Phase Transition and Phase Segregation in a Single Double-Stranded DNA Molecule.
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Journal Article↗
Biomedical subjects
K Yoshikawa
Publications and source records attributed to K Yoshikawa.
Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.
Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.
Cytosol↗
Association between genetic variants of mast-cell chymase and eczema.
BACKGROUND: Atopy is a common syndrome underlying asthma, rhinitis, and eczema, and is characterised by high immunoglobulin E (IgE) responses to common antigens. IgE and mast-cell chymase (MCC-a serine protease secreted by skin mast cells) have a key role in atopic or allergic inflammation of the skin. The gene for MCC is located within a cluster of genes for cellular proteases on chromosome 14q11.2. We aimed to identify variants of MCC and another gene within this complex, and assess whether there is a genetic association between variants of MCC and atopic disorders-particularly eczema. METHODS: We randomly selected 100 controls and recruited patients-100 in each group-with atopic asthma, non-atopic asthma, atopic rhinitis, and atopic eczema. PCR amplification was used to test genomic DNA for an association between allelic polymorphisms in MCC and a flanking gene (CGL1, for the cathepsin-G-like protein) on chromosome 14q11 and asthma, rhinitis, and eczema. FINDINGS: We found a significant association between a BstXI polymorphism in MCC and eczema (odds ratio 2.17 [95% CI 1.21-3.88], p = 0.009), but no association with atopic asthma, rhinitis, or non-atopic asthma. There was no association between an Mboll polymorphism in CGL1 and any of the atopic disorders. INTERPRETATION: These findings suggest that variants of MCC may be one source of genetic risk for eczema.
Adolescent↗
Formation of nucleation center in single double-stranded DNA chain.
The dynamic process of compaction of long double-stranded DNA, T4DNA (166 kbp), was studied by the observation for individual DNA using fluorescence microscopy. We have observed the process of folding of metastable elongated DNA into compacted form in polyethylene glycol solution. During the thermal Brownian motion, the transition from the unfolded state into the compacted state is initiated by the formation of a nucleation center in the DNA chain. This center then grows along the DNA chain until the entire individual DNA takes the compacted state. The greatest probability for the formation of a nucleation center is at the ends of the DNA chain. In addition to the ends, nucleation is observed only in the region containing the positions with relatively high GC content region along the DNA chain.
Bacteriophage T4↗
Functional magnetic resonance imaging (fMRI) during mental writing with phonograms.
Functional magnetic resonance imaging (fMRI) at 1.5 T using a gradient echo echo-planar sequence was employed to identify brain regions activated during the performance of a mental writing task using phonograms. Four regions were activated in all six subjects; the region surrounding the left intraparietal sulcus, the region surrounding the middle part of the left precentral sulcus and the posterior part of the left superior frontal sulcus, the region surrounding the right intraparietal sulcus, and the region surrounding either or both of the left and right cingulate sulci. The left intraparietal region was usually the most extensively activated. The results suggest that these four regions particularly the left intraparietal region, are essential in writing with phonograms. Since the left hemisphere was more extensively activated than the right, fMRI during the mental writing task is a good candidate for determining non-invasively which hemisphere is dominant for language.
Adult↗
Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma.
Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is unique in its ability to promote vascular permeability and endothelial cell growth, and its role in tumor development has received considerable attention. In this report, we describe the elevation of VPF/VEGF transcript expression in human hepatocellular carcinoma. Surgical samples of 23 patients with hepatocellular carcinoma were studied using reverse transcription-PCR analysis. The oligonucleotide primers were designed to amplify all four known splicing variants that could be expressed in the samples studied. Sixteen cases showed VPF/VEGF transcript expression in the tumor (16/23, 69.6%), whereas only 9 of the 23 patients showed it in the corresponding nontumoral part. There was no difference between the pattern of expression of VPF/VEGF isoforms in tumoral and nontumoral tissues. VPF/VEGF mRNA expression in the liver tumors was associated with fibrous capsule formation and septal formation (P < 0.05 respectively, Fisher's exact P test). In situ hybridization confirmed the presence of VPF/VEGF mRNA expression in tumor cells and less intensely in hepatocytes of nontumoral liver. We also found that VPF/VEGF expression in the tumor cell was increased in the area adjacent to necrotic regions (presumably hypoxic regions). As a regulator of vascular permeability and endothelial cell growth factor, VPF/VEGF may play an important role in the development of hepatocellular carcinoma.
Adult↗
High-performance liquid chromatographic determination of lipoamidase (lipoyl-X hydrolase) activity with a novel substrate, lipoyl-6-aminoquinoline.
An HPLC lipoamidase (lipoyl-X hydrolase) assay method has been developed, which uses a novel fluorescent substrate, lipoyl-6-aminoquinoline (LAQ). LAQ is synthesized from lipoic acid and 6-aminoquinoline (AQ) through lipoyl chloride as an intermediate and is conveniently purified by washing with chloroform-methanol. Mechanistic studies on the time-course, the dependence on enzyme and substrate concentrations were performed by using LAQ and a model enzyme (milk lipoamidase). Moreover, this method was successfully applied to the direct determination of the lipoamidase (LAQ hydrolase) activity in samples of human liver, milk, stools and porcine serum. Using this novel synthetic lipoyl substrate, we demonstrated that LAQ hydrolase was present in some specific tissues; LAQ hydrolase was solely present in the grey matter and not in the white matter in the human cerebrum. Furthermore, LAQ hydrolase activity was shown to increase in human liver cancer. Thus, this enzyme assay method is expected to be applicable to the tissue distribution study and also to the basic research on human diseases such as cancer.
4-Aminobenzoic Acid↗
Large discrete transition in a single DNA molecule appears continuous in the ensemble.
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Journal Article↗
Structure and expression of the mouse necdin gene. Identification of a postmitotic neuron-restrictive core promoter.
Necdin is a 325 amino acid residue protein encoded by a cDNA clone isolated from neurally differentiated embryonal carcinoma cells. In situ hybridization histochemistry revealed that necdin mRNA-containing cells in vivo coincided with postmitotic neurons in the mouse brain from early periods of neurogenesis until adulthood. To study the regulation of necdin gene expression, we have isolated and characterized the necdin gene from a mouse genomic DNA library. The necdin gene contains no intron, and its upstream region lacks canonical TATA and CAAT boxes. To assess promoter activity, the 5'-flanking sequence (844 base pairs) of the necdin gene was fused to the LacZ reporter gene and transiently transfected into retinoic acid-treated P19 embryonal carcinoma cells. Most of the transfectants expressing high levels of LacZ immunoreactivity were postmitotic neurons differentiated from P19 cells. Deletion analysis using luciferase reporter genes demonstrated that a neuron-restrictive core promoter is localized to positions -80 to -35, in which a G+C-rich domain and a putative binding site for transcription factors with PAS (per, arnt, and single-minded) dimerization domain are comprised. These results suggest that postmitotic neuron-restrictive expression of the necdin gene is mediated by the specific cis-acting elements and that this promoter is applicable to postmitotic neuron-targeted expression of various transgenic systems.
Amino Acid Sequence↗
Conformational dynamics of DNA during biased sinusoidal field gel electrophoresis.
DNA motion during biased sinusoidal field gel electrophoresis around the antiresonance condition was investigated by direct observation using fluorescent microscopy and Brownian dynamics simulation. Time development of the center of mass velocity, vx, and the principal value of the gyration tensor, R1, was measured at this antiresonance condition. The typical stretch-contract motion, which is observed in steady field and at high frequency field, is severely suppressed, and there are two or more dominant kinks that compete with each other. Analysis of the kink motion from the simulation results supports this picture.
Bacteriophage T4↗
Characteristics of endogenous uveitis in Hokkaido, Japan.
BACKGROUND: Etiological characteristics of endogenous uveitis vary around the world. There are few epidemiological reports on the etiology of uveitis from areas within Asia. We set out to examine the statistical data on uveitis in Japan. METHODS: We reviewed all the records of patients with endogenous uveitis who visited the Uveitis Survey Clinic of Hokkaido University Hospital in 1981 and 1994 and extended the survey to include new patients with uveitis seen over the past 3 years. RESULTS: Behçet's disease, sarcoidosis and Vogt-Koyanagi-Harada disease were the three most frequently diagnosed diseases in patients with endogenous uveitis in both 1981 and and 1994. The proportion of patients with unclassified disease entities decreased (from 38% to 30%) during the 13-year period from 1981 to 1994 as a result of the new disease categories established during this interval. Notable additions included human T-lymphotropic virus type 1-associated uveitis and tubulointerstitial nephritis and uveitis syndrome. Sarcoidosis is now the most frequent cause of endogenous uveitis in our clinic. CONCLUSION: Not only does the etiological basis of uveitis vary with ethnicity, but advances in clinical and basic research have changed the approach to the diagnosis of uveitis, altering the etiological profile over time.
Adolescent↗
Kaposi's sarcoma-associated herpesvirus (KSHV) sequences in premalignant and malignant skin tumors.
The novel herpesvirus-like DNA sequences, which were identified in AIDS-related Kaposi's sarcoma (KS) and designated KS associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), have been reported to be associated with various forms of KS. Here, we searched for the presence of KSHV sequences in various other skin lesions including premalignant or malignant skin tumors of a total of 69 clinical cases without immunodeficiency due to AIDS or following organ transplantation. Strikingly high rates of detection were obtained for premalignant Bowen's disease and malignant squamous cell carcinoma, accounting for 71.4% and 50%, respectively. A less frequent but as yet high incidence (33.3%) was scored for actinic keratosis, a premalignant epidermal disorder. In contrast, the frequency remained low (16.7%) for another type of proliferative skin lesions of extramammary Paget's disease and non-proliferative skin lesions (dermatitis, morphea, epidermal cyst and scar). These results suggest a close association of KSHV with at least some non-KS malignant and premalignant skin lesions in non-immunocompromised patients.
Acquired Immunodeficiency Syndrome↗
Differential localization of TGF-beta-precursor isotypes in psoriatic human skin.
Transforming growth factor-beta (TGF-beta) can act as a multi-functional regulator of both cell growth and differentiation. Three isoforms of TGF-betas, namely TGF-beta 1 TGF-beta 2 and TGF-beta 3, have been identified in human tissues. Previously we reported the expression of TGF-beta isoforms in normal human skin. However little is known about the role of TGF-beta isoforms in the pathogenesis of psoriasis. Using the TGF-beta precursor-specific antibodies to strengthen the specificity, we studied the immunohistochemical distribution of TGF-betas 1-3 in psoriatic skin. TGF-beta 2, which was found in the intercellular space of all the layers of the epidermis in normal human skin, was decreased in the psoriatic epidermis. The intensity of immunoreactivity has the tendency to decrease in the lower epidermis rather than in the upper epidermis of the transitional lesion. In contrast, TGF-beta 3 was present in the subepidermal area of the psoriatic skin as in the normal human skin. TGF-beta 1 was observed in neither epidermis nor dermis in both normal and psoriatic skin. Since TGF-beta is a potent growth inhibitor for human keratinocytes, the decrease of TGF-beta 2 in the epidermis of psoriatic skin may contribute to epidermal hyperplasia, a hallmark of psoriasis.
Humans↗
The folded state of long duplex-DNA chain reflects its solution history.
The higher-order structure of compacted single giant DNA induced by complexation with polypeptide (poly-Arg) in NaCl solution was investigated using fluorescence microscopy. As the poly-Arg concentration increased, the mean size of extended DNA chains gradually decreased. In the presence of excess poly-Arg, individual DNA chains collapsed into compact globules, and the degree of collapse of the DNA chains depended not only on the concentration of poly-Arg, but also on the time course of the addition of poly-Arg and NaCl, indicating that the structure of the collapsed DNA is not determined simply according to the minimum free energy. We discuss theoretically the presence of multiple-stationary states based on a consideration of simple kinetics in the process of binding. Depending on the past history, the number of poly-Arg and Na+ that bind to each DNA changes markedly. This interesting characteristic of long DNA is discussed in relation to the possible mechanism of self-regulation of gene expression in living cells.
Bacteriophage T4↗
Contrast agent for magnetic resonance angiography: experimental evaluation of superparamagnetic iron oxide composed of polysaccharide complex involving chondroitinsulfuric acid.
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Animals↗
Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor.
A mouse monoclonal antibody (IgG2b), 3C10, was produced against the truncated epidermal growth factor receptor (EGFR), encoded by the (type III) in-frame deletion mutation of 801 nucleotides of EGFR affecting the external domain, known to be expressed in some human glioblastoma. As this mutation newly generates a glycine residue at the fusion point, a 14 amino acid peptide around the fusion junction including this glycine was chemically synthesised and used for immunisation of (B6 x DBA/2) F1 mice. Flow cytometric analysis showed 3C10 antibody staining of a mouse NIH/3T3 transfectant (ERM5) with the type III EGFR deletion-mutant gene, but not one with wild-type EGFR. The antibody immunoprecipitated the truncated EGFR protein with a molecular mass of approximately 140 kDa from ERM5 cells. Immunostaining of glioblastomas revealed binding in the case with the type III EGFR mutation, the five other specimens without the mutation being negative despite overexpression of EGFR in some cases.
3T3 Cells↗
Total and segmental subcutaneous adipose tissue volume measured by ultrasound.
Total and segmental subcutaneous adipose tissue volumes (SATV) were estimated and compared in 13 healthy young women with a mean age of 21.4 yr. Total and segmental (head and neck, forearm, upper arm, trunk, thigh, and lower leg) SATV were estimated from subcutaneous adipose tissue layer thickness and the body surface area; the former by B-mode ultrasound and the latter by direct measurement. Consecutive magnetic resonance images (MRI) were obtained from head to toe (10 mm thickness), from which adipose tissue areas as well as total and regional SATV were determined. SATV of the upper arm and thigh estimated by ultrasound was significantly lower compared to that of MRI-measured SATV; however, there were no significant differences found in the other four segments. As a result, total estimated SATV obtained was on average 5.5% lower with ultrasound than with MRI-measured total SATV; however, there were no statistically significant differences between the two values. Significant strong correlations were observed between MRI-measured total and segmental SATV, and SATV estimated by ultrasound (r = 0.79-0.95) for all segments except head and neck. These results show the effectiveness of the ultrasound method for estimating SATV; however, the quantitative analysis tissue volumes would require correction.
Adipose Tissue↗
Successful treatment of molluscum contagiosum in the immunosuppressed adult with topical injection of streptococcal preparation OK-432.
A 37-year-old Japanese woman received an anticancer drug for 5 years following resection of mammary cancer and then developed widespread mollusca contagiosa, which we considered to be caused by immunosuppression induced by the chemotherapy. Because OK-432 (penicillin-treated and heat-treated lyophilized powder by a substrain of Streptococcus pyogenes A) was expected to be effective for immunosuppression, we tried its topical injection. The skin lesions disappeared almost completely within three months. OK-432 therapy is considered hopeful for treating viral skin diseases, even in immunosuppressed patients.
Adult↗