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Biomedical subjects

J Watanabe

Publications and source records attributed to J Watanabe.

At least 181 records · Page 10Linked to original sources
PubMed1996

Peri- and postnatal changes in reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase content in hepatocytes of rats.

To study the process of the expression of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase (EC 1.6.2.4) in the liver during development, the amount of enzyme in the cytoplasm of periportal and perivenular hepatocytes in sections cut from livers of male rats was measured during peri- and postnatal growth by quantitative immunohistochemistry with a video image processor. In livers of 19-day-old foetuses, the reductase content in the cytoplasm of periportal and perivenular hepatocytes was 0.16 microM and 0.20 microM, respectively. From the 19th day of gestation to 5 days after birth, the enzyme content increased markedly in the cytoplasm of periportal (288%) and perivenular hepatocytes (301%). Subsequently, the content in the cytoplasm of periportal hepatocytes increased slightly (46%) from 5 to 20 days of age, remained unchanged from 20 to 45 days of age, and increased slightly (15%) from 45 to 90 days of age. However, the content in the cytoplasm of perivenular hepatocytes increased progressively (125%) between 5 and 90 days of age. Thus, the amount of cytochrome P-450 reductase increases markedly in periportal and perivenular hepatocytes during the perinatal period, and subsequently the enzyme content increases gradually in periportal hepatocytes and progressively in perivenular hepatocytes. The present results also suggest that the divergence between cytochrome P-450 expression and the cytochrome P-450-dependent drug metabolic activity in hepatocytes during the perinatal period, found in previous studies, can be attributed to a low cytochrome P-450 reductase density in the membrane of endoplasmic reticulum of periportal and perivenular hepatocytes.

Animals↗
PubMed1996

Neuronal activity in monkey visual areas V1, V2, V4 and TEO during fixation task.

We analyzed 577 neurons recorded from visual areas V1, V2, V4, and the inferotemporal area (TEO) of macaque monkeys, which performed a visual fixation task and a spot-off-on (blink) test during the fixation period. Among these neurons, 35% were defined as task-related cells, because they gave responses at the task-start, fixation, or task-end periods but were unresponsive to the spot blink, which was physically identical to these stimuli. Blink-responsive cells accounted for 29% and task-unresponsive cells for 30% of the neurons. The task-related response was large and frequent in V4 (34%) and TEO (41%), but small and less frequent in V1 (31%) and V2 (27%). Other observations further demonstrated nonsensory activities in these areas: In some cells, response to the fixation spot was inhibitory, whereas light stimulation on the fovea was excitatory; some V1 and V2 cells had color-irrelevant responses, and some cells responded to the spot-off only when the monkey regarded it as a task-end cue.

Animals↗
PubMed1996

Preparation of liposomes that mimic the membrane of endoplasmic reticulum of rat hepatocytes.

To examine the interaction between biomembranes and membrane-bound proteins, large unilamellar liposomes have been required. In the present study, we prepared liposomes from a mixture of phospholipids having a phospholipid composition similar to that in the endoplasmic reticula (microsomes) of rat hepatocytes by eight different methods. The resulting liposomes were examined by a combination of the freeze-fracture-replica procedure with biochemical methods. The freeze-thawing method of Pick (1981) gave the best results; large unilamellar liposomes that mimic the membrane of endoplasmic reticulum were obtained. Liposomes made by this method are thus suitable for analysis of the interaction between the endoplasmic reticulum membrane and membrane-bound proteins.

Animals↗
PubMed1996

Clinicopathologic study on clear cell hepatocellular carcinoma.

In order to study the clinicopathologic characteristics of the clear cell variant of hepatocellular carcinoma (HCC), 215 consecutive cases measuring less than 5 cm in diameter were reviewed. The cases were divided into clear cell HCC (20 cases); focal clear cell HCC (77 cases); and non-clear cell HCC (118 cases). Clinical and pathological findings were compared among these groups. Clear cell HCC was moderately differentiated in 80% of cases and the incidence was not related to tumor size. The male to female ratio was 2.3:1, lower than the 6.9:1 of non-clear cell HCC. The association rate with liver cirrhosis was 90%, higher than the 59.3% of non-clear cell HCC. Three- and five-year survival rates, and no recurrence time were 54.5%, 33.3%, and 564 days, respectively, lower than the findings of 74.3%, 46.1%, and 770 days for non-clear cell HCC. But there is no significant difference in prognosis between both groups. Ultrastructurally, clear cells showed abundant glycogen storage and a variable degree of fat vacuoles, with a marked reduction of the number and size of organelles in the 8 cases examined. Non-clear cells of focal clear cell and non-clear cell HCC showed a moderate degree of glycogen storage in 85.7% and 28.6% of the seven cases examined from each group, with significant difference. It was concluded that clear cell HCC occurs mostly in the moderately differentiated form and is characterized by high female prevalence, high rate of association with liver cirrhosis, and has no significant difference in prognosis compared with non-clear cell HCC.

Adenocarcinoma, Clear Cell↗
PubMed1996

Relationship between immunostaining intensity and antigen content in sections.

We studied the relationship between staining intensity of immunohistochemical reaction and antigen content in sections. Alpha-fetoprotein (AFP) and albumin in sections cut from livers of newborn, 5-, 10-, 20-, and 60-day-old rats were examined as examples. First, we compared average immunostaining intensity (sum of specific absorbance in pixel/number of pixels) measured by image processing (IP), with antigen content measured by immunochemical assay to determine whether the intensity is proportional to antigen content. The intensity of AFP was proportional to the antigen content, whereas that of albumin was not. Subsequently, the antigen preservation test was carried out to determine whether the intensity was decreased by fixation and, if so, which type of decrease (proportional or disproportionate) occurred. Thereafter, antigen content in the same portion in the same immunostained section was measured by the microphotometric (MP) method followed by the IP method, because the MP method gives a low average antigen content when a decrease in antibody binding occurs in sections, whereas the average antigen content measured by the IP method is unchanged. The intensity of AFP decreased primarily by a proportional decrease in antigenicity during fixation. However, the intensity of albumin decreased not only by a proportional decrease during fixation but also by a disproportionate reduction in antibody binding during immunostaining or before fixation. The results indicate that AFP content in sections is measurable by quantitative immunohistochemical methods, whereas albumin content is not.

Albumins↗
PubMed1996

Macromolecule-macromolecule interaction in drug distribution. V. Effects of plasma proteins on uptake of fractionated [3H]heparin in isolated rat Kupffer cells.

Effects of plasma proteins such as alpha-globulin on the uptake of high molecular weight (HMWFH: 23000 Da) and low molecular weight fractionated [3H]heparin (LMWFH: 10000 Da) were examined in isolated rat Kupffer cells. alpha-Globulin (8 mg/ml) affected neither surface binding nor internalization of LMWFH by Kupffer cells, while it reduced both surface binding and internalization of HMWFH without affecting the fraction internalized, which was a ratio of internalized amount to the total association. The total associations of HMWFH were about four times larger than that predicted assuming only the unbound fraction is available for uptake, suggesting the participation of protein-mediated transport in the uptake of HMWFH in Kupffer cells. Based on the same assumption, the saturable initial uptake of HMWFH versus concentration profile in the presence of alpha-globulin (8 mg/ml) was also analyzed to further examine the suggested protein-mediated transport. The estimated dissociation constant of 487 nM was three times larger than that in in vitro binding experiments (168 nM) and the binding capacity of 0.155 was one third of the value in vitro (0.5), suggesting apparent reductions in both binding affinity and capacity. Thus, we demonstrated the involvement of protein-mediated transport in the uptake of fractionated heparin in Kupffer cells and kinetically characterized it as the apparent enhancement of dissociation.

Alpha-Globulins↗
PubMed1996

Dose-dependent gastrointestinal absorption of 5-fluorouracil in rats in vivo.

Dose-dependent gastrointestinal absorption of 5-fluorouracil (5-FU) was kinetically evaluated in rats in vivo by analyzing gastrointestinal disposition after oral administration, where a linear model assuming first-order gastric emptying followed by first-order intestinal absorption was fitted to remaining fraction versus time profiles for the stomach and small intestine to estimate the rate constants of gastric emptying (kg) and intestinal absorption (Ka). With an increase in dose from 1.5 nmol/rat (low dow) to 15 mumol/rat (high dose), the Ka decreased from 5.95 to 0.55 min-1, suggesting the involvement of carrier-mediated transport. This study is the first to demonstrate the dose-dependent gastrointestinal absorption of 5-FU in vivo, though it has long been suggested in situ and in vitro. Meanwhile, at both the low and high doses, the Kg values, which were unaffected by dose (0.069 and 0.082 min-1, respectively, for the low and high doses), were smaller than the Ka values by an order of magnitude or more and the recovery of 5-FU was negligible, compared with that of inulin (a nonabsorbable marker), in the most distal segment of ileum. These results suggest that, regardless of dose, 5-FU is highly absorbable in a gastric emptying-limited manner. Thus, well-publicized bioavailability problems (low and erratic) of 5-FU may be attributable to extensive and variable first-pass metabolism rather than poor and variable gastrointestinal absorption.

Animals↗
PubMed1996

Macromolecule-macromolecule interaction in drug distribution. IV. Molecular weight dependency in the interaction of fractionated [3H]heparin with plasma proteins.

Molecular weight dependency in the interaction of fractionated [3H]heparin (FH) with plasma proteins was evaluated by determining the protein binding of low molecular weight fractionated [3H]heparin (LMWFH: 7000 Da) and high molecular weight fractionated [3H]heparin (HMWFH: 16000 Da) by ultrafiltration and the effects of plasma proteins on the uptake in rat hepatocytes in primary culture. The unbound fractions of LMWFH were 0.5 and 0.8 in the presence of alpha-globulin and albumin, respectively, and were about 10 times larger than those of HMWFH, 0.04 and 0.1, suggesting a reduction in binding with a decrease in molecular weight. However, while the uptake of LMWFH was reduced by these proteins by the extents similar to bound fractions of LMWFH, the uptake of HMWFH was reduced by extents far smaller than bound fractions and comparable with those for LMWFH. Thus, it seemed that, while only unbound LMWFH is available for uptake, HMWFH bound to proteins is to some extent available for uptake (protein-mediated transport). The protein-mediated transport of heparin seemed to reduce with a decrease in molecular weight. It was also shown that the extended uptake of LMWFH was smaller than that of HMWFH not only in the absence of proteins but also in the presence of alpha-globulin, the major binding protein. The lower uptake of LMWFH is consistent with in vivo suggestion of lower hepatic accumulation.

Animals↗
PubMed1996

Uptake mechanism of fractioned [(3)H]heparin in isolated rat kupffer cells: involvement of scavenger receptors.

The uptake of fractionated [(3)H]heparin was examined to elucidate the uptake mechanism in isolated rat Kupffer cells. The equilibrium binding of fractionated [(3)H]heparin to Kupffer cells was concentration-dependent with the dissociation constant of 5.7 nM and the maximum binding capacity of 1.5 pmol/10(6) cells. Several ligands of scavenger receptors inhibited the binding of fractionated [(3)H]heparin to Kupffer cells competitively and also the internalization of heparin, suggesting the involvement of scavenger receptors in the uptake of fractionated [(3)H]heparin. Fractionated [(3)H]heparin was also suggested to be internalized according to first order kinetics with the apparent internalization rate constant of 0.010 min (-1). Lowering temperature from 37 to 4 degrees C reduced the fraction internalized from 33% to 6% without affecting the total association, while the fraction internalized at 25 degrees C was comparable with that at 37 degrees C. Metabolic inhibitors (2,4-dinitrophenol and rotenone), an inhibitor of receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of fractionated [3(H)]heparin. As known inhibitors of receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis did not affect the uptake of fractionated heparin, the scavenger receptor-mediated uptake is suggested to be ATP-independent and different from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, although for temperature dependency it showed the typical characteristics of receptor-mediated endocytosis.

2,4-Dinitrophenol↗
PubMed1996

Evaluation of the fractional absorption of D-xylose by analysis of gastrointestinal disposition after oral administration in rats.

As an efficient method for estimating the amount orally absorbed, the fraction of D-xylose absorbed in rats was estimated from the ratio of the fecally excreted proportion of D-xylose dose to that of polyethylene glycol (PEG) 4000 as a nonabsorbable marker (D-xylose/PEG 4000 ratio). The D-xylose/PEG 4000 ratio was demonstrated to be independent of the fecal excretion of D-xylose and PEG 4000 (or sampling period), suggesting that it can represent the fraction of the total dose remaining. This result was consistent with the theoretical prediction based on the assumptions that every small fraction of dose behaves in the same manner with regard to absorption and transit through the absorption site (small intestine), and that the gastrointestinal transit of PEG 4000 is identical with that of D-xylose. The fraction of D-xylose absorbed was estimated to be 95.9% by subtracting the remaining fraction (D-xylose/PEG 4000 ratio) from unity (100%). The D-xylose/PEG 4000 ratio, or drug/marker ratio in general, can be determined before fecal excretion is completed. Thus it can provide an efficient way for estimating the fraction of a drug absorbed that is stable in the gastrointestinal tract, such as D-xylose. Although this proposed method of estimating a fraction is generally not applicable unless a given drug is proved to be stable in the gastrointestinal tract, it can be an efficient screening method to identify poorly absorbable drugs. It is especially useful in basic studies and preclinical tests in laboratory animals.

Administration, Oral↗
PubMed1996

Molecular weight dependency in the uptake of fractionated [3H]heparin in isolated rat Kupffer cells.

The uptake of low molecular weight fractionated [3H]heparin (LMWFH, 10000 Da) was compared with that of high molecular weight fractionated [3H]heparin (HMWFH, 23000 Da) in isolated rat Kupffer cells. Several heparin analogs, including HMWFH and ligands of scavenger receptors, inhibited both the surface binding and internalization of LMWFH, suggesting the involvement of scavenger receptors in the uptake of LMWFH in isolated rat Kupffer cells as well as HMWFH, in spite of a large difference in molecular weight. Metabolic inhibitors (2,4-dinitrophenol and rotenone), receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of LMWFH. These results suggest that the scavenger receptor-mediated uptake of LMWFH is ATP-independent and different from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, in agreement with our previous results for HMWFH. The equilibrium binding of LMWFH to Kupffer cells was concentration-dependent with the dissociation constant (Kd) of 50 nM and maximum binding capacity (Bmax) of 2.3 pmol/10(6) cells. The dissociation constant of LMWFH was an order of magnitude larger than that of HMWFH (5.7 nM), suggesting a decrease in binding affinity to scavenger receptors with a decrease in the molecular weight of fractionated heparin. It was also shown that LMWFH is internalized by scavenger receptors according to first-order kinetics with an apparent internalization rate constant (Kint,app) of 0.0053 min-1, which is about half that for HMWFH (0.0118 min-1). Molecular weight thus appears to be one of dominant factors determining the uptake of fractionated heparin by scavenger receptors in Kupffer cells, and may partly explain the reported lower hepatic uptake of low molecular weight heparin than that of unfractionated heparin.

2,4-Dinitrophenol↗
PubMed1996

Intestinal brush border transport mechanism of 5-fluorouracil in rats.

The intestinal transport mechanism of 5-fluorouracil (5-FU) was investigated in the intestinal everted sacs and brush border membrane vesicles (BBMVs) of rats. In the everted sacs, the initial uptake of 5-FU was apparently Na(+)-dependent, in terms of the inhibition of the uptake by replacing the Na+ in the medium with K+, and also concentration-dependent in the presence of Na+, with a maximum transport rate of 0.74 +/- 0.24 nmol/min/cm and a Michaelis constant of 0.025 +/- 0.018 mM. Passive transport was also significant, with a membrane permeability clearance of 5.9 +/- 0.6 microliters/min/cm. The uptake of 5-FU was inhibited by pyrimidines (uracil and thymine) and 2, 4-dinitrophenol (DNP), a metabolic inhibitor, but not by purines (adenine and guanine), pyrimidine nucleosides (thymidine and uridine), L-alanine or D-glucose. These results suggest the involvement of carrier-mediated transport specific to pyrimidines in intestinal 5-FU transport, and this appeared to satisfy the criteria of Na(+)-dependent secondary active transport. However, in BBMVs, 5-FU uptake was independent of Na+, minimally dependent on concentration and not inhibited by thymine, though slightly inhibited by uracil. 5-FU uptake was also independent of pH, outward HCO3- gradient and valinomycin-induced K+ diffusion potential. Thus, the carrier-mediated transport of 5-FU, or presumably pyrimidines, may require some other factors, which are yet to be identified, in addition to Na+. Alternatively, Na+ may not be prerequisite, and something else may be required in the absence of K+, as suggested from an additional result in everted sacs that the replacement of NaCl in the medium with mannitol failed to inhibit 5-FU uptake.

Animals↗
PubMed1996

The role of extracellular cations in the development of myogenic contraction in isolated rat small arteries.

The purpose of this study was to determine the roles of extracellular cations (Na+, Ca2+ and K+), membrane K+ channels and Na+/K+ ATPase in the development of myogenic contraction (transmural pressure-induced contraction) in isolated rat skeletal muscle and mesenteric small arteries. The vessels were pressurized under no-flow conditions in a tissue bath. Lumen diameter was measured with a videomicroscopic system. Myogenic contraction was evoked by increasing the lumen pressure from 40 to 100 mmHg. The vessels demonstrated myogenic contraction in low-Na+ (Na+ 1.18 mmol/L) physiological salt solution (PSS), and this was abolished by removing Ca2+ or by applying nifedipine or nisoldipine (10 mumol/L). Neither tetraethylammonium (TEA, 1 mmol/L), Ba2+ (10 mumol/L) nor glibenclamide (1 mumol/L) affected the magnitude of the myogenic contraction. K(+)-free PSS and ouabain (0.1 mmol/L) partially depressed myogenic contraction. In conclusion, myogenic contraction was triggered by a cellular process that requires extracellular Ca2+, but not Na+ or K+. This triggering process is not affected by TEA, Ba2+ or glibenclamide.

Animals↗