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Biomedical subjects

K Hanada

Publications and source records attributed to K Hanada.

At least 19 recordsLinked to original sources
PubMed1999

Escherichia coli mutM suppresses illegitimate recombination induced by oxidative stress.

DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of lambdabio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of lambdabio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total lambdabio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

Bacteriophage lambda↗
PubMed1999

Correlation of skin phototype with facial wrinkle formation.

Facial wrinkle formation is a representative sign of photoaging. Skin can be divided into different skin phototypes (SPTs) depending on its sensitivity to solar radiation. We have determined the relationship between SPTs and deep and fine wrinkle scores on the faces of 230 Japanese subjects. This group was chosen because it is relatively easy to quantify acute and chronic sun damage in Japanese individuals. SPT in the Japanese subjects was classified into one of four groups, SPT-I to -IV according to Fitzpatrick's classification method. The subjects were further classified into indoor or outdoor workers. After photographing the outer canthus, deep and fine wrinkles were discriminated visually and scored from 1-11 and from 1-9, respectively. UV light was used as a photographic light source for clear depiction of the fine wrinkles. Higher scores were recorded for deep wrinkles in individuals with SPT-I, who are more sensitive to sunlight than those with SPT-III or IV, who are more tolerant to sunlight. However, the same tendency did not seem to be apparent for fine wrinkle scores. A high incidence of SPT-IV was seen in outdoor workers. Early detection of SPT-I in individuals who are sensitive to sunlight, may help to predict and prevent photoaging of deep wrinkles. Sensitivity to sunlight may play a part in determining an individual's choice of working environment.

Adult↗
PubMed1998

Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase.

Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin, but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lysenin-resistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wild-type cells. One mutant strain was severely defective in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable of de novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface sphingomyelin is essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient mutants. Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.

Acyltransferases↗
PubMed1998

Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes.

To understand biological function of IL-6 in the skin in vivo, we constructed a vector that strongly expressed human IL-6 in keratinocytes and introduced it into rat keratinocytes in vivo by the naked DNA method. The overexpression of IL-6 induced macroscopic erythema and histologically evident keratinocyte proliferation and lymphocytic infiltration in the treated area of rat skin. Since previous studies using IL-6 transgenic mice have not shown skin inflammation of these mice, our result provides the first evidence that IL-6 is related to the pathogenesis of inflammatory skin diseases. ELISA suggested that a certain degree of transgenic IL-6 expression in keratinocytes was required for inducing skin inflammation. Cytokine profile in rat keratinocytes after the gene introduction was examined by reverse transcriptase-PCR assay and revealed that gene expression of rat IL-1alpha and TNF-alpha showed no marked change until 24 h, whereas that of rat IL-6 and TGF-alpha increased with time. We then introduced and expressed the IL-6 mutant genes, which were designed to behave as IL-6Ralpha antagonists, and found that their ability to induce erythema was lower than that of the wild-type gene. Furthermore, preintroduction of some mutant genes delayed the erythema induced by postintroduction of the wild-type IL-6 gene, suggesting that the mutant forms of IL-6 prevent wild-type IL-6 from binding to IL-6Ralpha. This result indicates that keratinocyte gene therapy may be possible for inflammatory skin diseases using IL-6 mutant genes.

Animals↗
PubMed1998

Calretinin-like immunoreactivity in the regenerating periodontal ruffini endings of the rat incisor following injury to the inferior alveolar nerve.

Regeneration of calretinin (CR)-like immunoreactive (IR) nerve fibers was investigated in the periodontal ligament of the rat lower incisor following resection of the inferior alveolar nerve (IAN). In addition, the degeneration and regeneration processes of periodontal nerve fibers were examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker. In normal animals, the periodontal nerve fibers showing PGP 9.5-like immunoreactivity (LI) formed either periodontal Ruffini endings with expanded arborization and thin free nerve endings in the alveolar half of the ligament. Thick CR-IR nerve fibers also appeared in a dendritic fashion in the same region, but thin CR-IR nerve fibers were rarely observed. Five days following resection of the IAN, a major population of PGP 9.5-IR and all CR-IR nerve fibers disappeared except for some thin PGP 9.5-IR nerves in the periodontal ligament. Regenerated PGP 9.5-IR nerve fibers appeared around 7 days following resection, in contrast to a very small number of regenerated CR-IR nerve fibers. Around 14-21 days following resection, the number and terminal morphology of regenerated PGP 9.5-IR nerve fibers were comparable to those observed in normal animals, but the number of regenerated CR-IR nerve fibers was still smaller than that of normal animals. The number of regenerated CR-IR nerve fibers increased to return to normal by 56 days following injury. The delay of expression of CR-LI in the regenerated periodontal Ruffini endings suggests that functional recovery of periodontal Ruffini endings occurred after the completion of the regeneration of periodontal nerve fibers.

Animals↗
PubMed1998

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography.

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(-)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 - 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(-)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2-6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(-)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5-6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.

Animals↗
PubMed1998

The establishment of a preoperative diagnosis of pancreatic carcinoma using cell specimens from pancreatic duct brushing with special attention to p53 mutations.

BACKGROUND: Previously, the authors reported that 82% of cases of pancreatic carcinoma were positive for p53 in cytologic specimens obtained by selective endoscopic pancreatic duct brushing (SEPB). However, there was an extreme discrepancy between the authors' data of p53 overexpression using cytologic specimens and other reports using surgically resected specimens. In this study, the authors demonstrate that p53 positive cells precisely reflect its gene mutations, and also establish systematic procedures for the preoperative diagnosis of patients with pancreatic carcinoma. METHODS: The authors examined 44 cases of pancreatic carcinoma, 30 cases of chronic pancreatitis, and 9 cases of papillary adenoma. In all cases, pathologic diagnosis was made by surgery or autopsy. The conventional cytology and p53 immunocytology were performed simultaneously in the cell specimens obtained by SEPB. In the cases immunostained for p53, DNA was extracted selectively from p53 immunostained cells using a light microscope. p53 mutations in exons 5 to 8 were examined by direct sequencing. RESULTS: Forty of 44 pancreatic carcinomas (91%) were diagnosed correctly by the methods of conventional cytology associated with p53 immunocytology. p53 mutations were detected in 12 of 14 cases that were positive for p53 (86%). Four of six cases that were inoperable due to massive metastasis or invasion had the mutation at codon 273 (CGT to CAT) in exon 8. CONCLUSIONS: These results suggest that p53 immunocytology reflects its gene mutations precisely, and that the point mutation at codon 273 (CGT to CAT) of p53 may play an important role in the invasive potential and metastasis of pancreatic carcinoma.

Adenocarcinoma↗
PubMed1998

Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy.

Animals↗
PubMed1998

Carbonic anhydrase isozyme II immunoreactivity in the mechanoreceptive Ruffini endings of the periodontal ligament in rat incisor.

The present study describes the distribution of carbonic anhydrase isozyme II (CA II) in the lingual periodontal ligament of the rat incisor. Some thick nerve fibers in the nerve bundle displayed CA II-like immunoreactivity (LI) as well as non-neuronal elements such as osteoclasts. At the alveolar half of the lingual periodontal ligament of the incisor, thick CA II-like immunoreactive (-IR) nerve fibers showed a tree-like raminification, but thin and beaded CA II-IR nerve fibers were rare. Under the electron microscope, CA II-LI were diffusely localized in the axoplasm of the axon terminals surrounded by Schwann sheaths which were immunonegative for CA II. The cell bodies of the terminal Schwann cells associated with the periodontal Ruffini endings did not exhibit CA II-LI. The present immunohistochemical evidence indicates that CA II may participate in the regulation of the intra-neuronal ion in the periodontal Ruffini endings which are thought to be in a state of high neuronal activity.

Animals↗
PubMed1998

Functional analysis of CD82 in the early phase of T cell activation: roles in cell adhesion and signal transduction.

To define T cell co-stimulatory molecules that work in the early phase of T cell activation, we established monoclonal antibodies (mAb) that inhibit or enhance T cell activation by the histiocytic leukemia cell line U937. One of the mAb, 53H5, which recognized both T cells and U937, was identified to bind to CD82 by expression cloning. Functional analyses of CD82 revealed that 1) CD82 needs to exist on both T cells and U937 for the full activation of T cells; 2) CD82 expression is up-regulated on both T cells and U937 by stimulation such as CD3 ligation or treatment with phorbol 12-myristate 13-acetate; 3) overexpression of CD82 enhances both homotypic and heterotypic cell adhesion between T cells and U937; 4) CD82 signal co-stimulates T cells and the signal works synergistically with the CD28-mediated T cell co-stimulation signal; 5) in mixed leukocyte reactions using U937 as stimulator cells, CD82 overexpression on U937 correlates with the higher allogeneicity of U937 cells. These results indicate that CD82 co-stimulates T cells not only by sending intra-T cell signals that work synergistically with CD28 signals but also by inducing enhanced T cell-antigen-presenting cell interaction.

Antigens, CD↗
PubMed1998

Single local injection of recombinant fibroblast growth factor-2 stimulates healing of segmental bone defects in rabbits.

The effects of a single local injection of recombinant human fibroblast growth factor-2 on the healing of segmental bone defects were evaluated in rabbits. One month after the external fixator originally designed for this experiment was installed in the tibia of the rabbit, a 3-mm bone defect was created by an osteotomy in the middle of the tibia and 0, 50, 100, 200, or 400 microg of fibroblast growth factor-2 in 100 microl of saline solution was injected into the defect. Injection of the growth factor increased the volume and mineral content of newly made bone at the defect in a dose-dependent manner with significant effects at concentrations of 100 microg or greater. These significant effects were observed at 5 weeks and later. One hundred micrograms of the growth factor increased the volume and mineral content of newly made bone by 95 and 36%, respectively, at 5 weeks. These results indicate that a single local injection of fibroblast growth factor-2 stimulates the healing of segmental defects. We speculate that such an injection could be clinically useful for the healing of fractures even when the fracture gap is rather large.

Animals↗
PubMed1998

Stimulation of bone formation by intraosseous injection of basic fibroblast growth factor in ovariectomised rats.

The effect on intraosseous bone formation of a single local injection of recombinant human basic fibroblast growth factor into trabecular bones was examined in ovariectomised osteoporotic rats. Fibroblast growth factor (400 micrograms), or the vehicle alone, was injected into the ilium at 16 weeks after ovariectomy or a simulated operation. Bone mineral density in the ovariectomised rats increased to a level similar to the latter at 2 weeks and reached a maximum at 8 weeks. After 8 weeks, BMD decreased slowly and the value at 24 weeks was still higher than that in the ovariectomised rats. Fibroblast growth factor stimulated osteoid formation in the first 2 weeks, bone volume reaching a peak at 8 weeks. From 8 to 12 weeks, bone resorption increased, resulting in decreases in bone volume to the levels of the group with simulated operations at 24 weeks. Structural analysis at 8 and 24 weeks showed that ovariectomy decreased the continuity of trabeculae and the injection of fibroblast growth factor restored it to levels higher than, or equal to, those who had the simulated operation. The present study demonstrated that intraosseous fibroblast growth factor given to ovariectomised rats restored bone volume and quality to the levels of the rats who had a simulated operation only.

Animals↗
PubMed1998

Calcium pyrophosphate dihydrate crystal deposition disease after anterior cruciate ligament reconstruction.

We report the case of a 34-year-old woman who presented with calcium pyrophosphate dihydrate (CPPD) crystal deposition disease shortly after anterior cruciate ligament (ACL) reconstruction using a polyester artificial ligament (Leeds-Keio; Neoligaments, Leeds, England). The patient had earlier undergone a medial collateral ligament repair of a sprain to her right knee incurred while skiing. Nine years later, she underwent ACL reconstruction. Seventeen months after ACL reconstruction, calcification was observed on radiographs of the medial and lateral menisci. Based on these calcifications and polarized light microscopic findings of the joint fluid, the diagnosis was made of CPPD crystal deposition. CPPD deposition appeared to have resulted from intra-articular damage incurred during ACL reconstruction as well as prolonged anterior instability.

Adult↗
PubMed1998

Enantioselective tissue distribution of the basic drugs disopyramide, flecainide and verapamil in rats: role of plasma protein and tissue phosphatidylserine binding.

PURPOSE: The stereoselective distribution of three basic drugs, disopyramide (DP), flecainide (FLC) and verapamil (VP), was studied to clarify the relationship between the tissue-to-unbound plasma concentration ratio (Kpf) and drug lipophilicity and binding to phosphatidylserine phs), which are possible factors determining the tissue distribution of these drug enantiomers. METHODS: The drug enantiomer or racemate was administered to rats by intravenous constant infusion. Their concentrations in plasma and tissues were determined using enantioselective high-performance liquid chromatography. Plasma protein binding, and buffer-octanol and buffer-hexane containing PhS partition coefficients were also determined. RESULTS: The stereoselectivity of the tissue-to-plasma concentration ratio (Kp) was partly associated with that of serum protein binding. However, the Kpf value of R(+)-VP in the lung was significantly higher than that of S(-)-VP. A linear correlation was observed between the Kpf values of these drug enantiomers in brain, heart, lung and muscle, and their buffer-hexane containing PhS partition coefficients. The in vitro data for the binding of these drugs to PhS suggest that stereoselective binding of VP to PhS may correspond to its stereoselective tissue binding. CONCLUSIONS: Our findings provide some evidence for a role of tissue PhS in the tissue distribution of basic drugs with respect to stereoselectivity of drug enantiomers distribution.

Animals↗
PubMed1998

Immediate-type heat urticaria: report of a case and study of plasma histamine release.

We report a 68-year-old man who had immediate-type heat urticaria with systemic symptoms. Immersing his hand in water at 42 degrees C (heat challenge test) produced an urticarial response, with an increase in the plasma histamine level from 0.26 to 7.64 ng/mL. Administration of oral antihistamines alone did not suppress either the urticarial response or the increase in plasma histamine. However, a combination of antihistamines and desensitization improved the skin lesions and reduced the plasma histamine level. The heat challenge test subsequently provoked a negative response and there was no increase in plasma histamine level 3 months after starting the combination therapy. These results indicate that the histamine level reflected the result of the heat challenge test and the amelioration of the skin eruption.

Aged↗