at -240 nt upstream from the sky translation initiation codon. These observations suggest that the brt- and sky-types of mRNA are transcribed from a single sky gene by an alternative promoter usage.

Animals↗

PubMed1996

Prolactin-dependent growth and gamma-casein gene expression in Ba/F3 cells transfected with a long form of mouse mammary prolactin receptor.

Complementary DNA (cDNA) encoding a long form of prolactin receptor (PRL-RL) was cloned from mouse mammary gland by PCR using primers designed from the noncoding regions of previously reported rat ovarian PRL-RL cDNA. The nucleotide sequence encoding the extracellular and transmembrane domains of PRL-RL is completely identical to that of short forms of mouse PRL-R. The amino acid sequence deduced from the nucleotide sequence of mouse PRL-RL is 91% identical to that of rat PRL-RL. To address the question of whether or not the cloned mouse PRL-RL cDNA encodes a functional PRL-RL we transfected Ba/F3 IL-3-dependent murine pro-B lymphoid cells with the cDNA. By culturing the transfected cells in a medium which contained PRL in place of IL-3, we selected 5 PRL-dependent clones. All of these PRL-dependent clones, BaF/PD cells, expressed PRL-RL mRNA. In addition, BaF/PD cells expressed mammary-specific gamma-casein mRNA in response to PRL and dexamethasone. Based on these results, it was concluded that the mouse mammary PRL-RL cDNA cloned in this study is functionally active in mediating both PRL-dependent growth and mammary-specific gene expression.

Amino Acid Sequence↗

PubMed1996

Acute expression of the PRL receptor gene after ovariectomy in midpregnant mouse mammary gland.

In order to examine the time-dependent expression of the PRL receptor (PRL-R) gene at lactogenesis, the level of PRL-R mRNA was determined following ovariectomy in pregnant mouse mammary gland. Following reverse transcription, the quantity of mRNA was measured by the competitive polymerase chain reaction. The casein (a 22,000 molecular weight component) mRNA level was measured as a marker for milk synthesis. Following ovariectomy, the onset of abortion occurred mostly at 22-23 h and the level of casein mRNA began to increase at 12 h. The long and short forms of PRL-R mRNAs were detected in a molar ratio of 1:0.2 on day 12 of pregnancy. Eight h after ovariectomy, the long form of PRL-R mRNA began to increase, showing a bell-shaped profile with the highest peak at 16 h. The short form of PRL-R mRNA was at low levels and remained constant. The levels of the long form of PRL-R mRNA decreased similarly in the presence and absence of foster pups from 24 to 48 h. Conversely, casein mRNA were maintained at high levels by supplying foster pups. The level of the long form of PRL-R mRNA reached a maximum prior to abortion. The present experiments demonstrated that the acute expression of the PRL-R gene occurred in the mammary gland at lactogenesis.

Adrenalectomy↗

PubMed1996

Hormone-dependent expression of gamma-casein mRNA in mouse mammary epithelial cells cultured on floating collagen gels.

Mammary epithelial cells isolated from pregnant mice were plated on a collagen gel matrix and cultured in a serum-free medium supplemented with combinations of insulin, dexamethasone and prolactin (PRL). After the cells formed a monolayer, the collagen gel was detached from the culture dish and allowed to float in the medium. Quantification of gamma-casein mRNA by a competitive PCR method revealed that the cells on the floating gel accumulated considerably larger amounts of gamma-casein mRNA than the cells on the gel remained attached to the dish. Under these floating collagen gel culture conditions, addition of both dexamethasone and PRL to the insulin-supplemented basal medium maximally stimulated the accumulation of gamma-casein mRNA. These observations strongly suggest that the status of the extracellular matrix as well as hormones controls the differentiation of mouse mammary epithelial cells at the mRNA level.

Animals↗

PubMed1995

Cloning of cDNAs for the precursor protein of a low-molecular-weight subunit of the inner layer of the egg envelope (chorion) of the fish Oryzias latipes.

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.

Amino Acid Sequence↗

PubMed1995

Biochemical changes during growth and regression of pregnancy-dependent mammary tumors of GR/A mice.

The weights of pregnancy-dependent mammary tumors (PDMT) of GR/A mice continued to increase until parturition and decreased soon after delivery; however, mitotic indices in epithelial cells and stromal cells of PDMT reached a maximum plateau on Day 18-19 of pregnancy and decreased thereafter. Growth of PDMT in progesterone-treated mice on Day 15 of pregnancy was higher than that in 17 beta-estradiol-treated mice and no treatment controls. DNA fragmentation was observed in PDMT on Day 20 of pregnancy and just after parturition. Two-dimensional gel electrophoresis of PDMT extracts revealed that five and six protein spots appeared newly on Day 20 of pregnancy and just after parturition, respectively. N-terminal amino acid sequences of two of the protein spots were identical to that of alpha-lactalbumin. PDMT on Day 15 and 20 of pregnancy and just after parturition secreted matrilysin, one of the matrix metalloproteinases, which was identified by Western blotting. However, matrilysin was not found in hormone-independent autonomous mammary tumors of the mouse. Estrogen receptor and c-fos mRNA expression levels in PDMT were high on Day 15 of pregnancy but low on Day 20 of pregnancy and just after parturition. These findings suggest that regression of PDMT is caused by apoptosis, and new proteins expressed on Day 20 may participate in the process of regression.

Amino Acid Sequence↗

PubMed1994

Complete Nucleotide Sequence of Mouse Mammary Tumor Virus from JYG Chinese Wild Mice: Absence of Bacterial Insertion Sequences in the Cloned Viral gag Gene.

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.

Journal Article↗

PubMed1994

Identification of a mammary transforming gene (MAT1) associated with mouse mammary carcinogenesis.

We have developed an efficient in vitro transformation system using N-methyl-N-nitrosourea that allows us to study the role of hormones and growth factors in mouse mammary tumorigenesis. Utilizing this system, we reported earlier that mammary tumors induced in vitro with N-methyl-N-nitrosourea in the presence of mammogenic hormones (progesterone and prolactin) contain predominately an activated c-Ki-ras protooncogene with a G35 --> A35 transitional mutation in the 12th codon. Mammary tumors induced in the presence of another mitogen, lithium (Li), do not have a mutation in the c-Ki-ras protooncogene. By using an expression cloning system, a plasmid clone containing a 1.75-kb cDNA insert has been isolated from this group of tumors. Nucleic acid sequence analysis of the insert reveals that it has a short open reading frame of 61 amino acids and that it does not have sequence homology with any known gene. The gene, designated MAT1, can neoplastically transform NIH 3T3 cells and also the mammary epithelial cell line TM3. Expression of this gene occurs in normal mouse tissues including mammary gland and is overexpressed in the original mammary tumors as indicated by Northern blot analysis. In vitro transcription and translation of the clone shows a protein product of 6000 Da, which agrees with the predicted open reading frame.

3T3 Cells↗

PubMed1994

Identification of mouse mammary fibroblast-derived mammary growth factor as hepatocyte growth factor.

A growth factor for normal and neoplastic mouse mammary epithelial cells in primary culture was partially purified from the conditioned medium (CM) of mouse mammary fibroblasts. The N-terminal amino acid sequence of the major 33 kD protein separated on SDS-polyacrylamide gel electrophoresis under reducing conditions suggested that the growth factor is closely related to hepatocyte growth factor (HGF). To examine whether mammary growth factor is identical to HGF we cloned mouse HGF cDNA from mammary fibroblasts using PCR with primers designed from the previously reported human and rat HGF cDNA sequences. We then expressed the cloned mouse HGF cDNA in COS-1 cells. The CM from mouse HGF cDNA-transfected COS-1 cells stimulated the growth of mammary tumor cells in a manner similar to the CM from mammary fibroblasts. From these observations it is concluded that mammary growth factor produced by mammary fibroblasts is identical to mouse HGF.

Amino Acid Sequence↗