Gill tissue lipids of salmon (Salmo salar L.) presmolts and smolts from anadromous and landlocked populations.

Composition of membrane lipids from the gills of juvenile Atlantic salmon (Salmo salar) in presmolt and smolt phases of development was compared among anadromous and non-anadromous populations. Three stocks migrating from spawning rivers to either lake (landlocked stock), brackish water or full strength sea water were grown under common garden conditions, and gill lipids and their acyl and alkenyl chains were examined in February (presmolts) and at the end of May (smolts) by mass spectrometry and gas-liquid chromatography. The most remarkable changes upon transition from the presmolt phase to the smolt phase were: (i) increase in the cholesterol/phospholipid ratio, (ii) decrease in the abundance of phosphatidylinositol (PI) content, (iii) increase in the amount of sulfatides, (iv) increase in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species with two highly unsaturated acyl chains, and finally (v) convergence of interstock differences in PC and PE species composition towards a similar lipid composition. Increases in the gill membrane content of cholesterol and sulfatides are discussed as pre-adaptation of salmon gills for salt-secretion, which may occur by increases in membrane microdomains (rafts) harboring ion channels and pumps. The decreases of PI were likely related to adjusting the gill membrane permeability to ions by diminishing prostanoid production. The similarity of those changes among three salmon stocks and the convergence of initially (presmolt phase) different PC and PE species profiles between the stocks towards similar lipid composition suggests that smoltification process of the gill epithelium is largely similar in anadromous and landlocked populations.

Methotrexate maintains bone mass by preventing both a decrease in bone formation and an increase in bone resorption in adjuvant-induced arthritic rats.

We examined the effects of low doses methotrexate (MTX) and indomethacin (IND) on bone mass and turnover in normal male Sprague-Dawley rats and those with adjuvant-induced arthritis. Normal and the adjuvant (heat-killed mycobacterium)-injected rats, 6 weeks of age, were given MTX at daily doses of 0.05, 0.1, or 0.2 mg/kg body weight (BW) or IND at a daily dose of 1.0 mg/kg BW. Rats were killed at the start, or at 14 and 28 days. In normal rats, the administration of these agents did not change the lumbar and femoral BMD values, nor did the serum osteocalcin or urinary deoxypyridinoline (D-Pyr) levels. Lumbar trabecular osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS) were decreased in the rats given IND. In the arthritic rats, the administration of MTX did not prevent an early increase of paw edema in the adjuvant-injected limb, but late inflammatory edema was alleviated in the non-injected limb. However, MTX administration at a dose of 0.1-0.2 mg/kg BW maintained an age-dependent increase in the lumbar and femoral BMD values. While serum osteocalcin levels were decreased and urinary D-Pyr values were increased in the arthritic control rats, these bone markers remained at the levels of the normal rats. Decreases in mineral apposition rate (MAR) and bone formation rate (BFR/BS) and increases in the trabecular Oc.N/BS and Oc.S/BS values were prevented by MTX. While IND almost completely prevented inflammatory paw edema, it did not improve the parameters of bone formation. An increase in osteoclasts was prevented and the osteopenia in the lumbar and the femoral bone was only partially prevented by IND. These data suggest that MTX improves bone mass and turnover in the arthritic rat, in which several cytokines that affect bone cells are involved. An increase in bone resorption may be due to prostaglandins, but bone formation defect was suggested to be due to other cytokines such as IL-1, IL-6, and TNF-alpha in this model.

Effects of ZNC-2381, a new oral compound, on several hepatic injury models and on hepatocellular apoptosis in mice and rats.

The hepatoprotective effect of ZNC-2381 (1-(4-aminophenyl) methyl-3-(3-nitrophenyl)-1,3-dihydroimidazo[4,5-b]pyridine-2-one), a novel 2-one dihydroimidazopyridine derivative, has been evaluated in several experimental models of hepatic injury. In mice, oral ZNC-2381, administered at doses of 3, 10 or 30 mgkg(-1), 1 h before induction of hepatic injury with concanavalin A, dose-dependently inhibited increases in serum alanine aminotransferase (ALT) activity. Apoptosis of liver cells, as indicated by DNA fragmentation (nucleosome assay) and DNA-ladder formation (electrophoresis), was also inhibited dose-dependently. ZNC-2381 dose-dependently inhibited concanavalin A-induced increases in serum tumour necrosis factor (TNF)-alpha levels, and TNF-alpha mRNA expression in the liver. Oral ZNC-2381 also dose-dependently inhibited increases in serum ALT activity in mice with hepatic injury induced by Propionibacterium acnes and a bacterial lipopolysaccharide (LPS) or D-galactosamine-LPS, and in rats with D-galactosamine-induced hepatic injury. These results indicate that oral ZNC-2381 inhibits cytokine (TNF-alpha) production and cytokine-related hepatocellular apoptosis, and might thus prevent different types of hepatic injury.

Effects of chondroitin sulfate-C on articular cartilage destruction in murine collagen-induced arthritis.

The effects of chondroitin sulfate-C (CAS 25322-46-7, Chondroitin ZS Tab) on type II collagen (CII)-induced arthritis (CIA) in mice were evaluated. DBA/1J mice were immunized with bovine CII emulsified in Freund's complete adjuvant, followed by a booster injection 21 days later. Chondroitin sulfate-C at doses of 100, 300 and 1000 mg/kg was administered orally once daily beginning 14 days before initial immunization. An arthritis index and hind paw edema were examined from day 0 to day 49, when the mice were killed by ether anesthesia for histopathological examination. The delayed-type hypersensitivity (DTH) reaction, serum anti-CII antibody titer, and histopathologic characteristics of both synovitis and destruction of articular cartilage were analyzed. Both the arthritis index and the serum anti-CII antibody titer were reduced by treatment with chondroitin sulfate-C in a dose-dependent manner. Chondroitin sulfate-C (1000 mg/kg) significantly inhibited hind paw edema, synovitis and destruction of the articular cartilage, but not DTH reaction.

D-galactosamine-induced mouse hepatic apoptosis: possible involvement with tumor necrosis factor, but not with caspase-3 activity.

We investigated whether tumor necrosis factor (TNF) and caspase-3 activity are involved in the induction of hepatocellular apoptosis in D-galactosamine (D-GalN)-induced hepatotoxicity in mice. Acute hepatotoxicity was induced by the intraperitoneal injection of D-GalN into female BALB/c mice. D-GalN (0.75-3.0 g/kg) increased the serum glutamate pyruvate transaminase (s-GPT) activity and the percentage of liver DNA fragmentation, an indicator of hepatotoxicity, after 48 h, in a dose-dependent manner. Furthermore, after D-GalN (3.0 g/kg) administration, increased liver DNA fragmentation was detected biochemically at 24 h, then increased s-GPT activity accompanied by increased liver DNA fragmentation was observed after 48 h. The serum TNF (s-TNF) level and the TNF mRNA expression in the liver after D-GalN (3.0 g/kg, i.p.) administration were examined by an ELISA kit and reverse transcription polymerase chain reaction (RT-PCR), respectively, to investigate the relation between the s-GPT activity and liver DNA fragmentation. The s-TNF level and TNF mRNA expression in the liver after D-GalN (3.0 g/kg) administration were detected earlier than liver DNA fragmentation, then increased with time. However, there was almost no association of caspase-3 activity with the increase in liver DNA fragmentation. Increases in the s-TNF level, TNF mRNA expression and the percentage of DNA fragmentation in the liver and s-GPT activity were inhibited by dexamethasone (Dex; 0.4-2.5 mg/kg, i.p.) in a dose-dependent manner. Based on these findings, it was considered that the intracellular apoptosis signal in D-GalN-induced hepatotoxicity in mice did not depend on caspase-3 activity, and that other signals mediated by TNF may be involved.

Effect of a new non-steroidal anti-inflammatory combination of a histamine H2 antagonist and indomethacin on gastroduodenal mucosal membrane in rat.

The new non-steroidal anti-inflammatory drug (NSAID), N-(3-[3-(piperidinyl-methyl) phenoxy] propyl)-carbamoyl-methylthio]ethyl 1-(p-chlorobenzoyl) 5-methoxy-2-methyl-3-indolyl-acetate (CP 331, CAS 127966-70-5), a compound with a structure of an ester combining indomethacin (IM) and a histamine H2 antagonist, has been reported to have anti-inflammatory, analgesic and antipyretic effects. However, the influence of CP-331 on the gastroduodenal mucosa was not fully investigated. Therefore this study was undertaken to investigate the effect of CP-331 on the gastroduodenal mucosa membrane in rats. After single oral drug administration, the UD50 value (50% ulcerogenic dose) of CP-331 calculated from the incidence rate of gastric ulcer was higher than 1000 mg/kg; that for IM was 5.2 mg/kg. Moreover it was examined whether CP-331 had a preventive effect on NSAID-induced gastric damage. The results showed that the co-administration of CP-331 10-30 mg/kg prevented significantly the acute gastric mucosal injury caused by IM administration (20 mg/kg). CP-331 with anti-inflammatory activity does not cause gastric injury, moreover, because of its preventing and therapeutic effects on the damage to gastric mucous membrane induced by IM, CP-331 might be useful in the treatment of gastropathy caused by NSAID in clinic.

beta-Alanyl-L-histidinato zinc prevents hydrocortisone-induced disorder of bone metabolism in rats.

The preventive effect of beta-alanyl-L-histidinato zinc (AHZ) on osteopenia was investigated in rats treated with hydrocortisone. Rats received hydrocortisone (75 mg/kg body weight per day) s.c. for 30 days. The steroid treatment caused a significant increase in serum alkaline phosphatase activity and parathyroid hormone (PTH-c) level, while serum calcium, inorganic phosphorus, and zinc concentrations were not significantly altered. The femoral-diaphyseal alkaline phosphatase activity, deoxyribonucleic acid (DNA), and calcium contents were significantly decreased by the treatment of steroid, although the bone zinc content was not appreciably altered. When AHZ (10, 30, and 100 mg/kg per day) was administered p.o. for 30 days to rats giving the steroid, the dose of AHZ (30 and 100 mg/kg) completely prevented the increases in serum alkaline phosphatase activity and PTH-c level and the decreases in femoral-diaphyseal alkaline phosphatase activity, DNA, and calcium contents caused by the steroid treatment. The dose of AHZ (10, 30, and 100 mg/kg) significantly increased zinc content in the femoral diaphysis. Present results indicate that the dose of AHZ can prevent the disorder of bone metabolism caused by hydrocortisone treatment. AHZ may have a therapeutic role in the steroid-induced osteopenia.

Autoradiographic localization of human calcitonin sensitive binding sites in rat brain.

Using 125I-salmon calcitonin (sCT) as a ligand, in vitro autoradiography of rat brain outlined specific anatomical localization of human calcitonin (hCT) sensitive binding sites. The results presented herein show that there are hCT sensitive binding sites in the ventral part of the lateral septum among the sCT specific binding sites distributed throughout the diencephalon.

Methotrexate suppresses nitric oxide production ex vivo in macrophages from rats with adjuvant-induced arthritis.

We examined the effects of methotrexate (MTX) on the level of nitric oxide (NO) produced by peritoneal macrophages from rats with adjuvant-induced arthritis (AA) ex vivo. During the development of AA, paw swelling increased and LPS enhanced the capacity of peritoneal macrophages to produce NO and prostaglandin E2 (PGE2). MTX (0.1 mg/kg, p.o.) treatment for 21 days reduced the paw swelling, and inhibited the increased NO and PGE2 production. However, when MTX (0.1 mg/kg, p.o.) was administered to rats with established AA, these parameters were not significantly influenced. In normal rats, MTX (0.1 mg/kg, p.o.) treatment for 21 days did not change NO and PGE2 production of LPS-stimulated macrophages. On the other hand, macrophages from normal and AA rats cultured in the presence of MTX (1, 10 and 100 microM), were activated by LPS in vitro. MTX did not influence NO or PGE2 production by LPS-stimulated macrophages in normal and AA rats. By contrast, indomethacin (IM) (1.0 mg/kg, p.o.) treatment for 21 days reduced the paw swelling, and inhibited NO and PGE2 production in AA rats. IM inhibited significantly PGE2 production, but did not influence NO production by LPS-stimulated macrophages in vitro. These results suggest that MTX treatment reduces NO production in peritoneal macrophages in AA rats, and these actions of MTX may have an inhibitory effect without the modulation of PGE2.

Possible changes in expression of chemotaxin LECT2 mRNA in mouse liver after concanavalin A-induced hepatic injury.

The functions of leukocyte-derived chemotaxin 2 (LECT2), a novel liver-specific protein, are not well defined, especially after hepatic injury. The changes in expression of LECT2 mRNA were investigated after concanavalin A (Con A)-induced hepatic injury in mice. Serum glutamate pyruvate transaminase (s-GPT) activity and the percentage of liver DNA fragmentation, an indicator of hepatic apoptosis, increased 8 h after intravenous administration of Con A (13 mg/kg). Expression of LECT2 mRNA was reduced from 8-24 h after injection of Con A, but was detected again 48 h after recovery from hepatic injury. Expression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma mRNA was observed in liver 2 h after Con A injection. Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK), a caspase inhibitor, was administered to mice to investigate whether LECT2 was involved in apoptosis of liver cells after Con A injection. Z-VAD-FMK inhibited s-GPT activity and DNA fragmentation in the liver 8 h after Con A-induced hepatic injury, but did not prevent the reduction of LECT2 mRNA, or induction of TNF-alpha and IFN-gamma mRNA expression. When the relation between expression of LECT2, TNF-alpha and IFN-gamma mRNAs was examined 8 h after Con A injection in wild-type or immunodeficient (nu-/nu-) mice, the increase in TNF-alpha and IFN-gamma mRNA expression was found to be closely related to a reduction in LECT2 mRNA expression. These results suggest that the reduction in expression of LECT2 mRNA is not directly involved in apoptosis and may be inversely related to the expression of TNF-alpha and/or IFN-gamma mRNA in the injured liver.

Effect of beta-alanyl-L-histidinato zinc on bone metabolism in rats with adjuvant arthritis.

The effect of a new zinc compound, beta-alanyl-L-histidinato zinc (AHZ), on osteopenia was investigated in rats with adjuvant arthritis. Arthritis was induced in female rats by administering 1% Mycobacterium butyricum (MB) into the subplantar surface of the right hind paw. AHZ (10, 30 and 100 mg/kg body weight) was orally administered to MB-treated rats 28 times at 24-h intervals, and the rats were bled 24 h after the last administration. Treatment with MB caused a remarkable increase in paw volume and a corresponding decrease in the ratio of albumin per globulin in serum, indicating that the treatment induces inflammation. These alterations were not significantly changed by the administration of AHZ (10, 30 and 100 mg/kg). Serum calcium and zinc concentrations are significantly decreased in rats with adjuvant arthritis. These decreases were completely restored by the administration of AHZ (30 and 100 mg/kg). Furthermore, the inflammation-induced decreases in alkaline phosphatase activity and calcium content in the femoral diaphysis were clearly blocked by the administration of AHZ (30 and 100 mg/kg). Also, the larger doses of AHZ (30 and 100 mg/kg) produced a significant increase in femoral-diaphyseal deoxyribonucleic acid and in the zinc content in rats with adjuvant arthritis. These results suggest that AHZ has a stimulating effect on bone formation in the femoral diaphysis of rats with adjuvant arthritis, although the compound did not have an anti-arthritic effect.

Effects of chondroitin sulfate-C on bradykinin-induced proteoglycan depletion in rats.

Depletion of the proteoglycan content of articular cartilage was induced by injecting bradykinin (30-300 mumol/l, 50 microliters/knee) into the left knee articular cavities of rats 3 times a day for 2 days. The degree of the reduction in the intensity of histopathological safranin O staining was used as an index of proteoglycan depletion. Bradykinin reduced the cartilage proteoglycan contents of the knee joints of non-injected limbs in a dose-dependent manner and at 300 mumol/l markedly reduced these contents, but evoked no inflammatory changes. The extent of the reduction of the cartilage proteoglycan contents induced by bradykinin injection depended on the dose and injection frequency. Chondroitin sulfate-C (CAS 25322-46-7, Chondroitin ZS Tab) (30-1,000 mg/kg/day) administered orally to rats for 14 days inhibited the bradykinin-induced proteoglycan depletion of the articular cartilage in a dose-dependent manner. These results suggest that a reduction of the proteoglycan content of cartilage, like that associated with osteoarthritis, was induced by injecting bradykinin into the knee articular cavities of rats and chondroitin sulfate-C protected against this effect.

Anti-inflammatory, analgesic, and antipyretic effects and gastrointestinal toxicity of the new anti-inflammatory drug N-(3-[3-(piperidinylmethyl)phenoxy]propyl)-carbamoylmethylthio ]ethyl 1-(p-chlorobenzoyl) 5-methoxy-2-methyl-3-indolylacetate.

The anti-inflammatory, analgesic, and antipyretic effects and gastrointestinal toxicity of N-(3-[3-(piperidinylmethyl) phenoxy] propyl)- carbamoylmethylthio] ethyl 1-(p-chlorobenzoyl) 5-methoxy-2-methyl-3-indolylacetate (CP-331, CAS 127966-70-5), a new anti-inflammatory drug, were evaluated using indomethacin as a control. CP-331 exerted anti-inflammatory, analgesic and antipyretic effects on the models of carrageenin-induced paw edema, increased vascular permeability, ultraviolet light-induced erythema, granuloma proliferation, adjuvant arthritis, inflammatory pain, and yeast-induced fever. However, these effects were observed at a molar level similar to or higher than that of indomethacin. In addition, CP-331 influenced more markedly than indomethacin the delayed type hypersensitivity to sheep red blood cells. On the other hand, CP-331 did not damage the gastric mucosa even at a high dose of 1,000 mg/kg and also induced slighter damage to the intestinal mucosa than indomethacin. Thus, CP-331 exerted anti-inflammatory, analgesic, and antipyretic effects but without showing gastric toxicity, which is a common side effect of anti-inflammatory drugs. These results suggest the clinical applicability of this drug in the long-term therapy of inflammatory diseases such as rheumatoid arthritis.

[Evaluation of the vasodilating effect of naftidrofuryl oxalate using isolated canine and rat artery preparations].

Using isolated ring preparations of major arteries mainly of canine origin, we attempted to explore the mechanism of the vasodilating effect of naftidrofuryl oxalate (I) at the concentration of approximately 10 microM. 1) The resting tension of canine carotid, femoral, coronary, renal and basilar arteries were not affected by I. 2) A weak or no papaverine-like activity was noted on coronary, renal and basilar arteries contracted by KCl (25 mM) or U46619 (20 nM). Porcine endothelin (30 nM)-induced contraction in the basilar artery also showed no response to I. 3) I produced a relatively strong anti-serotonergic effect in the basilar and femoral arteries, and the minimum effective concentrations of I for pretreating these arteries were 0.3 and 0.1 microM, respectively. I failed, however, to affect 8-OH-DPAT-induced contraction of the basilar artery. 4) In a low concentration such as 1 nM, I was able to release the vasodilating factor from the carotid artery. 5) The oscillatory contractions which developed in the rat thoracic aorta with phenylephrine (10 microM) were not affected by I (approximately 0.1 microM). 6) Na oxalate (approximately 1 mM) produced none of the effects of I described in 2) approximately 4). Based on the results obtained, it is concluded that I would exert its vasodilating effect not only directly via an anti-serotonergic action but also indirectly via its secretagogue-like action.

Age-depending effects of methotrexate treatment on systemic bone turnover in experimental adjuvant arthritis.

Adjuvant arthritis was induced in rats in the growth stage (aged 6 weeks) and those in the mature stage (aged 4 months), and changes in the systemic bone turnover and the effects of methotrexate (MTX, CAS 133073-73-1) were compared. After induction of adjuvant arthritis, the paw edema ratio and the urinary deoxypyridinoline (u-Dpy) level increased in both age groups. No marked changes were observed in the serum osteocalcin (s-OC) level in either group. In the 6-week-old rats, arthritis completely inhibited the bone mass, and strength of the femur and lumbar vertebral body. The 4-month-old rats showed more marked changes than the 6-week-old rats in the bone mass and strength of the lumbar, vertebral body. MTX administration (0.05, 0.1 and 0.2 mg/kg/day) resulted in significant dose-dependent inhibition of arthritis-induced changes, and the effects of MTX were similar between the two age groups. MTX was useful at each age. These results suggest that 4-month-old rats with arthritis are more appropriate as a model for evaluation of drugs for bone metabolic turnover in human chronic rheumatoid arthritis.

[Influences of hypercholesterolemia on the vessel function of isolated rat thoracic aorta].

Mature male rats (SD strain, 8-week-old) were fed with a normal diet or a high cholesterol diet (HC: 1.5% cholesterol and 0.5% Na cholate in the normal diet) up to 8 weeks, and we examined how the vascular function level of the isolated thoracic aorta and the histological figures of some tissues including the aorta would change. 1) The contracting reactivity to phenylephrine (Phe, 10 microM) and the relaxing reactivity to acetylcholine (1 microM) measured thereafter remained unchanged during the period of aging and were not influenced by HC-feeding. The addition of L-arginine (Arg, 100 microM) did not affect the results. 2) The ability of the aorta to release NO and to relax, which was evaluated as the extent of the endothelium-dependent potentiation by NG-monomethyl-L-arginine (NMA) of the Phe contraction, did not change by HC-feeding up to 4 weeks, but appears to be attenuated after 8-week feeding. 3) The EC50 of NMA for the potentiation estimated without the addition of Arg remained unchanged, while the one in the presence of Arg gradually increased with aging but not with HC-feeding. 4) The histopathological study of the aorta and other tissues failed to detect any notable atherogenic changes in any of the HC-fed groups. The results indicate that under the experimental conditions employed, HC-feeding would not develop any significant atherogenic histopathological changes in the endothelium-smooth muscle preparation, but may induce some dysfunction in the NO-release mediated and auto-regulatory function of the vascular tone.

Effects of a novel hepatoprotective drug, ZNC-2381, on fas-induced hepatocellular caspase-3 activity and apoptosis in mice.

We examined the effects of ZNC-2381 (1-(4-aminophenyl)methyl-3-(3-nitrophenyl)-1,3-dihydroimidazo[4,5-b] pyridine-2-one), a new oral hepatoprotective agent, on hepatocellular caspase-3 activity and apoptosis induced by anti-mouse Fas antibody (anti-Fas ab) in mice. Oral ZNC-2381, administered at doses of 10, 30 and 100 mg/kg 1 h before inducing hepatic injury with anti-Fas ab, dose-dependently inhibited the increase in serum alanine aminotransferase (s-ALT) activity 8 h after injection of anti-Fas ab. Increases in DNA fragmentation (nucleosome assay) and caspase-3 activity in the liver 2 h after injection of anti-Fas ab were also inhibited by ZNC-2381 in a dose-dependent manner. As shown by histopathological examination, ZNC-2381 dose-dependently inhibited the appearance of TUNEL-positive apoptotic cells in the liver. Moreover, in studies in vitro, ZNC-2381 (1- 100 micromol/l) concentration-dependently inhibited increases in DNA fragmentation and caspase-3 activity caused by anti-Fas ab in isolated mouse hepatocytes. N- Acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-cho), a caspase-3-specific inhibitor, inhibited hepatocellular apoptosis caused by anti-Fas ab both in vivo and in vitro, as well as the increase in s-ALT activity in vivo. These results demonstrate that orally administered ZNC-2381 inhibits hepatocellular apoptosis induced by anti-Fas ab and presents the progression of hepatic injury. We propose that the mechanism of action of ZNC-2381 may involve blockade of the signal transduction pathway (caspase-3) of apoptosis mediated by anti-Fas ab.

Atomic force microscopy with carbon nanotube probe resolves the subunit organization of protein complexes.

Among many scanning probe microscopies, atomic force microscopy (AFM) is a useful technique to analyse the structure of biological materials because of its applicability to non-conductors in physiological conditions with high resolution. However, the resolution has been limited to an inherent property of the technique; tip effect associated with a large radius of the scanning probe. To overcome this problem, we developed a carbon nanotube probe by attaching a carbon nanotube to a conventional scanning probe under a well-controlled process. Because of the constant and small radius of the tip (2.5-10 nm) and the high aspect ratio (1:100) of the carbon nanotube, the lateral resolution has been much improved judging from the apparent widths of DNA and nucleosomes. The carbon nanotube probes also possessed a higher durability than the conventional probes. We further evaluated the quality of carbon nanotube probes by three parameters to find out the best condition for AFM imaging: the angle to the tip axis; the length; and the tight fixation to the conventional tip. These carbon nanotube probes, with high vertical resolution, enabled us to clearly visualize the subunit organization of multi-subunit proteins and to propose structural models for proliferating cell nuclear antigen and replication factor C. This success in the application of carbon nanotube probes provides the current AFM technology with an additional power for the analyses of the detailed structure of biological materials and the relationship between the structure and function of proteins.