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Biomedical subjects

M Mukai

Publications and source records attributed to M Mukai.

At least 91 records · Page 5Linked to original sources
PubMed2000

Evaluation of a surgical gamma probe for detection of 18F-FDG.

Because fluorine-18 fluorodeoxyglucose (18F-FDG) is an excellent tumor-localizing radio-pharmaceutical, a hand-held radiation detection probe capable of localizing an area with high 18F-FDG uptake would make radioguided surgery possible. In this laboratory study, we investigated the capability of a widely used intraoperative gamma probe with a cadmium zinc telluride (CdZnTe) detector for detection of 18F-FDG. For sensitivity tests, an 0.1- ml 18F-FDG preparation was made to act as a point source with radioactivities of 1.0, 2.0, and 3.0micro Ci (37 kBq, 74 kBq, 111 kBq). Relative transmission across the side wall of the probe and sensitivity at each source-to-probe distance were measured. For simulation studies, 2 l of 18F-FDG solution (0.02micro Ci, 0.74 kBq/ml) served as normal background. One ml of 18F-FDG was prepared to simulate tumors with radioactivities of 0.05, 0.1, 0.2, and 0.4micro Ci (1.85 kBq, 3.7 kBq, 17.4 kBq, and 14.8 kBq). The ratios of the radioactive concentration of tumor to that of the background were 2.5, 5, 10, and 20:1, respectively. The tested gamma probe was shown to be sensitive to 18F. The high-energy annihilation radiation was detected from the side wall of the probe despite application of a supplementary collimator. The count rate decreased markedly as the source-to-probe distance increased, owing to the effects of the inverse-square law. In the simulation studies, the probe detected a considerable amount of background activity. However, the measured count rate increased with the increasing source-to-background ratio. In our setting, the probe was capable of distinguishing the 18F-FDG source from the background when the source-to-background ratio was no less than 5:1. To make a surgical application feasible, however, collimation or shielding against high background radiation is necessary.

Fluorodeoxyglucose F18↗
PubMed1999

Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance raman spectroscopy.

FixL is a heme-based O(2) sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium meliloti FixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe(2+), Fe(2+)O(2), Fe(2+)CO, Fe(3+), Fe(3+)F(-), and Fe(3+)CN(-) states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain of Bradyrhizobium japonicum FixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15177-15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O(2) sensing.

Bacterial Proteins↗
PubMed1999

Inhibition of tumor invasion and metastasis by a novel lysophosphatidic acid (cyclic LPA).

Fetal calf serum (FCS) and 1-oleoyl lysophosphatidic acid (LPA) were previously found to be potent inducers of invasion (transcellular migration) in an in vitro system. A novel LPA, composed of cyclic phosphate and cyclopropane-containing hexadecanoic acid (PHYLPA), first isolated from myxoamoebae of Physarum polycephalum, and its synthetic derivatives (cLPA) were tested for their ability to inhibit tumor cell invasion and metastasis. Amoung these, Pal-cLPA, which has a palmitoyl moiety, was most potent in inhibiting invasion, with 93.8% inhibition at the concentration of 25 microM. Invasion in vitro by mouse melanoma cells (B16), human pancreatic adenocarcinoma cells (PSN-1), human lung cancer cells (OC-10) and human fibrosarcoma cells (HT-1080) was also inhibited by Pal-cLPA. The stimulation of MMI cells with LPA triggered F-actin formation, which was impaired by the addition of Pal-cLPA at invasion-inhibitory concentration. Pal-cLPA induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP) concentration in MMI cells. The addition of dibutyryl cAMP significantly abrogated LPA-induced invasion by MM1 cells and actin polymerization in the cells. The inhibition of MM1 cell invasion by Pal-cLPA may be ascribed to an increased level of cAMP. Pal-cLPA also suppressed invasion in vitro by MM1 cells induced by FCS dose dependently, without affecting proliferation. It also suppressed the pulmonary metastasis of B16 mouse melanoma cells injected into the tail vein of C57BL/6 mice. Thus, Pal-cLPA is effective in inhibiting invasion and metastasis of a variety of tumor cells.

Actins↗
PubMed1999

Design and synthesis of a globin fold.

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.

Amino Acid Sequence↗
PubMed1999

Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity.

The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.

Amino Acid Sequence↗
PubMed1999

Adsorption of Bovine Serum Albumin and Lysozyme on Hydrophobic Calcium Hydroxyapatites.

The adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) to oleyl phosphate(OP)-grafted calcium hydroxyapatite (OP-CaHAP) with different degrees of hydrophobicity, ranging the number of surface oleyl group per unit nm2 (nO) from 0 to 2.60, was investigated. The pronounced effects of the hydrophobic moiety of adsorbent on protein adsorption were observed. The saturated amount of adsorbed BSA (ns) was increased up to nO = 0.6 by an enlargement of hydrophobic interaction between hydrophobic CaHAP particle and proteins. However, ns decreased at nO >/= 1.3 by increasing the electrostatic repulsive force between negatively charged BSA and OP-CaHAP particles. On the other hand, the ns value of LSZ was continuously increased up to nO = 2.0 and saturated by increasing either the hydrophobic interaction or the electrostatic attraction of positively charged LSZ and negatively charged OP-grafted CaHAPs. The BSA adsorption experiment revealed that the effect of positively charged adsorption sites on the exposed ac or bc crystal faces (C-sites) of the CaHAPs is screened by the OP-groups grafted on their particle surfaces. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

A synthetic mimetic of CD4 is able to suppress disease in a rodent model of immune colitis.

CD4+ mucosal T cells mediate the intestinal inflammation in Crohn's disease and may serve as an important target for immune intervention. Here we assessed the therapeutic effect of a synthetic mimetic of CD4 designed to mimic both the sequence and conformation of the complementarity-determining region 3 of murine CD4 V1 domain (rD-mPGPtide) in a mouse colitis model using immunization with 2,4,6-trinitrobenzene sulfonic acid (TNB). i.v. administration of the rD-mPGPtide but not control scrambled peptide could suppress severe inflammation in the chronic colitis mouse model. After treatment with the rD-mPGPtide, a striking improvement of diarrhea and acute wasting disease was observed with decreased mortality. Serum anti-TNB antibody titers, CD45RBlowCD4+ T cells in the lamina propria and IFN-gamma mRNA expression in the mucosa were significantly decreased with the rD-mPGPtide treatment. Anti-CD4 antibody also suppressed disease by depletion of CD45RBhighCD4+ T cells in the colonic mucosa. The observation that the synthetically engineered analogue of murine CD4 inhibits inflammation in a rodent disease model by different mechanisms than anti-CD4 antibody suggests that a human version of this peptide has potential therapeutic utility in CD4+ mucosal T cell-mediated intestinal inflammation in Crohn's disease.

Animals↗
PubMed1999

Near-Infrared Diode-Laser Spectroscopy of the CCO Radical in the Ã(010)3Delta-;X(010)3Pi(b) Band.

The Ã(010)3Delta-;X(010)3Pi(b) band spectrum of the CCO radical has been studied in the region from 11,808 to 11,919 cm-1 by near-infrared diode-laser spectroscopy. A least-squares fit has been made using only part of the observed Ã(010)3Delta-;X(010)3Pi(b) band transitions to obtain molecular constants for the Ã(010)3Delta state, removing transitions with upper levels of irregular behavior. The spin-orbit parameter and the subband origin determined in the present analysis have been discussed in relation to higher order contributions to the Renner-Teller effect. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Peritoneal recurrence of colon cancer detected by positron emission tomography: report of a case.

Increased glucose metabolism has been reported to occur in association with colorectal cancer. As positron emission tomography (PET) using [18F]fluorodeoxyglucose is able to depict hypermetabolic sites, it can therefore be used to detect colorectal cancer. A 69-year-old male patient with a recurrent solitary liver metastasis from colon cancer underwent whole-body PET which revealed high [18F]fluorodeoxyglucose uptake in the lesion. Furthermore, PET revealed peritoneal metastases that had not been detected by conventional imaging methods. Consequently, PET proved useful in helping us to avoid performing unnecessary treatment for the liver metastasis. Although it is uncertain whether early identification of recurrence can prolong survival, it may help to prevent unnecessary treatments being carried out. Thus, the application of PET in carefully selected patients could be beneficial to the management of recurrent colorectal cancer.

Aged↗
PubMed1999

Involvement of small GTPases Rho and Rac in the invasion of rat ascites hepatoma cells.

Lysophosphatidic acid (LPA) triggers the invasion of a mesothelial cell monolayer by rat ascites hepatoma (MM1) cells. LPA also induces rapid morphological changes of MM1 cells, cell surface blebbing and pseudopodia formation. Pseudopodia formation is tightly correlated with cellular invasiveness. Clostridium Botulinum C3 exoenzyme and genistein abrogated the formation of blebs and pseudopodia together with the inhibition of invasion, indicating that GTPase Rho and certain tyrosine kinases are involved in both processes. MM1 cells expressing constitutively active Rho exhibited the invasion and the formation of blebs and pseudopodia in the absence of LPA. In contrast, MM1 cells expressing constitutively active Rac were not invasive in the absence of LPA, but were invasive in the presence of LPA. Their morphological response to LPA was almost the same as that of parental MM1 cells. Expression of dominant negative Rac suppressed the invasiveness to approximately 3% of that of parental MM1 cells, together with the inhibition of pseudopodia formation. Thus, Rho and Rac are cooperatively involved in both the invasion and the related morphological changes of MM1 cells. Rho activation is sufficient both for the induction of invasion and the morphological changes leading to the invasion, whereas Rac activation is necessary but not sufficient by itself. We propose that Rho activation is not mediated by Rac but the cooperation of both GTPases is essential to trigger the invasive behavior of MM1 cells.

Animals↗
PubMed1999

Phosphodiesterase type III inhibitor, cilostazol, inhibits colon cancer cell motility.

Metastasis of cancer cells is initiated by the cellular migration into extracellular matrix and surrounding vessels. We previously showed that elevation of cAMP levels in cancer cells suppressed trans-cellular migration in vitro. Drugs that can elevate cAMP levels in cancer cells effectively may be applied to prevent metastasis in cancer patients. Cilostazol, an oral anti-platelet drug, is a specific cAMP phosphodiesterase type III inhibitor and has been clinically used to treat thrombosis patients. In chemotaxis assay, cellular migration of human colon cancer cells, DLD- 1, was induced by 10 microg/ml of soluble fibronectin or 10% of fetal bovine serum (FBS). Treatment with cilostazol (50 microM) suppressed 92.3% or 84.6% of the migration in control cells, respectively. When DLD-1 cells were stimulated by soluble fibronectin in phagokinetic assay, migration assessed by the area of gold particle phagocytosis track was induced and cilostazol also decreased 67.3% of the cellular migration in control cells. Furthermore, in the trans-cellular migration assay, cilostazol suppressed cancer cell invasion induced by FBS. Thus, cilostazol can suppress colon cancer cell motility and might be effective as an anti-metastasis drug for cancer patients.

Animals↗
PubMed1999

Clinicopathological study of pheochromocytoma of the urinary bladder: immunohistochemical, flow cytometric and ultrastructural findings with review of the literature.

Pheochromocytomas usually arise from the adrenal medulla but may also arise from the carotid body, the retroperitoneum, the urinary bladder, and other locations. We report three cases of pheochromocytoma of the urinary bladder with clinicopathological, immunohistochemical, flow cytometric, and ultrastructural findings. Case 1, a 13-year-old boy presented with hematuria. He underwent partial cystectomy, 31 years later he presented with a tumor in the thoracic vertebra. Case 2, a 35-year-old woman presented with headache, nausea, vomiting, palpitations, and diaphoresis on evacuation. She underwent total cystectomy and regional lymph adenectomy. She survived for 10 years without recurrence or metastasis; however, she died from another disease. Case 3, a 31-year-old man presented with dysuria. He underwent total cystectomy and regional lymph adenectomy. The tumor metastasized to the lymph nodes, and the patient died after 4 years. The urinary bladder tumors in these three cases protruded into the lumen and invaded deeper than the middle of the muscle layer. The tumor of the urinary bladder, metastatic lymph nodes, and thoracic vertebra showed alveolar and trabecular patterns, and tumor cells were surrounded by capillaries. The tumor cells were moderate in size with ovoid nuclei and abundant eosinophilic cytoplasm that contained acidophilic granules reactive to Grimelius stain. Vascular invasion was observed in cases 1 and 2. Immunohistochemically, tumor cells showed reactivity for chromogranin, Leu 7, and S-100 protein. In each of the three cases, the DNA ploidy pattern on flow cytometry was aneuploid. Ultrastructural examination revealed several neurosecretory granules, rough endoplasmic reticulum, and a few mitochondria within the cytoplasm. It is difficult to determine whether pheochromocytoma of the urinary bladder is malignant on the basis of histological, immunohistochemical, and flow cytometric findings. Long-term follow up is necessary.

Adolescent↗
PubMed1999

The putative mechanism of thrombosis in antiphospholipid syndrome: impairment of the protein C and the fibrinolytic systems by monoclonal anticardiolipin antibodies.

The mechanism of thrombosis in patients with antiphospholipid syndrome is not clear. To investigate it, we examined the effect of monoclonal anticardiolipin (aCL) antibodies and beta2-glycoprotein I (beta2-GPI), which is required for formation of the aCL epitopes, on activated protein C (APC) and on fibrinolytic activity. First, APC activities were measured in the presence and absence of beta2-GPI or gamma M immunoglobulin (IgM) monoclonal aCLs (EY1C8 and EY2C9), or both, established from peripheral blood lymphocytes obtained from a patient with aCL. beta2-GPI exhibited a procoagulant activity by inhibiting APC activity as well as an anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL, and then only in the presence of beta2-GPI. The remaining tissue plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, and plasminogen activator inhibitor (PAI)-1 was measured by a chromogenic assay using the synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. beta2-GPI protected t-PA activity from inhibition by PAI-1. However, monoclonal aCLs (EY1C8 and EY2C9) inhibited the effect of beta2-GPI on fibrinolytic activity; that is, monoclonal aCLs inhibited fibrinolytic activity by elevating PAI-1 activity. Thrombosis in patients with aCL can be explained in part by both the inhibition of APC anticoagulant activity and the impairment of fibrinolytic activity by aCL.

Antibodies, Anticardiolipin↗
PubMed1999

Induction of indora expression in pole cells by the mesoderm is required for female germ-line development in Drosophila melanogaster.

In many animal groups, the interaction between germ and somatic line is required for germ-line development. In Drosophila, the germ-line precursors (pole cells) formed at the posterior tip of the embryos migrate toward the mesodermal layer where they adhere to the dorsolateral mesoderm, which ensheaths the pole cells to form the embryonic gonads. These mesodermal cells may control the expression of genes that function in pole cells for their development into germ cells. However, such downstream genes have not been isolated. In this study, we identify a novel transcript, indora (idr), which is expressed only in pole cells within the gonads. Reduction of idr transcripts by an antisense idr expression caused the failure of pole cells to produce functional germ cells in females. Furthermore, we demonstrate that idr expression depends on the presence of the dorsolateral mesoderm, but it does not necessarily require its specification as the gonadal mesoderm. Our findings suggest that the induction of idr in pole cells by the mesodermal cells is required for germ-line development.

Amino Acid Sequence↗
PubMed1999

Duration of antibodies against 24 kd protein of Rhipicephalus sanguineus extract in dogs infested with the adult ticks.

A 24 kd protein from Rhipicephalus sanguineus (Rs24p) which was common to larvae, nymphs, male and female whole body and salivary gland extract of males and female was detected specifically in the serum from dogs after repeated infestation with adult R. sanguineus. The duration of antibodies against Rs24p in dogs infested with adults was examined by Western blotting analysis. Anti-Rs24p antibody was detected in two of 4 dogs during the period of 40 days in the first infestation. In the second infestation, all dogs showed positive reaction against Rs24p, but the duration of the antibodies varied greatly among the animals.

Animals↗