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Biomedical subjects

M Yamato

Publications and source records attributed to M Yamato.

At least 19 recordsLinked to original sources
PubMed2001

Alteration of collagen IV in acutely deteriorated renal allografts.

BACKGROUND: The changes in the basement membrane occurring in acutely deteriorated renal allografts (ADR) have not been extensively investigated. Our purpose is to elucidate the alteration of collagen IV, a main constituent of the basement membrane in ADR. METHODS: Fifty biopsy specimens of ADR and 10 of chronic transplant nephropathy (CTN) were examined with two monoclonal antibodies specific for collagen IV. JK199 and JK132 are monoclonal antibodies that recognize triple helical collagen IV containing the alpha1 chain. JK199 recognizes all the basement membrane containing [alpha1 (IV)]2alpha2(IV), although JK132 reacts only with a limited portion of it. In the normal kidney, JK199 reacts with the mesangial matrix, the basement membrane of Bowman's capsule (BBM), and the tubular basement membrane, as well as with the glomelular basement membrane (GBM). JK132 reacts with the mesangial matrix, BBM, and the tubular basement membrane. RESULTS: In ADR, increased intensity of JK199 was observed in GBM, the mesangial matrix, BBM, the tubular basement membrane, and the interstitium. Increased intensity of JK132 was observed in the mesangial matrix, BBM, and the tubular basement membrane, but was not remarkable in GBM or the interstitium. In contrast, biopsy specimens of CTN showed increased intensity of JK132 in GBM, the mesangial matrix, BBM, the tubular basement membrane and the interstitium. CONCLUSION: These results suggest that collagen IV is up-regulated in ADR. Differential staining of collagen IV with JK199 and JK132 in GBM and the interstitium may contribute to diagnose CTN.

Adult↗
PubMed2001

Mut-Test to detect substances suppressing spontaneous mutation due to oxidative damage.

Since it has been considered that suppression of spontaneous mutation in cells is related to suppression of spontaneous carcinogenesis, it is significant to detect substances which suppress spontaneous mutation in bacterial cells such as Escherichia coli and Salmonella typhimurium in the environment. However, since the frequency of spontaneous mutation in bacteria is usually very low, generally 10(-8)-10(-10),it is difficult to determine significant suppressive ability of such substances on spontaneous mutation. A new method, Mut-Test, was developed by us, applying Luria & Delbruck fluctuation test, to detect substances which suppress spontaneous mutation using E. coli mutT mutant in which spontaneous mutation frequency due to oxidative damage is enhanced to approximately 500-1000 times of the wild type strain. Suppressive abilities of two hydroxyl radical scavengers: D(-)-mannitol and thiourea, were examined and clear positive results were obtained, suggesting that the radical scavengers are suitable as the positive control for the test. Using Mut-Test, suppressive abilities of four vitamins: L-ascorbic acid, beta-carotene, folic acid and riboflavin; 10 polyphenols: caffeic acid, ellagic acid, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, gallic acid, pyrocatechol, pyrogallol, quercetin and tannic acid which are recognized as antimutagens, were examined. Furthermore, the concentrations for 50% of suppressive abilities of five positive samples, L-ascorbic acid, folic acid, caffeic acid, pyrocatechol and pyrogallol were compared. Negative results were obtained in nine samples, riboflavin, tannic acid, etc. suggesting that their antimutagenic effect on cells may not be related to oxidative damage in cells.

Antimutagenic Agents↗
PubMed2001

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces.

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.

Animals↗
PubMed2001

Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces.

Here we demonstrate a novel cell coculture method without any apparent limitation in cell-type combinations that exploits thermally responsive polymer-grafted patterns to alter cell-cell and cell-surface interactions. Thermally responsive acrylamide polymer is first covalently patterned onto culture surfaces by masked electron beam irradiation. One cell type is then cultured to confluency at 37 degrees C. Reducing cell culture temperature below 32 degrees C selectively swells temperature sensitive polymer-grafted domains, detaching adherent cells only from these grafted patterns. Another cell type is then seeded over the same surface at 37 degrees C. These subsequently seeded cells adhere only to the now-exposed polymer-grafted domains. Initially seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then cocultured at 37 degrees C in well-ordered patterns.

Acrylic Resins↗
PubMed2001

Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method.

Because spontaneous host regeneration of damaged tissues is limited, novel therapeutics utilizing cultured cells with the aid of tissue engineering methods are promising alternatives for tissue replacement. One critical shortcoming is current requirement for invasive cell harvest from culture to fabricate cell-based devices. Although microglia that secrete neurotrophic factors are attractive candidates for novel cell transplantation therapy for damaged central nervous system tissue, the intact harvest of cultured microglia is presently not achievable. Therefore, primary microglia were plated onto culture surfaces grafted with the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). This surface undergoes rapid, reversible temperature-dependent changes in its hydration state and surface hydrophilicity. Microglia attached and proliferated on PIPAAm-grafted dishes at 37 degrees C. By reducing culture temperature, more than 90% of the cells spontaneously detached from the dishes within several minutes without trypsin or EDTA treatment. Recovered and replated microglia exhibited phenotypic properties comparable to those of primary microglia freshly isolated from brain. By contrast, less than 60% of the cells were harvested by trypsin digestion, and exhibited significant alteration of characteristic cellular properties as monitored by pathological states in vivo. This new technology exhibits utility for the preparation of cell sources required for cell transplantation as well as microglial function analysis.

Animals↗
PubMed2001

Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude.

Although cardiac myocytes adherent to tissue culture polystyrene (TCPS) dishes retain the spontaneous beating, the pulsatile amplitude is highly limited compared to that in vivo. One of the main reasons for the limited pulsation may be the interface between the cells and the TCPS surfaces. Release of these cells from rigid TCPS surfaces may augment their pulsatile amplitude. With this perspective, we have developed a novel cell manipulation technique to detach cultured cardiac myocytes from rigid surfaces and to rescue higher pulsatile amplitude of the cells using temperature-responsive culture dishes and discuss the possibility of improving this heart tissue model. Primary cardiac myocytes were cultured on the slightly hydrophobic dish surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide). Cells adhered and proliferated, forming confluent cardiac myocyte sheets in a fashion similar to those on ungrafted TCPS dishes. Decrease in culture temperature resulted in surface change of the polymer from slight hydrophobic to highly hydrophilic due to extensive hydration of the grafted polymer on the dishes. This results in release of cardiac myocyte sheets from the dishes without enzymatic or EDTA treatment. When no support was used, the detached cardiac myocyte sheets shrank to one-tenth size, which ceased their pulsation. When chitin membranes were used to support the confluent sheets to prevent cell shrinkage, the detached cell sheets could be transferred and readily adhered onto another virgin TCPS dishes. These transferred cell sheets preserved the similar cell morphology and pulsation to those before the detachment. When polyethylene meshes were used to support cell sheet transfer, detached cardiac myocyte sheets partially attached to the mesh threads. Then, the constructs were inverted and placed in another culture dish to prevent direct association to dish surfaces. Moreover, the cardiac myocyte sheets were reorganized to heart tissue-like structures by the unisotropic contraction orientated by the mesh threads, and the pulsatile amplitude increased more than 10 times higher. This technique would bring about new insight in tissue engineering as well as cultured heart model.

Animals↗
PubMed2001

Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature.

To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermo-responsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32 degrees C, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37 degrees C, as on the nongrafted culture dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20 degrees C without need for dispase. No cell remnants were observed on the dishes. Such cell sheet detachment was not observed on nongrafted dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted dishes. Transmission electron microscopy revealed that desmosomes were destroyed in dispase treatment but retained in low-temperature treatment. Use of thermo-responsive dishes was examined as a new tool for tissue engineering to achieve the preparation of artificial epithelium for cell transplantation as well as for the investigation of intact multilayered keratinocyte sheets.

Cell Culture Techniques↗
PubMed2000

Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion.

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.

Adenosine Triphosphate↗
PubMed2000

Rapid cell sheet detachment from poly(N-isopropylacrylamide)-grafted porous cell culture membranes.

Fabrication of functional tissue constructs using sandwiched layers of cultured cells could prove to be an attractive approach to tissue engineering. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheet and prevents functional damage. To accelerate the required culture substrate hydrophilic and hydrophobic structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform IR and electron spectroscopy for chemical analysis revealed that PIPAAm was successfully grafted to surfaces of porous membranes. Atomic force microscopy (AFM) results showed that PIPAAm-grafted membranes had smoother surfaces than ungrafted controls while retaining their porous structure. The mean roughness of PIPAAm-grafted and -ungrafted porous membrane surfaces determined by digital AFM autocalculation was 4.40 +/- 0.4 and 5.9 +/- 0.4 nm, respectively. Tissue culture polystyrene (TCPS) dishes grafted with PIPAAm were compared with PIPAAm-grafted porous membranes in cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-grafted TCPS surfaces compared to only 30 min to detach cell sheets from PIPAAm-grafted porous membranes. With porous membranes, the water accesses the PIPAAm-grafted surface from underneath and peripheral to the attached cell sheet, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cell sheet. With TCPS PIPAAm-grafted surfaces the water is supplied from only the periphery of a cell sheet, slowing detachment.

Acrylic Resins↗
PubMed2000

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets.

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.

Acrylic Resins↗
PubMed2000

Identification of high-risk breast cancer patients from genetic changes of their tumors.

To identify the genetic prognostic markers for breast cancer, we analyzed loss of heterozygosity (LOH) at 11p, 16q, 17p, 17q, and 18q, as well as amplification of the ERBB2, INT2, and MYC genes, in 131 patients with breast carcinoma, 49 of whom had lymph node involvement, but none of whom had distant metastases. Among the several chromosome arms tested, LOH at 17q was correlated with lymph node metastasis. Amplification of the ERBB2, MYC, and INT2 genes was found more frequently in tumors from patients with lymph node metastases than in tumors from those without lymph node metastases. Univariate analysis demonstrated that LOH at 17q and INT2 amplification were factors influencing disease-free survival (DFS). A multivariate analysis was performed on 89 tumors that were able to be evaluated for both LOH at 17q and INT2 amplification, and the results showed that patients who had tumors with these genetic changes were more likely to have a poor prognosis. The findings of this study suggest that investigating genetic changes, in addition to conventional clinicopathologic factors, may contribute to defining groups of breast cancer patients with differences in prognosis.

Blotting, Southern↗
PubMed2000

Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity.

We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.

Acrylic Resins↗
PubMed2000

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces.

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.

Acrylamides↗
PubMed2000

Both insulin sensitivity and glucose sensitivity are impaired in patients with non-diabetic liver cirrhosis.

Background: Careful nutritional support is required in patients with liver cirrhosis due to their glucose intolerance. To elucidate the mechanism of glucose intolerance in cirrhotics, we measured insulin secretion, whole body insulin sensitivity (SI), and glucose sensitivity (SG) in non-diabetic cirrhotics.Methods: Eight patients with compensated cirrhosis who showed normal fasting blood glucose levels and non-diabetic curves on a 75 g oral glucose tolerance test participated in this study. Four normal volunteers were selected as controls. After an overnight fast, glucose was injected intravenously at 300 mg kg(-1) in 2 min followed 20 min later by intravenous insulin at 0.02 U kg(-1) in 5 min. Sequential blood samples were drawn from 20 min before the glucose injection to 3 h post-injection, and plasma glucose and insulin levels were determined. Plasma glucose and insulin disappearance curves were analyzed using the minimal compartment model, and kinetic parameters, including glucose clearance (KG), insulin secretion, SI and SG, were estimated.Results: KG was slower in cirrhosis than in controls, although not significant (P=0.051). Insulin secretion was not different between the two groups. However, SI was significantly lower in cirrhotics (0.814x10(-4) min(-1) pM(-1); 0.572-1.403x10(-4) min(-1) pM(-1)) as compared to controls (1.643x10(-4) min(-1) pM(-1); 0.678-2.085x10(-4) min(-1) pM(-1)) (P=0.029). SG was also lower in the cirrhosis (0.0154 min(-1); 0.0071-0.0208 min(-1)) than in the control group (0.0211 min(-1); 0.0184-0.0260 min(-1)) (P=0.026).Conclusion: Both SI and SG are already impaired in non-diabetic cirrhotic patients even when KG is minimally delayed and insulin secretion has not yet been affected.

Journal Article↗
PubMed2000

Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface.

We developed a novel method to obtain designed shape cell sheets for tissue engineering. Shaping of cell sheets were achieved by the use of poly(N-isopropylacrylamide) (PIPAAm) and poly(N,N'-dimethylacrylamide) (PDMAAm) for temperature-responsive cell adhesive and cell nonadhesive domains, respectively. These polymers were covalently grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation with mask patterns. At 37 degrees C, human aortic endothelial cells (HAECs) attached, spread, and proliferated to make a monolayer only on PIPAAm-grafted domains. HAECs did not adhere on PDMAAm-grafted domains for more than 1 month even under the serum-supplemented condition. By reducing the culture temperature below 32 degrees C, PIPAAm changed to hydrophilic and HAEC sheets were detached from PIPAAm-grafted surfaces without any need of an enzyme such as trypsin. Cell-cell junctions were retained in the recovered cell sheets and easily moved to virgin TCPS dishes with the aid of hydrophilically modified polyvinylidenefluoride membranes as a supporter during the transfer. Moved cell sheets rapidly adhered onto the dish surfaces, and the supporter was easily peeled off from the cell layers. HAEC sheets transferred to new dishes revealed the identical shape and size to those before transfer. This novel technique is the only way to create, harvest, and transfer designed shape cell sheets and would have promising applications in tissue engineering.

Acrylamides↗