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Biomedical subjects

A I Negami

Publications and source records attributed to A I Negami.

15 recordsLinked to original sources
PubMed1995

Serum human hepatocyte growth factor in human menstrual cycle and pregnancy: a novel serum marker of regeneration and reconstruction of human endometrium.

Serum concentrations of human hepatocyte growth factor (hHGF) were measured in normal menstrual cycles and during uncomplicated pregnancies. In the normal menstrual cycle, the concentrations of hHGF increased through the mid and the late luteal phases to reach the highest peak during the menstrual phases, followed by a gradual decline during the follicular phase toward the trough levels seen in the ovulatory and the very early luteal phases. During pregnancy, serum hHGF concentrations increased continuously from the late luteal levels and constituted 4 distinct peaks. By immunostaining, c-Met protein, a receptor for hHGF, was localized not in the stromal but in the epithelial layer of the endometrium. In cultured isolated endometrial cells, hHGF stimulated the proliferation of both the epithelial and the stromal cells. It is likely that hHGF is involved in the repair or reconstruction process of the endometrium after menstrual shedding and implantation.

Cell Division↗
PubMed1994

Morphological characterization of glomerular endothelial cells cultured on a basement membrane matrix.

We characterized the morphological expression of glomerular endothelial cells (GEC) cultured on a basement membrane matrix, Matrigel. GEC cultured on this matrix formed abundant networks of branching and anastomosing cords of cells, and formed capillary-like structures with a lumen and interdigitating junctional processes. In contrast, GEC cultured on fibronectin-coated dishes had elongated spindle shapes without any interdigitating junctional processes. These findings suggest that the culture system with Matrigel is useful for inducing and maintaining characteristics close to in vivo GEC.

Animals↗
PubMed1993

Therapeutic effects of danazol on endometrial cells may be activated by the presence of extracellular matrix.

OBJECTIVE: To demonstrate the direct effect of danazol on the endometrium in the presence of various types of extracellular matrix. STUDY DESIGN: Human endometrial cells (epithelial and stromal cells) were cultured over various extracellular matrix in the absence or presence of danazol. The effects of danazol on the growth of endometrial cells and the specific glandular structure were studied. RESULTS: Danazol (10(-7) mol/L) inhibited endometrial cell growth, and in the presence of Matrigel, the same danazol concentration demonstrated a stronger inhibitory effect on epithelial cell growth. Danazol inhibited the formation of the specific glandular structure. Its effect depended on the culture substrates used and was reversible. Danazol did not inhibit growth of cells cultured over type V collagen substrate, whereas growth of those grown over type IV collagen was greatly inhibited. CONCLUSION: The results indicate that a distinctive extracellular matrix around ectopic endometriotic tissue modulates cell growth and differentiation. Endometrium associated with a normal extracellular matrix is apparently protected from therapeutic drugs, whereas endometriotic tissue growing over a deranged extracellular matrix would be sensitive to therapeutic drugs, such as danazol.

Cell Differentiation↗
PubMed1992

[Implantation model in vitro].

Implantation is thought to be interactive and synchronized process between mother and embryo, however, the mechanisms of the early implantation process remain unelucidated. Therefore, the effectiveness of in vitro fertilization and embryo transfer is still poor. As a result; immunohistochemically, type IV collagen and other extracellular matrix components were detected mainly below the epithelial layer. Type I and III collagens were detected diffusely in the stromal layer. In the lower stromal layer and superficial myometrial layer, type V collagen was detected. Human endometrial epithelial cells performed the re-epithelialization following glandular formation. Rabbit endometrial cells performed also re-epithelialization following the fold formation. The stromal cells were invaded into the inner layer of the folding. Estrogen added to the culture media stimulated the glandular formation. Progesterone administration after estrogen priming did not affect the glandular formation, however, the proliferation of the superficial epithelium and the re-epithelialized area were increased. Rabbit blastocysts successfully attached and implanted into the reconstructed endometrium. The development of the implanted embryos was morphologically normal. Human cultured trophoblast cells attached, invaded and penetrated into the extracellular matrix components. On the other hand, using type V collagen coated dishes, trophoblast cells could invade the stromal layer, however, type V collagen layer did not permit the trophoblast cells to invade into the collagen layer. In vivo, type V collagen, expressed in the lower stromal layer and the surface of the myometrium, may play a role to maintain the early embryo in the decidual compartment. EGTA inhibited the attachment of the blastocysts. Anti-laminin antibody and RGDS peptide, attachment domain of laminin, also inhibited the implantation. These findings suggested that the Ca2+ dependent process was necessary for the attachment between the trophoblasts and the endometrial cells, and then the implantation process was triggered after the attachment to the laminin in basement membrane. The endometrial tissue, obtained from the infertile patient, was cultured on the basement membrane extract with serially obtained maternal serum. Addition of the maternal serum after proper administration of pFSH, high estrogen conditions were made in the culture dishes. These conditions increased the height of the glandular structure and relatively decreased the area of the surface epithelium. Decreased the area of surface epithelium affected the rate of the attachment. Endometrial cell culture system associated with functional and morphological characteristics was established. Serial observation of the endometrial cells in this system revealed the rabbit and the human implantation process, and the embryo-endometrial interaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Effects of prolactin on cultured human endometrial cells.

We report the effects of PRL on cultured human endometrial cells (epithelial cells and stromal cells) and discuss the role of PRL in implantation. Essentially similar responses to PRL in cell attachment and growth were obtained both in epithelial and stromal cells: a low concentration of PRL (3-30 ng/ml of PRL) enhanced cell attachment and growth. In contrast, the presence of a high concentration of PRL (over 100 ng/ml) resulted in a decrease in cell attachment and growth in normal culture conditions. Morphological analysis of epithelial glandular reformation on basement membrane extracts indicated that PRL had dual effects on endometrial epithelial cells: as a stimulator in low concentrations and an inhibitor in high concentrations. These data suggest that PRL may influence the functions of human endometrial cells in a dual way depending on its level and plays an important role in the implantation process.

Basement Membrane↗
PubMed1989

Gland and epithelium formation in vitro from epithelial cells of the human endometrium.

As is often the case with cell culture, normal human endometrial epithelial cells show a low, squamous shape with flattened nuclei and lack three-dimensional morphology in in-vitro culture systems. Here we report the first well-differentiated epithelial culture system using basement membrane extracts (BME) from an Engelbreth-Holm-Swarm tumour (EHS tumour). In this model, BME regulated the reconstruction of glandular formation and subsequent reformation of the epithelium by epithelial cells. An electron microscopic study clearly indicated that there were two distinct types of cells grown on the substrate. The first, having a high columnar shape, formed the glandular epithelium, and the second, having a cuboidal shape, covered the surface of the BME. Study of epithelial reformation indicated that the regeneration of superficial epithelium could occur, following the development of the glandular formation, in a helix-like pattern. Total protein secretion was greater when the cells were grown on the BME than on plastic. Thus, normal human endometrial epithelial cells cultured on the BME assumed a phenotype and morphology characteristic of those in vivo.

Adult↗
PubMed1989

[A morphological study of glandular formation and epithelialization from normal human endometrial epithelial cells in vitro].

We employed basement membrane extracts (BME) prepared from Engelbreth-Holm-Swarm tumor for in vitro culture of normal human endometrial cells (E-cells) and observed a process of glandular formation followed by epithelialization. E-cells isolated enzymatically were cultured in BME-coated culture dishes and morphological observations were carried out. The results were as follows: 1) E-cells cultured on BME had a globular shape, grew in a helix-like pattern, and formed a tubular glandular structure, rising perpendicularly above the BME surface. 2) Following the glandular formation, 2 weeks after the beginning of the culture, E-cells on the top of the glandular structure spread out in all directions and formed an epithelial-like monolayer cell sheet on the surface of the BME mimicking superficial luminal epithelialization. 3) Glandular E-cells were columnar in shape, had numerous surface microvilli, and faced a lumen which was sealed by a tight junction complex of cells. Superficial E-cells were cuboidal in shape, with mild polarized organelles, nuclei, and abundant microvilli and the surface was entirely covered with BME. These results indicated that this culture system using a BME could be useful in studying the physiology of the human endometrium in vitro and the mechanism of reproduction.

Adult↗
PubMed1989

[The study of bioenergetics of mouse pregnant uterine muscle by magnetic resonance spectroscopy (MRS)].

To investigate the bioenergetics of uterine muscles in vivo, we examined the energy state of mouse preterm uterus by means of magnetic resonance spectroscopy. Full-term mouse uterus contained ATP, PCr, phospho-di and mono ester (PDE and PME) and inorganic phosphate (Pi). The oxytocin-induced uterine muscle contraction peaks level and positions changed. Multiple peak analysis indicated a muscle contraction induced increase in the Pi concentration and decrease in the PCr concentration. The peak position of Pi was shifted in the contractive state also, indicating that the intracellular pH was lower than in the non-contractive state and this low pH level was recovered within several minutes. There was no change in the AMP peak height in the contractive and non-contractive states. These data indicated that the energetics of mouse uterine muscle was maintained by the ATP-PCr system and acidosis of muscle was recovered within several minutes at rest. The constant AMP peak levels may indicate that phosphorylase is not regulated by AMP, but the phosphorylated phosphorylase kinase and pH levels in the contractive and non-contractive states also may indicate that phosphorylase kinase is not regulated by proteolysis or by the intracellular pH level but by the elevated intracellular calcium ion and calmodulin system.

Animals↗
PubMed1986

Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids.

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712-719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme. Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2-4-fold, and the approximate Ka values of this reaction were 10 micrograms/ml, 6.3 micrograms/ml and 30 micrograms/ml respectively. The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2-fold. When fully autophosphorylated, about 12-13 mol phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors. In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30-40-fold at pH 6.8 and 2.5-3-fold at pH 8.2. Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 X 10(-5) M to 8 X 10(-7) M. With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.

Adenosine Triphosphate↗
PubMed1985

Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase.

Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.

Animals↗