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Biomedical subjects

H Iwata

Publications and source records attributed to H Iwata.

At least 109 records · Page 6Linked to original sources
PubMed2001

Functional comparison of the single-layer agarose microbeads and the developed three-layer agarose microbeads as the bioartificial pancreas: an in vitro study.

In this study, the insulin secretory characteristics of the microencapsulated hamster islets were studied during long-term culture. The hamster islets were encapsulated as single-layer agarose microbeads or three-layer agarose microbeads with agarose and agarose containing poly(styrene sulfonic acid) (PSSa), respectively. The influence of PSSa on the function of the rat islets microencapsulted in three-layer microbeads was primarily monitored. The aim of this study was to examine the influence of the PSSa on the in vitro function of the islets encapsulated in the agarose/PSSa microbeads compared with single-layer agarose microbeads during long-term culture. The microbeads were cultured for 30 days in medium of Eagle's MEM at 37 degrees C in 5% CO2 and 95% air. The basal insulin secretion into the culture medium was measured daily during the first 12 days and two times per week until 30 days. The microbeads were subjected to static incubation test on the 10th, 20th, and 30th day during culture. The basal insulin secretion level of the agarose/PSSa microbeads was significantly higher than that of single-layer agarose microbeads. The static incubation tests revealed a similar pattern of insulin secretion from both microbeads when they were exposed to high glucose challenge. In the static incubation test, both could significantly increase insulin release to more than 6.61 times (stimulation index) in response to high glucose stimulation and could significantly decrease when glucose concentration returned from high glucose to low glucose on the 10th, 20th, and 30th day of culture. This study demonstrated that the hamster islets enclosed in agarose/PSSa hydrogel not only continuously secreted basal amounts of insulin, but also maintained their response to high glucose stimulation similar to the agarose microbeads. The above results together with those of our previous in vivo study suggest that the three-layer microbeads (agarose/PSSa) are well suitable for xenotransplantation of islets for the clinical application.

Animals↗
PubMed2001

The influence of the anticomplement synthetic sulfonic polymers on the function of pancreatic islets: an in vitro study.

In a previous experiment, we demonstrated the anticomplementary efficacy of poly(stryrene sulfonic acid) (PSSa) and poly(2-acrylamido-2-methyl propane sulfonic acid) (PAMPS). The aim of this study was to examine their influence on the function of pancreatic islets in vitro. In this study, after culturing the rat islets with RPMI-1640 culture medium containing different concentrations of soluble PSSa or PAMPS for 24 h at 37 degrees C, we performed morphological and functional examination of the rat islets. We found that the islets maintained their normal morphology regardless of whether they were in the PSSa or PAMPS groups when the concentrations of soluble PSSa or PAMPS in the media were below 1 g/dl. In the static incubation study, the islets cultured in the PAMPS groups showed significantly high insulin secretory response to glucose challenge but those in the PSSa groups lost the response when the concentrations of soluble PSSa or PAMPS in the media were below 1 g/dl. The PAMPS not only had strong anticomplementry effect, but also maintained the good insulin secretory capacity of the islets. These results indicated that PAMPS is a promising bioartificial material for future clinical application of biohybrid artificial pancreas preparation. It is well suitable for xenotransplantation experiments.

Animals↗
PubMed2000

Antithrombogenicity of cultured endothelial cell-detached surface.

To examine the antithrombogenicity of cultured endothelial cell-detached surface, a simple hybrid vascular model tube consisting of a glass tube and endothelial cells was constructed. To detach the endothelial cells from the inner surface of the model tube, a steady shear stress of 2 or 8 N m(-2) was imposed onto the surface of endothelial cell monolayer by means of a coaxial double cylinder rotational-type apparatus. Coagulation of blood in contact with the endothelial cell-detached surface was examined using a damped oscillation rheometer. Coagulation of whole blood in the cell-detached tube occurred at about 40 min, which was almost the same as that in the endothelial cell-coated tube. A few platelets without shape change adhered to the endothelial cell-detached surface. These data suggest that the endothelial cell-detached surface may exhibit antithrombogenic and anticoagulant surfaces. Biochemical analyses showed that the glass surface, where endothelial cell was detached, was covered with components such as collagen type IV that is considered to be produced from the endothelial cells on the glass surface.

Journal Article↗
PubMed2000

Nitric oxide via macrophage iNOS induces apoptosis following traumatic spinal cord injury.

To investigate the pathophysiological mechanisms involved in post-traumatic impairment of the spinal cord, we analyzed expression patterns of the inducible nitric oxide synthase (iNOS) gene following acute injury of rat spinal cord using a weight drop technique. PCR analysis revealed that iNOS mRNA appeared at 3-12 h after injury and declined thereafter. Immunohistochemical analysis showed that iNOS-positive cells invaded the lesioned area through the perivascular space at 6 h after injury. The population of these cells peaked at 24 h and then declined to disappear 3 days after injury. The iNOS-positive cells were also stained with ED-2 but not with ED-1 or OX-42, indicating that these cells were macrophages and/or perivascular cells. In parallel with the appearance of iNOS-positive cells, other cells emerged that were positively stained by the terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) assay. TUNEL-positive cells were scattered in the lesioned area 1 day after injury, but some in the surrounding area close to iNOS-positive cells. Administration of L-Ng-nitro-arginine methylester, a competitive inhibitor of NOS, resulted in a reduction of TUNEL-positive cells in the lesioned area. These results suggest that nitric oxide generated by iNOS of macrophages and/or perivascular cells plays a significant role in eliminating damaged cells from the lesioned area by apoptosis.

Animals↗
PubMed2000

Triparental origin of Damask roses.

Damask roses are one group of old rose varieties and a key material in old European rose improvement in the 19th century. To clarify the origin of Damask roses, we selected four varieties as the oldest Damask varieties and examined the relationship between the Damask varieties and their putative ancestors at the molecular level. Randomly amplified polymorphic DNA analysis of the Damask varieties proved that they had an identical profile, indicating they were established from a common ancestor. They have never been allowed to reproduce sexually; their reproduction depends entirely on vegetative propagation. We identified three Rosa species, R. moschata, R. gallica and R. fedschenkoana, as parental species of the original hybridization that contributed to forming the four oldest Damask varieties by sequencing the internal transcribed spacer of ribosomal DNA. We also found that all the four oldest Damask varieties had chloroplasts derived only from R. moschata, as judged from psbA-trnH spacer sequences. This triparental origin of the four oldest Damask varieties can explain some morphological characteristics of the four oldest Damask varieties, like fruit shape, leaf color and the 'Moss' character.

Base Sequence↗
PubMed2000

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation.

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.

Acetylation↗
PubMed2000

TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation.

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.

Base Sequence↗
PubMed2000

Increase in neurotrophin-3 expression followed by Purkinje cell degeneration in the adult rat cerebellum after spinal cord transection.

Changes in brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) contents following thoracic spinal cord transection were investigated in the cerebral cortex, hippocampus, and cerebellum of rats. The NT-3 content became significantly elevated at 3 days after transection only in the cerebellum and gradually declined to the control level by 6 days after the injury, remaining unchanged in the cerebral cortex and hippocampus. No significant change in the BDNF content was observed in any of the regions tested. Immunohistochemical analysis showed that the labeling indicating NT-3-like immunoreactivity was intensified in both cerebellar granule and Purkinje cells 3 days after the injury. The number of Purkinje cells with aggregation of chromatin around the nuclear membrane and swelling of the cytoplasm and/or organelles gradually increased with time starting 4 days after the injury, demonstrating morphological changes indicative of necrosis. However, no abnormal morphology was found in cerebellar granule cells at any time examined. We suggest that it is reasonable that increased NT-3 stimulated the death of Purkinje cells, because 1) the degeneration was necrosis, which is known to be accelerated by neurotrophins under certain pathological conditions, and 2) the increase in NT-3 occurred prior to Purkinje cell degeneration. Therefore, our present results may imply that spinal cord injury-induced NT-3 accelerates injury rather than alleviates degeneration of Purkinje cells.

Animals↗
PubMed2000

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) following traumatic spinal cord injury.

We investigated the temporal and spatial expression patterns of the GDNF gene after subjecting rats to an acute contusion injury of the spinal cord using the weight drop technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that GDNF transcription in the spinal cord began to increase within 30 min after injury and peaked within 3 h. Immunohistochemical analysis showed GDNF immunoreactivity to be present mainly in microglia and macrophages 1 day after injury, but not in neurons or astrocytes. This immediate upregulation of GDNF gene expression may be a component of an inflammatory process and probably exerts a protective effect on neurons following spinal cord injury (SCI).

Animals↗
PubMed2000

Effects of micro-encapsulation on morphology and endocrine function of cryopreserved neonatal porcine islet-like cell clusters.

BACKGROUND: For the success of clinical islets transplantation, the development of a long-term storage method is necessary. However, the structure of digested islets is scanty for culture and cryopreservation. In this study, the effect of micro-encapsulation to cryopreserved porcine islet-like cell clusters (ICCs) was investigated. METHODS: The ICCs prepared from neonatal pigs by collagenase digestion and culture technique were cryopreserved and micro-encapsulated in 5% agarose membranes. After cryopreservation, ICC cultured without encapsulation (group A) and cultured with encapsulation (group B) were assessed by comparison with no cryopreserved ICC (control) both in vitro by static incubation test and in vivo in a xenotransplantation study. RESULTS: Micro-encapsulation was able to maintain the fine morphology and the number of ICCs of group B after 7 days of culture. There were not significant differences in insulin secretion of group B and control on day 1 and 7 of culture (1 day:11+/-0.99, 7 days: 5.30+/-1.08 microU/ICC/hr NS versus control). On day 7 of culture, the retrieval rate of group B (105.2+/-9.8%) is obviously higher compared with group A (63.0+/-6.3%). In the xenotransplatation model, the ICCs of group B showed long survival time (7.9+/-0.4 weeks) and good transplantation effect. CONCLUSION: Our study suggests that micro-encapsulation is one of the useful method for cryopreserved ICC to maintain the fine morphology and effectively recover the endocrine function.

Animals↗
PubMed2000

Study of complement activation on well-defined surfaces using surface plasmon resonance.

It has been accepted that covalent immobilization of C3b on artificial materials is the most important step to initiate the complement activation. However, there are few studies that have directly demonstrated covalent immobilization of C3b on artificial surfaces. In this study, model thin layers were prepared by the self-assembled monolayer method to produce a surface covered with hydroxyl or methyl groups using mercaptododecane (CH(3)-SAM) and mercaptoundecanol (OH-SAM). Interactions of the complement system with the model surfaces were studied using a surface plasmon resonance instrument. The OH-SAM immobilized C3b, resulting in activating of the complement system through the alternative pathway in Veronal-buffered saline, but this surface did not activate the classical pathway. However, the OH-SAM could not activate the alternative pathway in Veronal-buffered saline containing 10 mM EGTA and 2 mM MgCl(2) that is believed not to interfere with the activation of the alternative pathway. The hydrophobic CH(3)-SAM surface could not activate the classical pathway, but activated the alternative pathway, although the extent was small.

Journal Article↗
PubMed2000

In situ RT-PCR detection of CYP1A mRNA in pharyngeal epithelium and chondroid cells from chemically untreated fish: involvement in vertebrate craniofacial skeletal development?

Knowledge about the expression sites of cytochrome P450 1A (CYP1A) mRNA is crucial for a better understanding of the physiological function of CYP1A. We investigated the cellular localization of CYP1A mRNA in chemically untreated fish by use of an in situ reverse transcription-polymerase chain reaction (IS RT-PCR) technique. The fathead minnow (Pimephales promelas) was formalin-fixed, and paraffin-embedded. Sections (5 microm) were treated with trypsinogen. Following reverse transcription of CYP1A mRNA, the cDNA was amplified in situ by PCR with specific primers. Detection of the amplicons was accomplished by a second PCR performed with digoxigenin-labeled dUTP. CYP1A mRNA expression was detected in cytoplasm of chondroid cells surrounding hyaline cartilage in gill arches. This result was consistent with that of immunohistochemical analysis with a CYP1A1-specific monoclonal antibody. CYP1A mRNA also was found in stratified squamous epithelium of the pharynx and gill arches, but no staining was detected in those cells by immunohistochemical analysis. The results indicate that IS RT-PCR is an effective/sensitive technique for localizing low level CYP1A expression. In addition, the sites where we identified expression of CYP1A are targets of retinoic acid, sonic hedgehog and Hox genes, suggesting that functional CYP1A in vertebrates could participate in craniofacial skeletal development through involvement in the retinoic acid signaling cascade.

Amino Acid Sequence↗
PubMed2000

Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Amino Acid Sequence↗
PubMed2000

Progenitor endothelial cells on vascular grafts: an ultrastructural study.

The morphology of progenitor endothelial cells on vascular graft surfaces is addressed in this report. Such cells were seen to attach to intima-expressed CD34 and Flk-1 antigen and showed positive 5-bromo-2-deoxyuridine (BrdU) uptake. We examined CD34 and Flk-1 antigen-expressing endothelial progenitor cells three-dimensionally using confocal laser scanning microscopy (CLSM). Under detailed CLSM observation, through an ameboid-form cell, these progenitor endothelial cells changed from a globular to a flattened form. We also investigated these morphological changes using scanning electron microscopy. From these results, progenitor endothelial cells were observed not only near the advancing edge of endothelium, but also around the developing intimal site. Their form also changed from globular to flattened as observed in the CLSM results. These morphological changes were seen more frequently near the advancing edge and around the developing intimal site. They attached directly to vascular prosthesis fibers and likewise covered the graft luminal surface. Progenitor endothelial cells in any form had a common surface structure. We conclude from our results that progenitor endothelial cells can attach to graft fibers directly without clotting and directly cover the graft luminal surfaces.

Animals↗
PubMed2000

Effects of PEMF on a murine osteosarcoma cell line: drug-resistant (P-glycoprotein-positive) and non-resistant cells.

After pulsed exposure of Dunn osteosarcoma cells (nonresistant cells) to Adriamycin (ADR) at increasing concentrations and single-cell cloning of surviving cells, ADR-resistant cells were obtained. These resistant cells expressed P-glycoprotein and had resistance more than 10 times that of their nonresistant parent cells. Compared to the nonresistant cells not exposed to pulsing electromagnetic fields (PEMF) in ADR-free medium, their growth rates at ADR concentrations of 0.01 and 0.02 micrograms/ml, which were below IC50, were 83.0% and 61.8%, respectively. On the other hand, in the nonresistant cells exposed to PEMF (repetition frequency, 10 Hz; rise time, 25 microsec, peak magnetic field intensity, 0.4-0.8 mT), the growth rate was 111.9% in ADR-free medium, 95.5% at an ADR concentration of 0.01 micrograms/ml, and 92.2% at an ADR concentration of 0.02 micrograms/ml. This promotion of growth by PEMF is considered to be a result of mobilization of cells in the non-proliferative period of the cell cycle due to exposure to PEMF. However, at ADR concentrations above the IC50, the growth rate tended to decrease in the cells not exposed to PEMF. This may be caused by an increase in cells sensitive to ADR resulting from mobilization of cells in the non-proliferative period to the cell cycle. The growth rate in the resistant cells exposed to PEMF was significantly lower than that in the non-exposed resistant cells at all ADR concentrations, including ADR-free culture (P</=0.0114). Therefore, this study suggests that PEMF promotes the growth of undifferentiated cells but progressively suppresses the growth of more differentiated cells, i.e., PEMF controls cell growth depending on the degree of cell differentiation. This study also shows the potentiality of PEMF as an adjunctive treatment method for malignant tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Synthetic absorbable film for prevention of air leaks after stapled pulmonary resection.

Staple-line reinforcement buttresses made of bovine pericardium (BP), expanded polytetrafluoroethylene (ePTFE), and so on have been shown to be effective in preventing air leaks after stapled lung volume reduction operations, and some of them have been clinically utilized. However, each buttress suffers at least one disadvantage such as risk of viral infection and chronic inflammation. A new buttress was made using a poly(L-lactic acid-co-epsilon-caprolactone) film (L/C film) and its effectiveness as a staple-line reinforcement was examined by performing lung volume reduction operation on a canine model. Soft tissue responses to the buttress were compared with those to the BP strip and the absorbable behavior was studied. The L/C film buttress was flexible and thin enough to easily cut. Death of dogs, infection, acute and prolonged air leaks, and any complications related to its use were not observed. The tissue responses to the film were more mild and favorable than those to BP. The L/C film was absorbed after the staple line was covered by a connective tissue. The results described above suggest that the buttress made of an L/C film is a promising staple-line reinforcement material.

Absorption↗