Biomedical subjects
Publications and source records attributed to M Tsujimoto.
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The gene encoding dipeptidase was cloned from Acinetobacter calcoaceticus ATC 23055. Determination of the nucleotide sequence revealed that the gene had an open reading flame of 1,050 bp coding a protein of 350 amino acids. The deduced amino acid sequence showed 48.8% similarity to human renal dipeptidases and conserved two amino acid residues identified in human and pig renal dipeptidases as essential ones for the catalytic activity. Purified recombinant enzyme expressed in Escherichia coli did not hydrolyze the unsaturated dipeptide, glycyldehydrophenylalanine. On the other hand, it preferentially hydrolyzed dipeptides having a D-amino acid, when compared with those having an L-amino acid at the C-terminal. Furthermore, it could not hydrolyze tripeptides. These results indicate that the dipeptidase produced by A. calcoaceticus ATC 23055 has a unique substrate specificity and preferentially hydrolyzes dipeptides having a D-amino acid at the C-terminal.
We reported a 53-year-old female who was admitted due to partial loss of consciousness. She had been diagnosed as old pulmonary tuberculosis and diabetes mellitus. She was diagnosed as diabetic keto-acidosis on admission. We isolated Escherichia coli in the blood, transtracheal aspiration (TTA) and from the urine. We have experienced 6 cases where the same bacteria was isolated from the blood and TTA at the same time. In all 6 cases, we have found single bacteria in the blood and a few other bacteria in TTA. Blood culture is the most certain method to detect the origin of infectious diseases. But the compromised host, as in this case, has multifocal infections in many cases. In order to understand the pathological aspects of the infection, we must obtain many kinds of samples and as many as possible.
We measured the chemiluminescent activity (CL-index) in both the whole blood and isolated neutrophils from 12 patients with acute respiratory infection (7 cases; pneumonia, 4 cases; in the exacerbated phase, chronic lower tract infection, and one; acute bronchitis) two times per each case: before and after chemotherapy. Before the initiation of chemotherapy, neutrophil and whole blood CL was high but whole blood CL was higher. After the completion of chemotherapy, whole blood CL was decreased more significantly than neutrophil CL. There was no correlation between neutrophil CL and whole blood CL. However the neutrophil CL-index.N (neutrophil numbers x neutrophil CL-index) was correlated with the whole blood CL. Thus, we think the number of neutrophils is a critical factor for phagocytic function of neutrophils as determined by CL.
The concentrations of interleukin-5 (IL-5) in serum and in IL-2-stimulated lymphocyte culture medium (L-IL2-CM) prepared from patients with reactive eosinophilia were measured by enzyme-linked immunosorbent assay. The serum IL-5 concentration was increased in 16 of 34 cases. The concentrations of IL-5 in L-IL2-CM were increased in 8 of 21 patients.
We report a case of idiopathic brain stone occurring in the posterior fossa. A 72-year-old woman with a huge calcified mass in the posterior fossa on plain craniogram was referred to us. She had suffered from intractable tinnitus and mild cerebellar ataxia for twenty years. CT scan revealed a dense bony mass in the posterior fossa. Angiogram only showed an avascular huge mass in the posterior fossa. MRI clearly revealed the shape, heterogenous composition and anatomical relation to the surrounding structure of the mass. The mass was totally removed uneventfully under ABR and SEP monitoring. The patient was discharged with improvement of subjective symptoms. The mass was histologically diagnosed as an idiopathic brain stone because its components were mainly calcified tissues and collagen fiber of unknown etiology; no viable cells were found in any of the specimens. Idiopathic brain stones are very rare and only eight cases have been reported in the literature. The etiology and clinical symptoms of this condition are briefly discussed.
Six cases of pineocytoma, which had developed in the parenchyma of the adult pineal body, were examined immunohistochemically and under an electron microscope, after the malignancy of each case had been determined using our classification. One case was rated as grade 1 showing a lobular structure and resembling the normal pineal body. Two cases were rated as grade 2 without a lobular structure but with pineocytomatous rosettes (P-rosettes). Two cases were rated as grade 3 without P-rosettes but with few mitotic figures. One case was rated as grade 4 with marked cellular pleomorphism, numerous mitotic figures and necrotic foci. When examined immunohistochemically, neuron-specific enolase was positive but glial fibrillary acidic protein was negative in all cases. Under an electron microscope, all cases showed abortive synapses, and clear or dense core vesicles. These findings allow us to make two conclusions. First, pineocytoma is always a tumor of neuronal lineage, regardless of their grade of malignancy. Second, the grade 4 pineocytoma should be distinguished from the type of tumor classically called "pineoblastoma." That is, the former seems to be a biologically dedifferentiated tumor, while the latter seems to be biologically undifferentiated tumor.
A 69-year-old man was admitted because of sudden onset of consciousness disturbance. Neurological examination on admission revealed slightly disturbed consciousness, sensory aphasia and right hemiparesis. CT scan disclosed a hematoma in the left posterior temporal lobe. Left carotid angiograms showed dural AVF of the transverse/sigmoid sinus fed mainly by the left occipital and posterior auricular arteries. On the angiograms the left sigmoid sinus was completely occluded. This was associated with retrograde venous flow into the cortical veins, the superior petrosal sinus and the contralateral traverse sinus. After stabilizing the condition, we totally resected the dural AVF including the left transverse sinus. The postoperative course was uneventful. Histopathological examination of the surgically resected specimen revealed that the dural A-V fistula per se existed in the sinus wall.
Platelet-activating factor (PAF) acetylhydrolase is the key enzyme in PAF inactivation. We recently purified one isoform of the enzyme from a bovine brain soluble fraction and revealed it as a heterotrimeric enzyme consisting of 29-, 30-, and 45-kDa subunits. Among them, the 29-kDa subunit possesses an active serine residue since diisopropyl fluorophosphate (DFP), an inhibitor of the enzyme, labeled only this subunit (Hattori, M., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 18748-18753). In the current study, we cloned the cDNA for the 29-kDa catalytic subunit. The predicted sequence of 232 amino acids is unique and is not homologous with those of any other proteins reported so far. When transfected into either Escherichia coli or COS7 cells, the cDNA produced PAF acetylhydrolase activity in both types of cells, indicating that this subunit alone is enough for catalysis. The recombinant 29-kDa protein was also inhibited and labeled by DFP. Furthermore, we isolated and sequenced the [3H]DFP-labeled peptide fragment, revealing that Ser47 is the active serine residue. The sequence surrounding it is different from the consensus sequence of the serine esterase family. Interestingly, the sequence of about 30 amino acids located 6 residues downstream from the active serine site exhibits significant homology to the first transmembrane region of the PAF receptor. These data demonstrate that the catalytic subunit of brain PAF acetylhydrolase is a novel type of serine esterase.
Platelet-activating factor (PAF) is involved in a variety of biological and pathological processes and PAF acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is widely distributed in plasma and tissue cytosols. One isoform of PAF acetylhydrolase present in bovine brain cortex is a heterotrimer comprising subunits with relative molecular masses of 45K, 30K and 29K (ref. 4). We have now isolated the complementary DNA for the 45K subunit. Sequence analysis revealed a striking identity (99%) of the subunit with a protein encoded by the causative gene (LIS-1) for Miller-Dieker lissencephaly, a human brain malformation manifested by a smooth cerebral surface and abnormal neuronal migration. This indicates that the LIS-1 gene product is a human homologue of the 45K subunit of intracellular PAF acetylhydrolase. Our results raise the possibility that PAF and PAF acetylhydrolase are important in the formation of the brain cortex during differentiation and development.
A possible interaction between tumor necrosis factor-alpha (TNF) and other cytokines/growth factors in stimulating the production of nerve growth factor (NGF) in Swiss 3T3 cells was studied. TNF's stimulatory activity on fibroblast NGF production was synergized by interleukin-1 alpha (IL-1 alpha), IL-1 beta and interferon-gamma (IFN-gamma), but was antagonized by transforming growth factor-beta (TGF-beta). The most remarkable synergistic effect was observed between TNF and IL-1 alpha/beta; as little as 0.003 ng/ml of IL-1 beta markedly enhanced TNF's stimulatory activity on NGF production in the cells. These findings reinforce the idea that TNF, in concert with IL-1 alpha/beta, plays an essential role in regulating the regeneration of peripheral nerves following injury through an indirect mechanism by which it stimulates NGF production in fibroblasts.
Blood lymphocytes from patients with eosinophilia are known to produce interleukin-5 (IL-5) with appropriate stimulation in vitro. To determine whether blood lymphocytes from these patients produce IL-5 in vivo, we tested the IL-5 mRNA expression in blood lymphocytes immediately after separation by reverse-transcriptase polymerase chain reaction (RT-PCR) method. We found that lymphocytes from eosinophilic patients expressed IL-5 mRNA, but lymphocytes from normal volunteers did not express the lymphokine. These findings suggest that in patients with eosinophilia, peripheral blood lymphocytes produce IL-5 in vivo.
To study the role of IL-5 in allergic airway hyperreactivity, the time course for the production of cytokines, the infiltration of inflammatory cells and the onset of airway hyperreactivity after three inhalations of antigens were studied in mice. The effect of the soluble alpha-chain of murine recombinant interleukin-5 receptor (sIL-5R alpha) on these phenomena was also examined. Whereas IL-5 and IL-4 were produced in significant amounts, IL-1, IL-2 and gamma-interferon (gamma-IFN) were not detected even after three antigen inhalations. Monocytes and eosinophils but not neutrophils increased significantly after the third antigen exposure. The airway responsiveness to acetylcholine increased after the third aeroantigen-challenge. sIL-5R alpha, administered after each antigen-challenge, suppressed BAL eosinophilia with little effect on airway hyperreactivity.
Recombinant human bone morphogenetic protein-2 (rhBMP-2) was expressed in silkworm larvae, and a milligram quantity of the protein was purified and characterized. The expressed rhBMP-2 was biologically active in terms of induction of alkaline phosphatase activity in MC3T3-E1 cells and ectopic bone formation in mice. On SDS-polyacrylamide gel electrophoretic analysis, the purified protein showed a 16 kDa band under reducing conditions and a 30 kDa band under non-reducing conditions. The silkworm-expressed rhBMP-2 was glycosylated and susceptible to endo-beta-N-acetylglucosaminidase F (endo F) and endo H, but resistant to endo D. Deglycosylated rhBMP-2 treated with endo F retained its biological activity. These results suggest that rhBMP-2 exists as a dimer and disulfide bond(s) are responsible for the dimerization. Moreover, sugar chains have no direct effect on the biological activity of the protein. The availability of a quite large amount of rhBMP-2 has allowed us to study the biological function of this interesting factor in detail.
In the present study, were examined the inhibitory effect of erythromycin on the biofilm formation by a temperature-sensitive mutant (Ts25) of Pseudomonas aeruginosa N-42 on the surface of Ishikawa cells which can produce a mucin-like glycoprotein physicochemically similar to mucoproteins produced in the human airway. Usually, 38-45 microcolonies (biofilms) were formed after 10 days of incubation in cultures of Ishikawa cells and P. aeruginosa Ts25. Erythromcin suppressed the adhesion to Ishikawsa cells of P. aeruginosa and its subsequent biofilm formation at doses as low as 0.2 microgram/ml. Erythromycin also exhibited the suppressive effect on the production of glycoproteins by Ishikawa cells at doses higher than 1 microgram/ml and the production of elastase and exoenxyme A from P. aeruginosa at doses higher than 2 micrograms/ml. These results suggest that erythromycin can inhibit the biofilm formation in culture of human epithelial cells and P. aeruginosa and the in-vivo bacterial biofilm formation may be attributed to host cell-derived factors rather than bacterial products.
We performed transtracheal aspiration (TTA) in 1165 patients, who were suspected to have bronchopulmonary infection, from December 1978 to March 1993. We isolated pathogens from TTA in 806 patients (69.2%). We isolated H. influenzae (62 cases), S. pneumoniae (39 cases) and M. catarrhalis (24 cases) in patients with acute bronchitis, S. pneumoniae (65 cases), alpha-Streptococcus sp. (52 cases), H. influenzae (32 cases) and S. aureus (29 cases) in patients with pneumonia or lung abscess and H. influenzae (174 cases), S. pneumoniae (84 cases), P. aeruginosa (81 cases) and M. catarrhalis (42 cases) in patients with chronic lower respiratory tract infection. Anaerobic bacteria isolated from TTA included Peptostreptococcus sp. (19 cases), Bacteroides sp. (19 cases) and others. Mycoplasma pneumoniae was isolated from TTA in 8 patients with pneumonia without other organisms. Virus isolated from TTA included Rhinovirus (6 cases) and others. These results suggest that various pathogens affect the pathogenesis of bronchopulmonary infection. Therefore, we must diagnose the bronchopulmonary infection by the correct methods such as TTA.
We performed a clinical study of 20 cases (33 episodes) of respiratory infections due to mucoid Pseudomonas aeruginosa by transtracheal aspiration (TTA) in the recent 10 years. There was only one pneumonia without underlying chronic lower respiratory infection (CLRTI) case positive for mucoid P. aeruginosa and others were all CLRTI among 33 TTA trials. In contrast, nonmucoid P. aeruginosa was recovered from 9 cases of respiratory infections without underlying CLRTI among 46 TTA trials. Monomicrobial infection of mucoid P. aeruginosa was 69.7%, and polymicrobial infection containing mucoid P. aeruginosa was 30.3%, and Haemophilus influenzae was the most frequent microorganism recovered with mucoid P. aeruginosa. The recovery rate of mucoid P. aeruginosa among P. aeruginosa-colonized cases was 56.3% in diffuse panbronchiolitis, and that was 42.9% and 40.0% in bronchiectasis and chronic bronchitis, respectively. Mortality due to pneumonia with nonmucoid P. aeruginosa was 46.1%, but there was no fatal pneumonia case with mucoid P. aeruginosa. In CLRTI, laboratory data were not remarkably different between mucoid and non-mocoid P. aeruginosa-colonized cases. Thus, these results suggest that mucoid P. aeruginosa is a more important organism in persistent infections in the lower respiratory tract compared with nonmucoid P. aeruginosa, and further investigations is required on the mechanism and clinical role of this infection.
We isolated the virus from the respiratory specimens of 16 patients (7.9%) when we investigated the bronchopulmonary infections of 203 adult patients by the viral isolation method for the diagnosis of the viral infection and transtracheal aspiration (TTA) for taking directly the specimen from the lower respiratory tract. The bronchopulmonary infections, isolated the virus, consisted of acute bronchitis (3 cases), pneumonia (8 cases) and the acute exacerbation of chronic lower respiratory infection (5 cases). The positive viral isolation consisted of Rhinovirus (11 strains), Herpes simplex virus I (3 strains), Parainfluenza virus III (1 strain) and Respiratory syncytial virus (1 stain). There were 9 transtracheal aspirates, 8 pharyngeal swabs and 5 nasal washings in the positive viral specimens. We experienced 5 cases who had the same virus isolated from both the upper respiratory tract and the lower respiratory tract. H. influenzae, P. aeruginosa and other bacteria were found in 14 of the 16 TTA cultures. These results suggest that the role of the viral infection is important either independently or relating the bacterial infection in the adult bronchopulmonary infections.