Biomedical subjects

S Kanamura

Publications and source records attributed to S Kanamura.

At least 19 recordsLinked to original sources

PubMed2000

Effect of phenobarbital on intralobular expression of CYP2B1/2 in livers of rats: difference in the expression between single and repetitive administrations.

Phenobarbital (PB) was shown to induce the major PB-inducible cytochrome P450 (CYP) isoforms, CYP2B1/2, in perivenular hepatocytes by a single injection, and in midzonal and periportal hepatocytes in addition to perivenular hepatocytes by injections of the same dosage once a day for 3 days in rat livers. The present study was undertaken to determine whether the spread of enzyme induction to midzonal and periportal hepatocytes is caused by the increase in total dose of the drug by repetitive injections or by the repetitive injections of the drug themselves. Male adult rats were administered PB by a single injection (80 mg/kg) or repetitive injections (20 mg/kg once a day for 4 days; a total dose of 80 mg/kg), and the molar content of CYP2B1/2 was measured by quantitative immunohistochemistry in the cytoplasm of perivenular, midzonal, and periportal hepatocytes. In addition, the molar content of total CYP in the cytoplasm was measured by microphotometry, and the expression of CYP2B2 mRNA was examined by in situ hybridization. When animals received the single injection, the isoforms and CYP2B2 mRNA increased markedly in perivenular hepatocytes, increased somewhat in midzonal hepatocytes, and remained unchanged in periportal hepatocytes. If animals received the repetitive injections, however, although the isoforms and the mRNA increased markedly in perivenular hepatocytes, they also increased markedly in midzonal hepatocytes and somewhat in periportal hepatocytes. These findings demonstrated that the enlargement of the sublobular area in which induction of the isoforms occurred was caused by the repetitive injections of PB themselves.

Analysis of Variance↗

PubMed2000

Lymph node metastases identified with mediastinoscopy in a patient with superficial carcinoma of the esophagus.

Superficial esophageal cancers limited to the lamina propria are not associated with lymph node metastases. Mediastinoscopic transhiatal esophagectomy was planned in a patient with widespread superficial cancer of the midthoracic esophagus. Sampling of the upper mediastinal lymph nodes revealed metastases. The operation was converted to a transthoracic esophagectomy with radical lymphadenectomy. Histopathologic examination of the resection specimen showed three metastatic lymph nodes, despite local invasion limited to the lamina propria. This is the first report of a patient with superficial esophageal cancer and lymph node metastases.

Carcinoma, Squamous Cell↗

PubMed2000

Cell biology of cytochrome P-450 in the liver.

Cytochromes P-450 (P-450) are members of a multigene superfamily of hemoproteins consisting the microsomal monooxygenase system with NADPH P-450 reductase (reductase) and/or reducing equivalents. Expression of many P-450 isoforms in hepatocytes is shown to be regulated at the level of transcription through interaction between cis-acting elements in the genes and DNA-binding (transacting) factors. Some isoforms of the CYP1A, 2B, 2E, and 3A subfamilies are regulated at the posttranscriptional level. For the topology of P-450 and reductase molecules in ER membrane of hepatocytes, models from stopped flow analysis and electron spin resonance are proposed. The densities of total P-450 and reductase molecules are revealed to be high enough to support the cluster model, suggesting that about ten P-450 molecules form an aggregate and surround one reductase molecule, and therefore the two enzymes form large micelles. ER proliferation after PB administration, which had been correlated with increase in P-450 level, is shown to be probably independent of the increase in P-450 level. There are considerable discrepancies among results reported on sublobular expression of various P-450 isoforms. Causes of the discrepancies are likely to be differences in experimental conditions of histochemical detection carried out and/or in species, strain, and/or sex.

Animals↗

PubMed2000

Rifampicin suppresses hepatic CYP2E1 expression and minimizes DNA injury caused by carbon tetrachloride in perivenular hepatocytes of mice.

BACKGROUND: Rifampicin has been shown to increase during activities of serum transaminases and to decrease in cytochrome P-450-mediated monooxygenase activities in livers of mice treated with carbon tetrachloride (CCl4). Although these findings suggest that rifampicin prevents hepatocyte damage caused by CCl4, detailed information on the protective effects is not available. METHODS: We injected first rifampicin and then CCl4 into mice and examined denaturation and fragmentation of hepatocyte DNA by in situ nick translation, in situ end labeling, and in situ hybridization. Furthermore, expressions of p53, a cytoplasmic marker for apoptosis, and bcl2, an anticell death factor, were examined immunohistochemically. In addition, a major ethanol-inducible P-450 isoform in liver homogenates or microsomes, CYP2E1, was examined by Western blotting, because the enzyme metabolizes CCl4 and forms free radicals to injure perivenular hepatocytes in which the enzyme is restrictedly expressed. RESULTS: Rifampicin prevented the denaturation and fragmentation of DNA caused by CCl4 in perivenular hepatocytes except for those located within two or three cell layers surrounding the central venule. Furthermore, CYP2E1 decreased in liver homogenates or microsomes from rifampicin-treated animals. It is therefore likely that rifampicin suppresses expression of CYP2E1 and protects CCl4-mediated DNA damage of hepatocytes by inhibiting formation of free radicals. In addition, perivenular hepatocytes except for those surrounding the venule showed negative immunoreaction for p53 and bcl2 in rifampicin+CCl4-treated animals. CONCLUSIONS: The drug did not alter the mechanism of cell death from necrosis to apoptosis and did not promote recovery of hepatocytes from CCl4-mediated damage.

Animals↗

PubMed1998

Culture of stromal cells derived from medullary cavity of human long bone in the presence of 1,25-dihydroxyvitamin D3, recombinant human bone morphogenetic protein-2, or ipriflavone.

We previously showed that stromal cells derived from bone marrow specimens formed at the fracture site of human long bone differentiated during culture to polygonal cells and spindle cells, and polygonal cells, but not spindle cells, produced calcified matrix. To clarify the origin of polygonal and/or spindle cells, and factors necessary for differentiation of marrow stromal cells to osteogenic cells, we cultured stromal cells derived from the normal (unfractured) medullary cavity (SCN) as well as stromal cells from the medullary cavity distant from the fracture site (SCF). After 3 weeks of primary culture and 2 days of secondary culture, the cells were cultured in medium containing 1,25-dihydroxyvitamin D3 (VD), recombinant human bone morphogenetic protein-2 (BMP), or ipriflavone (IF) for 3 weeks. For biochemical analysis, cells reaching confluence after 3 weeks of secondary culture were cultured with one of the factors for 3 days. Some of SCF cultured with VD or IF were transformed to polygonal cells, and showed high alkaline phosphatase (ALPase) activity and high osteocalcin and insoluble calcium production. Cloned polygonal cells from the SCF formed nodules and aggregates consisting of calcium. Other SCF cultured with VD or IF and SCF cultured with BMP were spindle shaped. Some spindle-shaped cells from SCF cultured with BMP or IF revealed high ALPase activity and high osteocalcin production, comparable with the spindle cells from the fracture site. However, spindle-shaped cells from SCF cultured with VD and other spindle-shaped cells from SCF cultured with BMP or IF showed low ALPase activity and low osteocalcin production. The results show that SCF probably contain at least three subpopulations: (a) cells that differentiate to polygonal cells by the influence of VD or IF; (b) cells that differentiate to the spindle cells by the influence of BMP or IF; and (c) cells that are not transformed by the influence of VD, BMP, or IF.

Adolescent↗

PubMed1998

Localization of xenobiotic-responsive element binding protein in rat hepatocyte nuclei after methylcholanthrene administration as revealed by in situ Southwestern hybridization.

Xenobiotic-responsive element binding protein (XRE-BP), a heterodimer of aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt), regulates the transcription of cytochrome P-450 1A1 gene (CYP1A1) through XRE in response to xenobiotic inducers. For a better understanding of the regulatory mechanism of CYP1A1 through XRE, localization of XRE-BP was examined in liver sections or isolated hepatocyte nuclei from control and 3-methylcholanthrene (MC)-treated rats by in situ Southwestern hybridization, using synthetic XRE as a probe, and was observed by confocal laser scanning microscopy and electron microscopy. Gel mobility shift assay and competitive binding assay showed specificity of the synthetic XRE probe. XRE-BP was exclusively localized in hepatocyte nuclei in liver sections from animals 3 hr after MC injection, whereas the protein was absent in hepatocyte cytoplasm in MC-treated animals and in hepatocyte nuclei and cytoplasm in control animals. In isolated hepatocyte nuclei, XRE-BP began to accumulate in the central region between 0.5 and 3 hr, showed a peak between 3 and 6 hr, decreased gradually between 6 and 72 hr, and disappeared at 72 hr after MC injection. The protein was scarce in peripheral and nucleolar regions of the nucleus. Therefore, XRE-BP is formed in the nuclei of hepatocytes after MC stimulation. In addition, XRE-BP was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals, although the protein was absent in the nuclei before the preincubation. These findings strongly suggest that AhR translocates from hepatocyte cytoplasm to the nucleus and forms XRE-BP in the nucleus after MC stimulation.

Animals↗

PubMed1998

Effect of 3-methylcholanthrene administration on expression of cytochrome P-450 isoforms induced by phenobarbital in rat hepatocytes.

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.

Animals↗

PubMed1998

Determination of accuracy of quantitative immunohistochemical results with an antigen-immobilized filter model system.

To determine accuracy of quantitative immunohistochemical results, serially diluted liver cell lysates from methylcholanthrene (MC)-treated rats containing cytochrome P-450 (P-450) 1A were immobilized on nitrocellulose (NC) filters and stained by the indirect immunoperoxidase method under saturation conditions. The stained filters were processed for image analysis and the relationship between the resulting immunostaining intensity due to P-450 1A and the antigen amount immobilized on the filters was examined. The relationship examined in the filters fitted with an exponential curve. Thus, the intensity due to P-450 1A is not simply proportional to the antigen amount. Subsequently, immunostaining intensity due to P-450 1A was measured in sections from control and methylcholanthrene (MC)-treated rats by image analysis with an image processor, and the antigen content in cell lysates from control and MC-treated animals was measured immunochemically by quantitative single radial immunodiffusion. Although immunochemically measured P-450 1A content in the lysates increased markedly (26 times), average staining intensity in sections increased slightly (2-3 times) after MC injection. However, when the exponential curve obtained from the filter binding assay was used as a standard curve to convert staining intensity in sections to molar content of P-450 1A, the resulting content was compatible with the immunochemical content. The divergence between the increase in immunochemically measured P-450 1A content and that in immunostaining intensity in sections after MC injection is therefore attributed primarily to the curvilinear relationship between the intensity and the content.

Animals↗

PubMed1997

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats.

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg-1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration.

Animals↗

PubMed1997

Quantitative analysis of endoplasmic reticulum and cytochrome P-450 in hepatocytes from rats injected with methylcholanthrene.

To examine whether the smooth endoplasmic reticulum (SER) proliferates in hepatocytes from animals treated with methylcholanthrene (MC) frequently used as an inducer for the enzymes of the microsomal mono-oxygenase system, we estimated the area of (smooth and rough) ER per unit cytoplasmic volume by morphometry in periportal, midzonal and perivenular hepatocytes from rats injected with 25 mg/kg MC once a day for 3 days. In addition, immunostaining intensity of major MC-inducible cytochrome P-450 (P-450) forms (1A1/1A2) and total P-450 content in the cytoplasm of hepatocytes in the three zones were measured by microphotometry to ascertain whether P-450 is sufficiently induced in each sublobular zone by the administration. In spite of significant increase in the staining intensity of P-450 1A1/1A2 and amount of total P-450, the proliferation of SER (and RER) did not occur in the three-zone hepatocytes from rats injected with MC. In perivenular hepatocytes, constitutive forms of P-450 other than 1A1/1A2 decreased (to 10%) instead of marked increase in P-450 1A1/1A2 (about 20 times), while the constitutive forms decreased to 50% in midzonal hepatocytes and remained unchanged in periportal hepatocytes after MC administration. In addition, the present results show divergence between biochemical and immumohistochemical results previously reported on MC-inducible P-450 after MC administration to be due primarily to a curvilinear relationship between content and intensity.

Animals↗

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

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

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↗

PubMed1995

Endoplasmic reticulum proliferates without an increase in cytochrome P-450 in hepatocytes of mice treated with phenobarbital and cobalt chloride.

To determine whether endoplasmic reticulum (ER) proliferation in hepatocytes after phenobarbital (PB) administration relates closely to cytochrome P-450 (P-450) increase, we have measured the amount of total P-450 per unit cytoplasmic volume (P-450 content) by microphotometry and estimated the area of ER per unit cytoplasmic volume (ER area) by morphometry in periportal, midzonal, and perivenular hepatocytes of mice injected daily with PB (100 mg/kg), or with PB (100 mg/kg) plus cobalt chloride (50 mg/kg) for three days. After injection of PB, the P-450 content and ER area increased in hepatocytes of the three sublobular zones. In mice treated with PB plus cobalt chloride, however, the ER area increased, but the P-450 content decreased or remained unchanged in hepatocytes of the three zones. We conclude that cobalt chloride inhibits the increase in total P-450 but has no effect on the proliferation of ER of hepatocytes in mice treated with PB, indicating a dissociation of ER proliferation and P-450 increase after administration of PB.

Animals↗

PubMed1994

Absence of paravascular nerve projection and cross-innervation in interscapular brown adipose tissues of mice.

To determine whether a paravascular nerve projection or cross-innervation exists in the interscapular brown adipose tissue (BAT), the distribution of noradrenergic or peptidergic nerve fibers in intact or denervated interscapular BAT pads of mice was examined histochemically. Noradrenaline (NA) fibers were visualized by the glyoxylic acid condensation method, and neuropeptide Y (NPY) and substance P (SP) fibers were detected immunohistochemically. Numerous NA-positive fibers and a few NPY- or SP-positive fibers were observed around intralobular arterioles in intact BAT pads. Some NA-positive fibers and very few NPY- or SP-positive fibers were seen around interlobular arteries. NA- and NPY-positive fibers were also found around brown adipose cells in the parenchyme of BAT, whereas SP-positive fibers were absent around the cells. However, all sections cut from denervated BAT pads of unilaterally or bilaterally denervated animals showed a total absence of NA-, NPY- or SP-positive nerve fibers. Therefore, neither a paravascular projection of NA, NPY, and SP fibers to the BAT nor a cross-innervation of these nerve fibers between the left and right BAT pads exists in the mouse BAT.

Adipose Tissue, Brown↗

PubMed1994

Age-related changes in fibre number, fibre size, fibre type composition and adenosine triphosphatase activity in rat soleus muscle.

To study the aging of muscle fibres in red skeletal muscle, fibre number, fibre diameter and fibre type composition in the soleus muscle of male rats of 3, 12 and 24 months old were examined. The total number of muscle fibres remained unchanged, while average diameter increased slightly with increasing age. The staining intensity of myosin adenosine triphosphatase (ATPase) activity in the fibres decreased with advancing age. Therefore, observation on the basis of myosin ATPase histochemistry alone is not adequate to study the aging of muscle fibres. In the muscles of 24 month-old animals, four fibre types were recognized; 1) many (52%) type I-O fibres showing weak ATPase and succinate dehydrogenase (SDH) reactions with slight subsarcolemmal aggregates of diformazan (SAD); 2) some (33%) type M fibres showing weak ATPase and intense SDH reactions with marked SAD; 3) a few (12%) type O fibres showing weak ATPase and intense SDH reactions without SAD; and 4) very few (4%) type IIA fibres. Histochemical and morphometric results suggest that type I-O, type M and type O fibres are derived from type I, type I and type IIA fibres, respectively. Furthermore, no transitional fibres from type IIA to type I were observed. Therefore, age-related changes in fibre type composition in the muscle cannot be explained by the simple idea that most type IIA fibres are transformed into type I fibres.

Aging↗

PubMed1994

Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with a video image processor.

We have a quantitative light microscopic immunohistochemical method using video image processing. First, an antigen (NADPH-cytochrome P-450 reductase) content in homogenates of livers of rats was measured by enzyme immunoassay. Then frozen sections from rat livers were incubated with the anti-NADPH-cytochrome P-450 reductase antibody under saturation conditions by the indirect immunoperoxidase method. Subsequently, relative staining intensities in small portions and those in wide areas in the sections were measured with a video image processor. Finally, the resulting relative values obtained from the small portions were converted into absolute NADPH-cytochrome P-450 reductase contents using the results of enzyme immunoassay and the average relative staining intensity obtained from the wide areas in the sections. The reductase content in sections from rat livers measured by the image processing method coincided with the content measured by the microphotometric method using a nitrocellulose model system. The present image processing method is applicable to measurement of contents of antigens that can not be immobilized in model systems.

Animals↗

PubMed1993

Quantitative analysis of smooth endoplasmic reticulum proliferation in periportal, midzonal and perivenular hepatocytes of mice after administration of phenobarbital.

Smooth endoplasmic reticulum (SER) proliferation after phenobarbital (PB) administration has been described to occur predominantly in perivenular hepatocytes. We analyzed by quantitative electron microscopy changes in ER amounts in periportal, midzonal and perivenular hepatocytes from mice injected with 35, 50, 100 or 150 mg/kg of PB once a day for 3 days. The SER proliferated in midzonal hepatocytes in addition to perivenular hepatocytes even when low doses (35 or 50 mg/kg) were administered, and in hepatocytes of all three zones in case of high doses (100 or 150 mg/kg). Moreover, net proliferation of SER after administration of an amount of PB was not different in hepatocytes of any zones where the proliferation occurred, except animals injected with 100 mg/kg. The results suggest that there is no difference in SER producing capacity in hepatocytes of three zones when the cells have recognized the stimulation of PB.

Animals↗