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Biomedical subjects

K Kikuchi

Publications and source records attributed to K Kikuchi.

At least 91 records · Page 5Linked to original sources
PubMed2001

High sensitivity of a novel ELISA for anti-M2 in primary biliary cirrhosis.

The use of an ELISA for the detection of anti-M2, a specific autoantibody in primary biliary cirrhosis (PBC), has been common in Japan. However, there are some problems in the sensitivity of this ELISA, especially in PBC patients showing antimitochondrial antibody (AMA)-negative sera or low AMA titers by immunofluorescence. Recently, a new ELISA for anti-M2 was developed, using porcine heart mitochondrial protein as the antigen. We report here comparative studies of the new and the former anti-M2 ELISAs. Porcine heart mitochondrial protein was prepared and used as the antigen for the new ELISA for anti-M2. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this protein showed three major M2 antigen proteins. As the second antibody, peroxidase-conjugated anti-human mouse monoclonal IgM, in addition to monoclonal IgG, was included. The sera of 171 PBC patients were examined. As controls, we examined the sera of 167 non-PBC patients and the sera of 115 normal controls. The cut-off index was set at 10 U/ml, based on the results for the normal controls. No sera from the non-PBC patients or the normal controls were positive for anti-M2 by either the new or the former ELISA. However, the positivity rate for anti-M2 in PBC patients with the new ELISA was 78%; in contrast, that with the former ELISA was only 54%; this difference was significant (P = 0.00001). In particular, in 65 patients showing AMA titers of 1:20 or less, the positivity rate with the new ELISA was 51%; in contrast, that with the former ELISA was only 17%. As the sensitivity of the new ELISA is significantly higher than that of the former ELISA, especially for sera from patients showing AMA-negativity or low titers of AMA, the new ELISA is considered to be more effective than the former ELISA for use in anti-M2 screening assays in patients with PBC.

Animals↗
PubMed2001

Correlation of genetic etiology with response to beta-adrenergic blockade among symptomatic patients with familial long-QT syndrome.

Mutations in any of the five genes KCNQ1, KCNH2, KCNE1, KCNE2, and SCN5A can be responsible for familial long QT syndrome (LQTS), an arrhythmogenic disorder that entails a high risk of sudden death. beta-Adrenergic blocking agents are the first therapeutic choice, and 80% of patients treated with these agents show symptomatic relief; however the remaining 20% do not respond well. We previously performed a nationwide analysis of familial long QT syndrome (LQTS) in Japan and identified 32 mutations in the KCNQ1 and KCNH2 genes. In the present retrospective study, we found that patients carrying mutations in the KCNQ1 gene responded better to beta-adrenergic blocking agents than those with KCNH2 mutations (12 of 13 vs 1 of 5; P = 0.0077, Fisher's exact test). This is a good example of the power of genetic diagnosis to direct the selection of appropriate therapy for patients with diseases of heterogeneous genetic etiology.

Adolescent↗
PubMed2001

Relationship between serum lipoprotein(a) level and thrombin generation to the circadian variation in onset of acute myocardial infarction.

A high incidence of acute myocardial infarction (AMI) has been reported between 06:00 and 12:00 h. This may be related to an abnormality in hemostasis. An association has been founded between the serum lipid level and coronary atherosclerosis, as well as the serum lipid level and a hemostatic abnormality. We investigated the association between the time of AMI, the level of serum lipid, and of hemostatic factor. Of the 42 subjects evaluated retrospectively, 20 had experienced an AMI between 06:00 and 12:00 h (group A), while 22 had developed an AMI during some other period (group B). All patients received emergency coronary angiography, which identified a total occlusion of coronary artery in the proximal portion of the left antecedent branch. The serum level of several lipid factors and of hemostatic factors were compared between the two groups. Characteristics of patients were similar in both groups. The serum levels of lipoprotein(a) (Lp(a)) and of thrombin-antithrombin III complex (TAT) were higher in group A than in group B, respectively. The level of other factors were similar in both groups. Group A showed a significant correlation between the level of Lp(a) and TAT, with a tendency (not statistically significant), toward a positive correlation between Lp(a) and PAI-1, and a negative correlation between Lp(a) and t-PA. In a subgroup that experienced AMI in the early morning, a higher level of Lp(a) was associated with an elevation of TAT, a marker for thrombin generation, and with the level of fibrinolytic factor. This suggests that Lp(a) is closely related to the increase in the early morning incidence of AMI via a change in the prothrombotic state.

Antithrombin III↗
PubMed2001

Small molecule-based laser inactivation of inositol 1,4,5-trisphosphate receptor.

BACKGROUND: Chromophore-assisted laser inactivation (CALI) is a powerful method for the study of in situ protein function in cellular processes. By using CALI, it is possible to abrogate the function of a target protein with unprecedented spatial and temporal resolution. However, CALI has some limitations, which restrict wider biological application, owing mainly to the use of antibody for target recognition. To circumvent the limitations, we have developed small molecule-based CALI (smCALI). RESULTS: The inositol 1,4,5-trisphosphate receptor (IP3R) was selected as the target protein and a malachite green-conjugated IP3 analog, MGIP3, was used as a small-molecular probe. We examined the effect of MGIP3-based CALI on Ca2+ release via IP3R using permeabilized smooth muscle cells. When the cells were treated with MGIP3 followed by laser irradiation, the IP3-induced Ca2+ release rate was decreased in a concentration- and irradiation time-dependent manner. The effect was specific for IP3R, because the Ca2+ uptake function of the co-localized sarco/endoplasmic reticulum Ca2+-ATPase was not affected. CONCLUSIONS: IP3R was specifically inactivated by smCALI using MGIP3. The efficiency of inactivation was calculated to be substantially greater than that of antibody-based CALI. The efficient and specific inactivation of IP3R would allow us to obtain an insight into spatiotemporal roles of IP3R in various cell functions. Our results may be considered to be a first step for a wider application of smCALI as a useful method to study spatiotemporal protein functions.

Animals↗
PubMed2001

Perfluorocarbon-mediated aeration applied to recombinant protein production by virus-infected insect cells.

Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells. The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer. The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture. On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC-medium interface. These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system. Overall mass-transfer coefficient K(L) for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures. The K(L) value in the PFC-mediated system was 2.60x10(-3)cms(-1), 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC-medium and medium-air interfaces, respectively. However, the K(L) values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces. DO behavior in the cultures was well explained by the model calculation using the determined K(L) values and oxygen-consumption rates of viable cells. This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24-0.5ppm (3-7%) in the insect cell/baculovirus expression system.

Journal Article↗
PubMed2001

HBe seroconversion and HBs mutation among Japanese hepatitis B virus carriers.

It has been reported in Germany that seroconversion to anti-HBe or anti-HBs is frequently associated with genotype changes of hepatitis B virus (HBV) from genotype A to genotype D. We previously reported that the HBeAg-negative state in Japan was significantly more common in patients infected with genotype B HBV than those infected with genotype C HBV. To determine whether the high prevalence of genotype B in the HBeAg-negative state is due to a change from genotype C to genotype B, 72 pairs of serum samples before and after HBe seroconversion were examined for nucleotide sequences in the S gene (amino acids 42-164) among Japanese HBV carriers. No one was identified to have undergone genotype change during HBe seroconversion. A total of 71 codon mutations were observed. Sixty-two of these 71 codon mutations (87.3%) were non-synonymous. Genotype B HBV had no mutational hot spots. In genotype C, there was a mutational hot spot at amino acid 126 of the S protein, and it was suggested that Thr126 before HBe seroconversion was more susceptible to becoming an asymptomatic carrier after HBe seroconversion than Ile126. In conclusion, genotype changes during HBe seroconversion were not found to be common in Japan.

Journal Article↗
PubMed2001

False positive reaction in ELISA for IgM class anti-M2 antibody and its prevention.

Anti-M2 of anti-mitochondrial antibodies is recognized as the specific autoantibody detected in sera from patients with primary biliary cirrhosis (PBC). The IgG class and IgM class of this antibody can be separately measured using each ELISA. In the present study, false positive reactions were found in some sera from non-PBC patients such as acute hepatitis A, syphilis and rheumatoid arthritis using the IgM anti-M2 ELISA. They showed an increase of polyclonal IgM, and positivity for IgM anti-cardiolipin or rheumatoid factors, respectively. So, we developed a means to prevent these false positive reactions. First, dilutions of test sera at 1:1000-fold were carried out in addition to the original method at 1:100-fold. Secondly, some blocking reagents were added into the buffer system. By serum dilution, non-specific bindings disappeared in most samples other than showing an increase in polyclonal IgM. Moreover, the addition of suitable blocking reagents such as fetal bovine serum (FBS) and skimmed milk into the buffer system could prevent these non-specific bindings. From these findings, the procedure of optical serum dilution and the addition of suitable blocking reagents successfully prevented false positive reactions in this IgM anti-M2 ELISA.

Journal Article↗
PubMed2001

Definition of antigen specificity for antimitochondrial proteins detected by Western blotting using native mitochondrial proteins in primary biliary cirrhosis.

The major autoantigens to anti-mitochondrial antibody (AMA) in primary biliary cirrhosis (PBC) have previously been identified to be PDC-E2, BCOADC-E2, and OGDC-E2. However, analysis of these autoantigens to AMA cannot be examined using the two routine assays; immmunofluorescence and ELISA. Moreover, there are some problems in specificity and sensitivity in these routine assays. So, analysis with Western blotting using native mitochondrial protein as the antigen is required; it allows the identification of the molecular weights for the proteins which react with AMA in patients' sera. However, since the antigen-proteins used are not unified, molecular weights of AMA corresponding proteins vary among laboratories. In the present study, as the first step to help address this issue, we investigated the antigen specificity of protein bands detected by Western blotting using our in-house bovine and porcine heart mitochondrial proteins. Three major recombinant mitochondrial proteins were prepared. The antigen specificity was examined by the absorption tests preincubated with the three recombinant mitochondrial proteins. The molecular weights of developing our bovine and porcine heart mitochondrial proteins using SDS-PAGE were multiple protein bands including 74, 52, 50, and 43 kDa protein bands. Of them, the 74, 50, and 43 kDa protein bands were absorbed with preincubations of recombinant PDC-E2, BCOADC-E2, and OGDC-E2 protein, respectively. AMA specificity of these three major proteins with our Western blotting was confirmed.

Journal Article↗
PubMed2001

Functional properties of the rat phosphatase 1alpha promoter.

The aim of this study was to investigate the functional properties of the promoter of the protein phosphatase 1alpha catalytic subunit. Luciferase plasmids with different fragments of the rat catalytic subunit of the protein phosphatase 1alpha promoter ranging from -3.7 kbp to -59 bp were transiently transfected into cells by the calcium-phosphate precipitation method. The promoter activity was determined in the absence and presence of inotropic agents which influencing the cAMP-depending pathway. The basal transcriptional activity decreased at fragment -124 bp and shorter fragments. To identify regions of regulatory importance we investigated the cAMP-dependent influence on the transcriptional activity. Stimulation of the complete promoter region with forskolin (1-100 microM) for 6 h led to a concentration-dependent decrease of transcriptional activity. Moreover, regions shorter than 3.7 kbp were inhibited by forskolin (10 microM). Short time stimulation (10 min) with forskolin (10 microM) increased the transcriptional activity of only the 3.7 kbp fragment. The effects were antagonized by Rp-cAMPS, a specific antagonist of protein kinase A, indicating cAMP-dependent effects. The results provide evidence for cAMP-dependent regulation of the protein phosphatase 1alpha promotor.

Animals↗
PubMed2001

Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides.

The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the alpha carbon atom. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay.

Citrulline↗
PubMed2001

Environmental effects on the functions of the stratum corneum.

The stratum corneum (SC) is such an efficient barrier that only 2-5 g per h per cm2 of transepidermal water loss (TEWL) occurs in normal skin. The SC also plays another important role at the skin surface in keeping our skin smooth and flexible by binding water. We exposed a simulation model of in vivo SC to various, excessive physical insults in vitro, such as irradiation with 1 J per cm2 of UVB, 50 J per cm2 of UVA, or 3000 rad of X-ray, heating at 90 degrees C for 3 min, freezing at -196 degrees C for 60 s or repeated placement in an extremely dry or humid condition. None of them could cause any permanent change in the SC functions. Only the application of chemical agents such as lipid solvents or a detergent or the affliction of trauma resulted in a functional derangement of the SC. Because the viable skin tissues are more vulnerable to the effects of the environment than the SC, most of the abnormalities of the SC functions developing after environmental insults are secondarily caused by enhanced epidermal proliferation induced under the influence of underlying inflammation. These functional abnormalities were found to be demonstrable with biophysical measurements long after the disappearance of skin redness, the clinically observable sign of inflammation. The SC abnormalities in inflamed skin are also detectable as a change in the content of chemical mediators. For example, the ratio between proinflammatory IL-1 and its receptor antagonist (IL-1ra) whose production by epidermal keratinocytes is markedly enhanced by various proinflammatory stimuli, showed a deviation towards an excess of the latter in inflammatory skin. Facial skin that is always exposed to the environment is unique in that its SC shows such a deviation in the IL-1/IL-1ra ratio suggestive for the presence of mild inflammation even in normal individuals.

Dermatitis↗
PubMed2001

Patterns of basal cell keratin 14 expression in Bowen's disease: a possible marker for tumour progression.

BACKGROUND: Bowen's disease is a well-established in situ malignancy of the epidermis. The keratin expression in Bowen's disease has been studied in many reports. However, the patterns of keratin (K) 14 expression in each case have not been closely examined. OBJECTIVES: To investigate if the pattern of expression of K14 has a relationship with tumour progression, we analysed the expression patterns of K14 in relation to the nature of tumour cells, comparing tumour cells in direct contact with the dermis, tumour cells separated from the dermis, and tumour cells invading into the dermis. METHODS: Twenty-seven tissue sections from 22 patients were stained with anti-K14 antibody, as well as with antilaminin and periodic acid-Schiff (PAS) staining to evaluate the conditions of the basement membrane. Staining patterns of K10 and integrin beta1, and their relationships with proliferating cell nuclear antigen (PCNA) and Ki-67 staining patterns, were also examined. RESULTS: Tumour cells with no, or with obscured, basement membranes always showed positive staining for K14, while those with continuous (intact) basement membranes usually did not. Of 10 sections showing dermal involvement of Bowen's disease, five were K14 positive and five were K14 negative. All of these K14-positive sections with dermal involvement showed negative or obscured laminin and PAS staining. Most of the sections having K14-negative tumour cells with dermal involvement showed K14-positive lining cells with continuous staining with laminin and PAS-positive basement membranes. K10 was reciprocally expressed with K14 in most of the sections. Integrin beta1 was expressed in the basal layers of non-tumour epidermal cells, but not in tumour cells. Ki-67 and PCNA were expressed at high frequencies in tumour cells, clearly demarcating tumour cells from non-tumour cells. CONCLUSIONS: Tumour cells separated from the dermis by lining cells were K14 negative with PAS- and laminin-positive basement membranes around them; tumour cells without lining cells were K14 positive with or without continuous basement membranes. K14 expression may be a marker of tumour progression in Bowen's disease.

Basement Membrane↗
PubMed2001

A novel heat stress-responsive gene in the marine diatom Chaetoceros compressum encoding two types of transcripts, a trypsin-like protease and its related protein, by alternative RNA splicing.

We investigated the change of mRNA expression patterns in the laboratory-grown diatom Chaetoceros compressum under heat-stress conditions by mRNA arbitrarily primed (RAP) RT-PCR. Cells grown at 20 degrees C were subjected to heat treatment at 30 degrees C for 15 min and subsequently maintained at 20 degrees C for 8 h. Four genes including HI-5 were detected as heat stress-responsive genes by fingerprint analysis of RAP RT-PCR. Cloning for full-length cDNA sequences of HI-5 transcripts and related genomic DNA analysis revealed that two types of mRNA, HI-5a and HI-5b, were transcribed from the single HI-5 gene. While the HI-5a protein contained a catalytic domain characteristic to trypsin-like proteases, the HI-5b protein lacked this domain due to an insertion in the associated mRNA of 112 nucleotides; this insertion sequence contained a stop codon near the central region. Quantitative RT-PCR was performed to investigate the changes in expression levels of the two types of mRNA following heat treatment. The HI-5b transcripts were constitutively expressed in both unstressed and heat-stressed cells. In contrast, the number of HI-5a transcripts markedly increased in cells immediately after heat stress, reaching levels 19-fold higher at 8 h after heat stress than that in unstressed cells. These results suggest that RNA splicing plays a key role in heat stress-dependent expression of the HI-5a and HI-5b transcripts from the single HI-5 gene in the diatom.

Alternative Splicing↗
PubMed2001

beta-Catenin gene mutation in human hair follicle-related tumors.

beta-Catenin, a multifunctional protein related to the adherens junction and to signal transduction, is a key molecule of cell proliferation, and it is central to epithelial architecture, regulating the polarity of cells and tissues. beta-Catenin stabilization may play a key role in epidermal signaling leading to hair development, and its aberrant activation may be implicated in formation of hair tumors. Several investigators have shown that pilomatricomas are frequently associated with beta-catenin mutation. In this study, we confirmed beta-catenin gene (CTNNB1) mutation in human pilomatricomas (100% frequency) from which adequate DNA could be obtained for gene analysis. A novel mutation, D32N, was found in one case of pilomatricoma. A preliminary immunohistological study revealed prominent beta-catenin staining in basophilic cells of pilomatricomas, especially in nuclei. Benign tumors which were considered to be derived from hair matrix or hair follicles, and other benign skin tumors, were also investigated. beta-Catenin mutations were not detected in any of the these tumors. These results seem to indicate that hair matrix cells are key players in hair development. Investigation into gene abnormalities of hair-follicle tumors may elucidate the cause of their neoplastic transformation, and may provide a suggestion for the mechanism of hair development.

Adolescent↗
PubMed2001

Regulatory effects of senescence marker protein 30 on the proliferation of hepatocytes.

Senescence marker protein 30 (SMP 30) is preferentially expressed in the liver. One of its remarkable functions is the protection of cells against various injuries by enhancement of membrane calcium-pump activity. We analyzed the role of SMP 30 in hepatocyte proliferation. SMP 30 expression was decreased initially, then increased along with hepatic regeneration, after carbon tetrachloride (CCl4) administration. SMP 30 expression was decreased in the necrotic phase and then gradually increased. Its increase was slightly delayed just after the mitotic phase. These results lead us to speculate that mitoses of hepatic cells induce enhanced SMP 30 expression. In contrast, administration of lead nitrate (LN) as a hepatic mitogen induced a more stable increase of SMP 30 expression. To estimate the effect of SMP 30 on cell proliferation, we evaluated hepatic mitosis in wild-type and SMP 30-deficient knockout (KO) mice after CCl4 administration. We found an increase in mitotic numbers in hepatocytes of KO mice. This result suggests that SMP 30 has a suppressive effect on cell proliferation. Suppressive activity of SMP 30 cDNA was shown in cultured hepatoblastic cells. Our results suggest that SMP 30 performs a regulatory function in liver regeneration.

Animals↗
PubMed2001

Hypoxic ventilatory depression in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes.

We describe a case of a 21-year-old man with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) who presented with hypoxic ventilatory depression. He had chronic hypoventilation, which was not explained by weakness of respiratory muscles. His hypercapnic ventilatory response was not impaired. In contrast, hypoxic ventilatory depression was observed in the isocapnic progressive hypoxic response test. After exposure to hypoxic conditions, his respiratory frequency decreased and tidal volume was unchanged. The hypoxic ventilatory depression was partially blocked by pretreatment with aminophylline. In conclusion, we need to be careful with patients with MELAS who are hypoxaemic because a vicious circle of hypoxia and hypoventilation can occur.

Adenosine↗
PubMed2001

Twenty-seven-year follow-up of arrhythmogenic right ventricular dysplasia.

This case report describes clinical features, especially of surface ECG changes, observed for 27 years in a patient with arrhythmogenic right ventricular dysplasia (ARVD). The course of this patient was characterized by progressive deterioration of right ventricular function and progression of delayed potentials (so-called epsilon waves) following QRS complexes. However, the relation between ventricular arrhythmias and ECG changes or the degree of right ventricular abnormality was difficult to discern.

Adult↗