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T Fuwa

Publications and source records attributed to T Fuwa.

At least 55 records · Page 3Linked to original sources
PubMed1990

Internalization of EGF in perfused rat liver is independent of the degree of receptor occupancy.

The internalization rate of epidermal growth factor (EGF) by the perfused rat liver was evaluated to determine whether the internalization rate constant depends on the degree of receptor occupancy. A tracer concentration of 125I-labeled EGF (30 pM) alone or 125I-EGF plus unlabeled EGF (20 nM) was infused into the liver in the single-pass perfusion system at 37 degrees C. At various times (2-20 min), the perfusion medium was switched to medium of pH 3.0, and the radioactivity of 125I-EGF emerging into the outflow (surface-bound EGF) and remaining in the liver (internalized EGF) was determined. At the tracer condition, less than 0.1% of the surface receptor was occupied by 125I-EGF for 20 min perfusion. When excess unlabeled EGF (20 nM) was present in the perfusate and the bulk of the cell surface receptors was occupied, the density of the cell surface EGF receptor after 20 min dropped to 14% of the initial value. The internalization rate constant, defined as the probability of an occupied receptor being internalized per minute, was calculated from the slope of a plot of the amount of internalized EGF vs. the integrated amount of surface receptor-bound EGF with time. The internalization rate constants calculated from the plots with and without unlabeled EGF in the perfusate were 0.21 and 0.33 min-1, respectively, and did not differ significantly (P greater than 0.1). These results indicated that the internalization rate constant of the EGF receptor complex is independent of the degree of receptor occupancy in the liver.

Animals↗
PubMed1990

Response of A431 experimental human solid xenograft to mitomycin C in combination with human epidermal growth factor in mice.

We previously demonstrated that the antitumor efficacy of various antitumor agents such as 5-fluorouracil and cisplatin against experimental solid tumors was enhanced by pre- or simultaneous administration of human epidermal growth factor (hEGF). In the present study, the combined therapy by hEGF and mitomycin C (MMC) as an antitumor agent was studied in A431 solid tumor-bearing mice to determine the dosage schedule of hEGF. When MMC alone was injected intraperitoneally (2 mg/kg) every 7th day to the tumor-bearing mice, tumor weights increased to 2138 +/- 285 mg from 282 +/- 41 mg during 22 d. Tumor weight in every day treatment of hEGF alone for 21 d increased to the same extent in the treatment by MMC alone. On the other hand, the increase of the solid tumor weight in the every day treatment and in the every 7th day treatment of hEGF, in combination with the every 7th day administration of MMC, were as follows; from 282 +/- 41 mg to 1522 +/- 357 mg (71.2 +/- 16.7% of MMC alone) and from 280 +/- 44 mg to 1245 +/- 150 mg (58.2 +/- 7.0% of MMC alone), respectively, demonstrating a greater antitumor potency of MMC in the combination with the every 7th day treatment of hEGF. Both combined therapies did not affect the toxicity of MMC as evaluated by decrease in nontumorous body weight. Single subcutaneous administration of hEGF to A431 tumor-bearing mice caused the decrease of the binding capacity of hEGF to A431 tumor cells by 80% 24 h after the administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Decrease in the number of receptors for epidermal growth factor in the liver of D-galactosamine-intoxicated rats.

Hepatic transport of epidermal growth factor (EGF) was studied in D-galactosamine-intoxicated rats by the multiple-indicator dilution (MID) method. The extraction ratio of 125I-labeled EGF in the intoxicated rats, obtained from a model-independent analysis of the dilution curves, decreased to 45% of the control values. A distributed two-compartment model was fitted to the dilution data by nonlinear least-squares regression, and the kinetic parameters, kon.PT (product of on-rate constant and receptor density), koff (off-rate constant) and ks (sequestration rate constant) were determined. The values of kon.PT and ks in the intoxicated rats decreased to approximately one-half and one-third of those in the control rats respectively. Similar decreases in the kon.PT and ks values in the intoxicated rats were also observed for the transport of 125I-labeled insulin, a positive control, into the liver. The 125I-labeled EGF binding experiment at equilibrium using liver homogenates revealed that the intoxication reduced the receptor density (PT) to one-third of the control values, whereas the equilibrium dissociation constant (kd) did not change significantly. The activities of Na+,K+-ATPase, cytochrome P-450 and glutathione S-transferase decreased in the intoxicated rats to 70-80% of the control values. The number of nuclei per unit area of tissue slices was also reduced to 70% of the control. Thus, the extent to which the enzyme activities and the number of nuclei decreased in the intoxicated liver was smaller than that of the number of EGF receptors. It is concluded that the reduction of EGF receptors cannot be explained by the "intact hepatocyte hypothesis" but rather by the functional change of hepatocytes induced by the administration of D-galactosamine.

Animals↗
PubMed1989

Preparation of EGF labeled liposomes and their uptake by hepatocytes.

The preparation of epidermal growth factor (EGF) bearing liposomes (EGF-liposomes) and their uptake by rat hepatocytes were described. EGF was bound to the liposomal surface by the disulfide bridge linkage using a heterobifunctional cross-linking reagent, N-hydroxysuccinimidyl-3-(2-pyridyl-dithio) propionate. EGF-liposomes were taken up in a significant amount by rat hepatocytes in an EGF receptor mediated manner, and their uptake was dependent on the amount of labeled EGF coupled to the liposomal surface. These results raise the possibility that the EGF-liposomes may be an effective drug carrier to the cells having EGF-receptors like some cancer cells.

Animals↗
PubMed1989

Polypeptide chain fold of human transforming growth factor alpha analogous to those of mouse and human epidermal growth factors as studied by two-dimensional 1H NMR.

The 1H NMR spectrum of human transforming growth factor alpha (TGF-alpha) was analyzed almost completely by the sequential assignment method using two-dimensional NMR techniques. On the basis of the nearly complete sequence-specific resonance assignment, secondary and tertiary structures of human TGF-alpha in solution (pH 4.9, 28 degrees C) were determined to satisfy the upper limits of proton-proton distances derived from nuclear Overhauser effect experiments. Although human TGF-alpha and mouse epidermal growth factor (EGF) share 27% homology in amino acid sequence, the backbone chain folds in the two growth factors are quite similar. The structure and function of TGF-alpha is well characterized by the "mitten model" previously proposed for mouse EGF. The gross shape of the TGF-alpha molecule resembles a mitten. TGF-alpha interacts with the receptor as a mitten would grasp an object. However, there is an appreciable structural difference between the two growth factors in the back of the mitten that is formed by the N-terminal polypeptide segment. This is consistent with the evidence that the backs of these molecules are not involved in the receptor binding.

Amino Acid Sequence↗
PubMed1989

Kinetic analysis of the elimination process of human epidermal growth factor (hEGF) in rats.

Pharmacokinetic study of human epidermal growth factor (hEGF) in rats was performed in vivo. The hepatic extraction ratio (EH) of [125I]hEGF, determined from the difference between the artery and the hepatic vein plasma concentrations at steady state, was 0.19. The hepatic clearance (CLH:7.56 ml/min/kg body wt), calculated by multiplying EH by the hepatic plasma flow rate (QP,H), was approximately 70% of the total body clearance (CLtot: 10.8 ml/min/kg body wt), which was determined from the steady-state arterial plasma concentration and the infusion rate. These results indicated that the liver is the main organ responsible for the removal of [125I]hEGF from the systemic circulation in rats. The renal extraction ratio (ER) of [125I]hEGF was half of that of [14C]inulin; this may have resulted from the plasma protein binding of [125I]hEGF, which was approximately 50% as determined by the charcoal adsorption method and the equilibrium gel-filtration method. The renal clearance (CLR:2.65 ml/min/kg body wt), calculated by multiplying ER by the renal plasma flow rate (QPR), was approximately 17% of the CLtot (15.6 ml/min/kg body wt), indicating a minor contribution of CLR to CLtot compared with that of CLH to CLtot. The CLR of [125I]hEGF calculated from the urinary excretion data was one-tenth of that calculated from the plasma concentration difference between the femoral artery and the renal vein at steady state. These results suggest that the bulk of [125I]hEGF cleared from the plasma by the kidney may have been metabolized further in the renal tubules before appearing in the urine.

Animals↗
PubMed1989

Protection of liver microsomal membranes from lipid peroxidation by garlic extract.

Garlic extract, the ethanol-soluble fraction of garlic, prevented formation of thiobarbituric-acid-reactive substances and fluorescent substances during lipid peroxidation of rat liver microsomes. Lipid peroxidation increased the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene labelled to the microsomes while this increase was prevented by the garlic extract. It thus seems probable that the garlic extract serves to maintain membrane fluidity. These effects were dependent on its concentration and particularly prominent on exceeding a certain concentration of garlic extract. These results suggest its possible role of protecting the membranes from lipid peroxidation.

Animals↗
PubMed1989

Epidermal growth factor receptor-mediated selective cytotoxicity of antitumor agents toward human xenografts and murine syngeneic solid tumors.

Severe toxic side effects of antiproliferative agents limit their clinical usefulness as antitumor drugs. Recently we observed that the antitumor efficacy of various antitumor agents (5-fluorouracil, tegafur, adriamycin, mitomycin C, cyclophosphamide, and cisplatin) against experimental solid tumors was enhanced by prior or simultaneous administration of human epidermal growth factor (EGF). However, coadministration of EGF did not enhance the toxicity of antitumor agents as measured by LD50 and body weight loss. The above selective potentiation of efficacy of the antitumor agents by human EGF can be characterized as follows. In a dose-dependent manner, human EGF enhanced the efficacy of an antitumor agent (5-FU) treatment against human epidermoid carcinoma A431 transplanted sc in athymic nude mice [ED50 = 2.9 (0.2-49.7, 95% confidence interval) microgram/kg, sc]. Various degrees of enhancement were also observed against other experimental tumors transplanted sc. The degrees of enhancement were directly proportional to the numbers of human EGF binding sites present on tumor cell plasma membrane (threshold of binding site density = 1.5 X 10(3) sites/cell) using 5-FU or cisplatin as an antitumor agent, thus suggesting that the binding of EGF to the receptors on tumor cells is an essential process in enhancing the susceptibility of tumor cells to antitumor agents. Normal cells including intestinal epithelial and bone marrow cells are endowed with fewer EGF binding sites (less than 10(3) sites/cell). This may explain partially the absence of EGF-enhanced cytotoxicity by antitumor agents toward normal cells.

Animals↗
PubMed1989

Antitumor-promoting activity of garlic extracts.

Garlic extract was proved to inhibit one of the earliest phenomena caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, in vitro; i.e., the enhancement of phospholipid metabolism. And also the first stage of tumor promotion in two-stage mouse skin carcinogenesis in vivo was suppressed by the treatment with garlic extract. Thus, garlic extract seems to be effective to inhibit initial events caused by TPA type tumor promoters in vitro and in vivo.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Kinetic analysis of in vivo receptor-dependent binding of human epidermal growth factor by rat tissues.

Kinetic analysis of the tissue distribution of human epidermal growth factor (hEGF) in rats was performed in vivo. The plasma disappearance half-life of [125I]hEGF was prolonged by coadministration of unlabeled hEGF, indicating saturation of the mechanism for hEGF removal from the systemic circulation. To analyze the contribution of each tissue to the uptake of hEGF, the amount of [125I]hEGF taken up by each tissue was determined after coadministration of various amounts of unlabeled hEGF. Kinetic analysis of the data yielded the following results. (1) Among the tissues examined, the distribution of [125I]hEGF to the liver, kidney, small intestine, stomach, and spleen was much greater than that accounted for by the distribution to the extracellular space of each tissue. (2) The binding (or uptake) of hEGF by these tissues showed remarkable saturation, which may represent the receptor-dependent binding (or uptake) mechanism. (3) The apparent binding (or uptake) clearance per gram of tissue at the low dose (in the range of first-order kinetics), defined with regard to the arterial plasma concentration, was greatest in the kidney, followed by the liver and small intestine. The larger binding (or uptake) clearance of the kidney compared with that of the liver can be attributed to the higher plasma flow rate (per gram of tissue) in the kidney. However, the intrinsic ability to take up hEGF was much greater in the liver than that in the kidney. The hepatic binding (or uptake) of hEGF at the low dose was almost limited by the hepatic plasma flow rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor.

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.

Aminocaproates↗
PubMed1988

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of 125I-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [ln (inulin/EGF) versus time] of the dilution curve for EGF exhibited an upward straight line initially for a short period of time (approximately equal to 10 sec), whereas the ratio plot [ln (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer approximately equal to 30 micrograms) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kappa on), the dissociation rate constant of EGF from the EGF-receptor complex (kappa off), and the sequestration rate constant of the complex. The kinetic parameters [the dissociation constant (Kd = kappa off/kappa on) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones--cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.

Algorithms↗
PubMed1988

Comparison of specific binding of human epidermal growth factor (EGF) to sinusoidal and bile canalicular membranes isolated from rat liver.

The binding characteristics of human epidermal growth factor (EGF) were compared between highly purified canalicular (CMV) and sinusoidal (basolateral) rat liver plasma membrane (SMV) preparations. The dissociation constants (2-3 nM) for these membranes were comparable, while the binding capacity for CMV was approximately half that for SMV. The binding capacity for CMV was too high to be accounted for only by the contamination with sinusoidal membranes, since the measurements of specific activities of various enzymes (Na+,K+-ATPase, alkaline phosphatase, and leucine aminopeptidase) indicated that the extents of the cross contamination with other membrane fractions were at most 10%. Although the physiological function of specific binding of EGF to bile canalicular membrane domain remains to be determined, it may have a role in biliary excretion of EGF. The specific binding of EGF to bile canalicular membranes from rat liver was identified for the first time.

Animals↗
PubMed1987

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells.

Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 microgram/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 microgram/ml insulin.

Animals↗