Effects of long-term administration of methionine on vascular endothelium in rabbits.

BACKGROUND AND AIM: We studied the effects of long-term methionine administration on the vascular endothelium of Japanese white rabbits. METHODS AND RESULTS: Eleven rabbits were divided into a control group (n = 6) and a methionine-fed group (n = 5), and reared for 22 weeks. Blood samples were collected at baseline and after 22 weeks for the measurement of serum homocysteine and cysteine, serum lipids and serum superoxide dismutase activity. At the end of experiments, the animals were sacrificed, and the thoracic aorta was removed for the measurement of isometric tension and histopathological examination. The blood samples taken from the methionine group in the 22nd week showed slight but significant increases in serum homocysteine and cysteine levels (Hcy: 13.7 +/- 1.4 vs 21.0 +/- 4.9, p < 0.01; Cys: 241.6 +/- 37.8 vs 342.6 +/- 35.0, p < 0.01). In the isometric tension experiments, the methionine group had a significantly decreased (p < 0.01) vasodilatation reaction induced by acetylcholine, an endothelium-dependent vasodilator. The histopathological examination (immunostaining in response to eNOS and tissue factor) showed significant increases in endothelium expression in the methionine group before atherosclerotic changes appeared. CONCLUSIONS: The above results suggest that vascular endothelial dysfunction played an important role in the atherosclerosis occurring after excess methionine feeding.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Three-dimensional analysis of morphological aspects of the human saccular macula.

The 3-dimensional shape of the human saccular macula and its orientation in the skull were quantitated in this study. The semicircular canals and saccular maculae were reconstructed 3-dimensionally on a computer from 3 human temporal bones. The 380 to 522 triangles in the entire area of the saccular macula were made by drawing lines between 2 adjacent points every 100-pm width of the saccular macula in each section. The angles between each triangle and each estimated standard axis in the skull obtained were calculated. This information will provide standard data regarding the 3-dimensional morphological aspects of the saccular macula for further investigations of the function of the sacculus. It was determined that the 3-dimensional shape of the saccular macula was not a plane, but was a curved surface like that of an ellipsoid. It is thought that this shape is necessary in order for the saccular macula to detect wide-range linear acceleration.

Cyclosporin A causes a hypermetabolic state and hypoxia in the liver: prevention by dietary glycine.

Acute cyclosporin A (CsA) treatment inhibits mitochondrial respiration, yet effects of chronic treatment remain unclear. Accordingly, the effects of chronic CsA on oxygen metabolism in perfused rat liver and isolated mitochondria were investigated. Basal rates of oxygen uptake of around 120 micromol/g/h in isolated perfused livers from vehicle-treated controls were elevated about 1.6-fold by chronic CsA treatment. In the presence of ammonium chloride, a substrate for urea synthesis, oxygen uptake was about 150 micromol/g/h and was increased about 1.7-fold by CsA, indicating that chronic CsA treatment causes a robust hypermetabolic state in the liver. In isolated mitochondria, state 3 rates of oxygen uptake were increased about 1.6-fold by chronic CsA treatment. Since significant increases in oxygen consumption could cause hypoxia, the hypoxia marker pimonidazole was given. Pimonidazole binding in the liver was increased about 3-fold by chronic CsA. Moreover, intracellular calcium in Kupffer cells isolated from vehicle-treated rats was not altered by CsA addition; however, in cells isolated from chronic CsA-treated rats, CsA increased intracellular calcium about 15-fold and prostaglandin E(2) (PGE(2)) production 3.5-fold. Importantly, dietary glycine (5%) largely blocked chronic CsA-induced activation of Kupffer cells, blunted production of PGE(2), prevented the hypermetabolic state, and minimized tissue hypoxia. Taken together, it is concluded that chronic CsA treatment causes a hypermetabolic state leading to hypoxia and injury to the liver. It is hypothesized that CsA activates Kupffer cells and increases production of PGE(2), which alters mitochondria leading to a hypermetabolic state. Glycine inhibits activation of Kupffer cells thus preventing liver injury.

Immunohistochemical and electron microscopic study of extrinsic hepatic reinnervation following orthotopic liver transplantation in rats.

BACKGROUND/AIMS: Because little has been known about the morphological and functional consequences of liver transplantation on hepatic autonomic nerves, we examined the time-course of extrinsic hepatic innervation at the level of the porta hepatis of liver allografts. METHODS: Orthotopic liver transplantation was performed using male Lewis rats. Crosscut tissue specimens were obtained postoperatively for up to 6 months from the porta hepatis of transplanted livers, and processed for immunohistochemical staining for protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP-43), and for transmission electron microscopy (TEM). RESULTS: Extrinsic nerve fibers at the porta hepatis stained positively for PGP 9.5 throughout the entire study period. In contrast, the immunoreactivity of GAP-43 was negative at postoperative day (POD) 1 and 2. GAP-43-positive nerves were first observed to appear in the porta hepatis at POD 3. The immunoreactivity of GAP-43 remained positive thereafter until 3 months post-OLT, and became negative in all the specimens at 4 months post-OLT. Transmission electron microscopy demonstrated a small number of regenerating axons existing among many degenerating axons at POD 3. At 3 months post-OLT, most regenerating axons had been fully ensheathed by the cytoplasm of Schwann cells, although their density remained at a lower level compared with normal. CONCLUSION: The results of this study suggest that liver allografts become extrinsically reinnervated, with the regenerating axons reaching the hepatic hilus 3 days after transplantation. The process of extrinsic hepatic reinnervation is considered to almost terminate 4 months after transplantation in rats.

Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin.

BACKGROUND: Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol-induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS). METHODS: Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor (TNF)-alpha was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western analysis. RESULTS: Ethanol levels exhibited a cyclic pattern in ethanol-treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3-fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS-induced intracellular Ca2+, and TNF-alpha all were increased. These phenomena were blocked by antibiotics. CONCLUSIONS: Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.

How is the liver primed or sensitized for alcoholic liver disease?

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-alpha gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFalpha gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.

An anatomical study of the medial canthus using a three-dimensional model.

Opinions on the direction and insertion of the muscle and tendon of the medial canthus not only differ depending on the reporter, but, to date, have lacked objectivity. The direction and insertion of the muscle and tendon of the medial canthus have, therefore, not been clear to surgeons operating on the medial canthus. In order to fully grasp the anatomy of this construct three-dimensionality, we constructed a 3D model of successive sections of the medial canthus in a frontal direction using five cadavers, and then studied this model. The pretarsal part of the orbicularis oculi muscle is formed from a single muscle bundle of both the upper and lower eyelids, and runs into the medial palpebral tendon. This muscle bundle further branches off along the outside of the lacrimal sac, internally. It surrounds the back of the lacrimal sac without entering it. The preseptal part of the orbicularis oculi muscle consists of a single muscle bundle for both the upper and lower eyelids. The muscle fibers on the side of the skin run into the medial palpebral tendon. The muscle fibers posterior to this muscle bundle run into tendinous fibers, and, in all of the upper eyelids examined, they stop at the lacrimal fornix. In three out of the five lower eyelids examined the muscle fibers stop at the anterior surface of the lacrimal sac, while in the remaining cases they run into the medial palpebral tendon, as with the muscle fibers on the side of the skin. The medial palpebral tendon traverses the anterior surface of the lacrimal sac in an internal direction without branching off anteroposteriorly.

Scanning electron microscopic study on double and single eyelids in Orientals.

The conventional theory is that Occidentals have a terminal insertion of the levator aponeurosis at the anterior portion, resulting in a double eyelid, whereas in Orientals this fiber is not present, and therefore results in a single eyelid have been anatomically demonstrated. However, there have been more than a few reports indicating that the anatomical difference between a single eyelid and double eyelid in Orientals cannot be explained by this theory. Therefore, in order to verify the direction of the levator aponeurosis in the eyelids of Orientals, we observed Japanese eyelids using a scanning electron microscope (SEM). As a result of three-dimensional, cross-sectional observations using SEM, we were able to confirm the existence of a branch of the levator aponeurosis that runs through the layer of the orbicularis oculi muscle and connects with the levator aponeurosis in the double eyelid, as in the occidental eyelid. This was not seen in the single eyelid. It is thought that this new anatomical finding will become an important fundamental for double eyelid operations in Orientals.

Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat.

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-kappaB (NF-kappaB) activation, liver tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g. kg(-1). day(-1)) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 +/- 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 +/- 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-kappaB activity, and TNF-alpha expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-kappaB activation and TNF-alpha mRNA expression in the liver.

Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF-kappaB activation and cytokine production.

Kupffer cells play a significant role in the pathogenesis of several liver diseases; therefore, a potential therapeutic strategy would be to inactivate the Kupffer cell with a gene-delivery system. Although recombinant adenovirus provides robust, transgene expression in parenchymal cells, whether adenovirus transduces Kupffer cells is unclear. Thus, the purpose of this study was to evaluate this possibility. In animals infected with adenovirus, Kupffer cells were identified positively to express adenoviral transgenes by immunohistochemical techniques and Western blot analysis, indicating that Kupffer cells are transduced in vivo. Indeed, isolated Kupffer cells were transduced in vitro with recombinant adenovirus in a dose-dependent manner. Moreover, adenoviral transduction of Kupffer cells was blocked by inhibitors of alphaVbeta5 integrin, the co-receptor for adenovirus binding, supporting the hypothesis that adenovirus transduces Kupffer cells via an alphaVbeta5 integrin-dependent mechanism. Indeed, it is shown here that Kupffer cells express alphaVbeta5 integrins. In a functional assay, infection of isolated Kupffer cells with adenovirus containing superoxide dismutase or IkappaB alpha super-repressor blunted LPS-induced nuclear transcription factor kappa B (NF-kappaB) activation and tumor necrosis factor alpha (TNF-alpha) production but not IL-10 production. Moreover, superoxide production was blocked by expression of superoxide dismutase. These data support the hypothesis that LPS-induced NF-kappaB activation and TNF-alpha production in Kupffer cells are oxidant-dependent. These findings suggest that Kupffer cell-targeted approaches may be a potential therapeutic strategy against many inflammatory diseases including early alcohol-induced liver injury.

Evaluation of beta-blocker therapy in patients with dilated cardiomyopathy--Clinical meaning of iodine 123-metaiodobenzylguanidine myocardial single-photon emission computed tomography.

BACKGROUND: Patients with heart failure show signs of cardiac sympathetic dysfunction such as elevation of blood norepinephrine (NE) level, as a result of reduction in the number of sympathetic nerves, decrease in myocardial NE content, accelerated NE turnover or spillover of NE, and NE reuptake disorder at sympathetic nerve endings. In dilated cardiomyopathy (DCM), iodine 123-metaiodobenzylguanidine (MIBG) used clinically as a tracer for imaging of the sympathetic function was found to be useful in evaluation of severity and prognosis. METHODS AND RESULTS: A total of 143 (123)I-MIBG myocardial single-photon emission computed tomography (SPECT) images were taken at successive intervals on 58 patients with DCM (mean age 54 +/- 11 years), as well as before and after therapy to determine the severity of DCM and the therapeutic effect of beta-blocker. Patients were divided into group A (n = 20), in which left ventricular ejection fraction (LVEF) improved by 10% or more within 6 months after the administration of beta-blocker, and group B (n = 20), in which there was less than a 10% change in LVEF. After (123)I-MIBG myocardial SPECT was taken, the washout rate for the entire left ventricle was calculated from early and delayed images. The estimations of extent score and severity score were based on the polar map prepared from short axial images taken from 17 healthy volunteers (mean age 35 +/- 5 years). There was a significant correlation between LVEF and (123)I-MIBG findings (extent score, severity score, and washout rate) obtained before and after beta-blocker therapy. After beta-blocker therapy, LVEF and (123)I-MIBG findings significantly improved in group A. On the other hand, no change occurred in (123)I-MIBG findings in group B. There was no significant difference in LVEF between group A (32.1% +/- 8.0%) and group B (29.5% +/- 7.2%). Also, there was no significant difference in the washout rate between group A (54.4% +/- 10.9%) and group B (52.9% +/- 7.2%). Comparison of (123)I-MIBG images before beta-blocker therapy of group A and group B revealed that the extent score (26.5 +/- 16.0 vs 44.5 +/- 21.1, respectively; P <.01) and the severity score (24.9 +/- 21.9 vs 58.2 +/- 35.2, respectively; P <.01) on the basis of the early (123)I-MIBG image was significantly lower for group A. CONCLUSIONS: From the above findings, patients with DCM in which (123)I-MIBG uptake is high on early images were expected to show improvement in cardiac function by beta-blocker therapy. Findings also suggested that (123)I-MIBG was useful for examining the severity of DCM, determining the applicability of beta-blocker therapy, estimating the maintenance dosage of beta-blocker, and evaluating prognosis.

A potential role of bradykinin in angiogenesis and growth of S-180 mouse tumors.

Angiogenesis is an important event in tumor growth. We evaluated the contribution of endogenous bradykinin to tumor-associated angiogenesis and tumor growth using pharmacological approaches in mice bearing sarcoma 180 cells. The weight of implanted tumors increased in parallel with increased hemoglobin contents (a parameter to evaluate angiogenesis) over a 20-day experimental period. Daily administration of bradykinin B2-receptor antagonists, Hoe140 (0.1 and 1 mg/kg per day, local injection) or FR173657 (30 mg/kg per day, p.o.), significantly suppressed the increment in angiogenesis and tumor weight, but a B1-receptor antagonist, desArg10-Hoe140 (1 mg/kgperday), did not. Administration of a plasma kallikrein inhibitor, soybean trypsin inhibitor (3 mg/site per day), significantly suppressed angiogenesis and tumor growth. In contrast, bradykinin-degrading enzyme inhibitors, captopril and phosphoramidon (500 microg/site per day), enhanced angiogenesis and increased tumor weight. Our results suggest that bradykinin, produced by plasma kallikrein or plasma kallikrein-like enzymes, promote tumor-associated angiogenesis and tumor growth in vivo.

Adenylate cyclase/protein kinase A signaling pathway enhances angiogenesis through induction of vascular endothelial growth factor in vivo.

We previously reported that endogenous prostaglandins (PGs) may increase cAMP facilitated angiogenesis through the induction of vascular endothelial growth factor (VEGF) in rat sponge implantation models. In the present experiment, we tested whether or not adenylate cyclase / protein kinase A (AC/PKA)-dependent VEGF induction enhanced angiogenesis in this model. Topical daily injections of 8-bromo-cAMP enhanced angiogenesis in a dose-dependent manner. Forskolin, an activator of AC, also facilitated angiogenesis as did amrinone, an inhibitor of phosphodiesterase. VEGF induction was confirmed by the increased levels in the fluids in the sponge matrix after topical injection of 8-bromo-cAMP. Immunohistochemical investigation further revealed the VEGF-expressed cells in the sponge granulation tissues to be fibroblasts, and the intensity of positive reactions was enhanced by 8-bromo-cAMP, forskolin and amrinone. Angiogenesis without topical injections of the above compounds was suppressed by SQ22,536, an inhibitor for AC, or H-89, an inhibitor for PKA, with concomitant reductions in VEGF levels. Daily topical injections of neutralizing antibody or anti-sense oligonucleotide against VEGF significantly suppressed angiogenesis. PGE2-induced angiogenesis was suppressed with SQ22,536 or H-89. These results suggested that AC/PKA-dependent induction of VEGF certainly enhanced angiogenesis and that pharmacological tools for controlling this signaling pathway may be able to facilitate the management of conditions involving angiogenesis.

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo.

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.

Endothelial cells contain a glycine-gated chloride channel.

Glycine inhibited growth of B16 melanoma tumors in vivo most likely because of the inhibition of angiogenesis. Here, the hypothesis that the anticancer effect of glycine in vivo is due to expression of a glycine-gated Cl- channel in endothelial cells was tested. First, the effects of glycine on vascular endothelial growth factor-induced increases in intracellular Ca2+ concentration in a bovine endothelial (CPA) cell line were studied. Vascular endothelial growth factor (1 ng/ml) increased intracellular Ca2+ concentration, with peak values reaching 141 +/- 11 nM. Glycine blunted this increase dose dependently. Furthermore, the inhibitory effects of glycine were prevented by 1 microM strychnine, a glycine receptor antagonist, or when cells were incubated in Cl(-)-free buffer. Moreover, glycine increased influx of 36Cl into CPA cells approximately 10-fold; this reaction was also strychnine sensitive. Furthermore, mRNA similar to the beta-subunit of the glycine-gated Cl- channel from spinal cord was identified in endothelial cells by reverse transcription-polymerase chain reaction. In addition, Western analysis using antibody for the glycine receptor demonstrated expression of the beta-subunit of the glycine receptor. Importantly, glycine diminished serum-stimulated proliferation and migration of endothelial cells. Collectively, these data indicate that the inhibitory effect of glycine on growth and migration of endothelial cells is due to activation of a glycine-gated Cl- channel. This hyperpolarizes the cell membrane and blocks influx of Ca2+, thereby minimizing growth factor-mediated signaling.

Structural analysis of red blood cell membrane with an atomic force microscope.

To evaluate the usefulness of the atomic force microscope (AFM) for structural analysis of biomedical samples and to determine suitable sample preparation methods for AFM observation, the membrane of human erythrocytes prepared by various methods for electron microscopy was examined by the AFM. Strand-like elevations with 20-50 nm in width, 30-80 nm in length and 3-5 nm in height were observed, which formed networks composed of squares, pentagons and hexagons on the cytoplasmic or back surface of the erythrocyte membrane. Using colloidal gold labelled antibody, this network was found to contain spectrin molecules. Therefore it was very likely that the undercoat molecules of the plasma membrane were imaged by AFM. A large number of gentle elevations 300-400 nm in diameter and 2 nm in height were found to be distributed uniformly on the extracellular or true surface of intact erythrocyte, presumably reflecting the presence of undercoat membrane skeleton on the cytoplasmic surface. However, no structure that seemed to be derived from glycocalyces was discernible on the true surface. Structure corresponding to the unit membrane or lipid bilayer structure observable by electron microscopy was not demonstrated in the cross-section of the membrane. In freeze-fractured samples, a large number of small particles that corresponded to the intramembranous particle were also demonstrated on the membrane halves. Since AFM allows depiction of the fine structures of biological samples with very simple sample processing at a resolution comparable to or exceeding that of SEM, imaging technology using AFM can be applied to obtain biomedical information. However, several problems have to be solved in future development of the equipment.