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H Tojo

Publications and source records attributed to H Tojo.

At least 109 records · Page 6Linked to original sources
PubMed1993

Purification and characterization of a phospholipase A2 from human ileal mucosa.

We purified a phospholipase A2 (PLA2) from human ileal mucosa to homogeneity. Its NH2-terminal amino acid sequence, amino acid composition, molecular weight, and elution behavior on reverse phase high-performance liquid chromatography were identical to those of human group II PLA2 purified from synovial fluid or spleen. The ileal PLA2 preferred anionic phosphatidylglycerol as substrate. On immunoblot analysis, human ileal mucosa gave more intense immunoreactivity with anti-human synovial fluid PLA2 antibody, at the same position as the purified enzyme, than the cecal mucosa. Northern blot analysis also showed that the level of group II PLA2 mRNA in the ileal mucosa was greater than that in the cecal mucosa. The enzyme was rather uniformly distributed over the colonic mucosa, from cecum to sigmoid colon. These results indicate that the ileal mucosa contains group II PLA2, and that its expression in the ileal mucosa was higher than that in the colonic mucosa.

Amino Acid Sequence↗
PubMed1993

Human fetal-to-adult globin gene switching in transgenic mice: persistent expression of the G gamma-globin gene in the Japanese HPFH.

We linked a 3.3 kb fragment containing the human gamma-globin gene together with a 5.5 kb fragment containing the human beta-globin gene. This construct was introduced into the germ line of mice. Analysis of the resulting transgenic mice showed that expression of the human gamma- and beta-globin genes were regulated as that of the mouse embryonic and adult globin genes and human gamma-to-beta globin switching was reconstructed in the mice. By replacing the gamma-globin gene in the construct with modified counterparts, this transgenic mouse system enables us to analyze cis-acting elements essential for erythroid-specific and developmental stage-specific expression of the test gene under the condition in which human beta-globin gene expression is regulated. In this system expression of the G gamma-globin gene bearing the point mutation found in a Japanese patient of hereditary persistence of fetal hemoglobin (HPFH) (1) persisted at a equivalent level to beta-globin expression in fetal and adult mice.

Aging↗
PubMed1993

Purification and characterization of guinea pig gastric phospholipase A2 of the pancreatic type.

Guinea pig gastric mucosa and juice contained exceptionally high phospholipase-A2 activity, whereas the activity in the pancreas was only minimal. Phospholipases A2 were purified to homogeneity from these three tissues. Structural evidence, including the sequence of the NH2-terminal 41 residues, the amino-acid composition and the molecular mass (13902 +/- 3 Da) determined accurately by mass spectrometry, showed that the gastric mucosa enzyme belongs to the pancreatic type. An unique feature of the sequence is the substitution of Phe for the hitherto invariant Tyr28 in the calcium-binding loop of pancreatic phospholipases A2. The affinity of the guinea pig enzyme for Ca2+ in the presence of substrate was, however, identical to that of the rat enzyme with Tyr28, suggesting the interaction of a phenolic hydroxyl group of the Tyr with its neighboring residues is not significantly linked to the binding of Ca2+. The NH2-terminal sequences and immunochemical properties of the enzymes purified from the gastric juice and pancreas were identical to those of the gastric mucosa enzyme. The distribution of cells immunoreactive with anti-(gastric PLA2) immunoglobulin in the stomach was quite similar to that of the chief cells. Unlike in pancreas of other animals, the prophospholipase A2 was not detectable in gastric mucosa or juice homogenates treated with diisopropyl fluorophosphate or in column effluents during purification under acidic conditions. An appreciable prophospholipase-A2-activating activity was not detectable in gastric mucosa extracts at low pH relevant to gastric juice, using rat prophospholipase A2 as substrate. This opposes the activation of secreted proenzyme in the gastric juice.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of phospholipase A2 from guinea pig gastric mucosa, its induction by carbachol and secretion in vivo.

A cDNA encoding phospholipase A2 (PLA2) was cloned from guinea pig gastric mucosa using a rat pancreatic-PLA2-cDNA fragment as a probe. The cDNA contains an open reading frame sufficient to encode the entire amino-acid sequence of a PLA2-precursor protein consisting of 146 amino acids, including a putative 16-residue signal peptide and a 6-residue activation peptide at the NH2-terminus. Its nucleotide sequence exhibits 70% similarity to that of rat pancreatic PLA2 cDNA. The deduced amino-acid sequence has all the typical pancreatic PLA2 characteristics, with the exception of the substitution of Phe for Tyr at position 28 in the calcium-binding loop of the mature enzyme. When an expression vector containing the PLA2 cDNA was transfected into COS-7 cells, a major portion of the proenzyme was secreted into the culture medium. Northern-blot analysis showed the mRNA was present in guinea pig lung and pancreas at much lower levels than in the stomach. The effect of carbachol, a muscarinic acetylcholine agonist, on the secretion of gastric PLA2 and on its mRNA level in the gastric mucosa were examined. PLA2 secretion into the gastric juice was maximal 30 min after the subcutaneous administration of carbachol (0.4 mg/kg). It also increased the PLA2-mRNA level in the tissue, the maximal mRNA level being delayed about 15 min compared with that in PLA2 secretion. These results suggest that vagal stimuli may contribute to PLA2 secretion and its compensatory synthesis, and that the secreted PLA2 may participate in the digestion of dietary and biliary phospholipids in the small intestine of guinea pig.

Amino Acid Sequence↗
PubMed1993

Heinz body hemolytic anemia induced by DQ-2511, a new anti-ulcer drug, in dogs.

DQ-2511, a new anti-ulcer drug, was administered to beagle dogs for 4 weeks to investigate the mechanism whereby this drug induced hemolytic anemia and its reversibility in comparison with beta-acetylphenylhydrazine. Hemolytic anemia accompanied by an increase in the number of cells containing Heinz bodies that was preceded by a marked decrease in blood-reduced glutathione concentration was observed in dogs receiving 600 mg/kg of DQ-2511, but only a slight increase in the methemoglobin level was noted. beta-Acetylphenylhydrazine, however, caused hemolytic anemia accompanied by marked increases in both Heinz body-containing cells and methemoglobin concentration, but the blood-reduced glutathione concentration was not decreased consistently with the formation of Heinz bodies. Hemolytic anemia disappeared after a 4-week recovery period in the dogs that received DQ-2511. These results suggest that decreases in reduced glutathione in erythrocytes play an important role in the anemia and Heinz body formation induced by DQ-2511, but not by beta-acetylphenylhydrazine.

Administration, Oral↗
PubMed1993

Serum pancreatic phospholipase A2 and prophospholipase A2 in acute pancreatitis and after endoscopic retrograde pancreatography.

Serum immunoreactive pancreatic phospholipase A2 (IP-PLA2) levels and the proportion of active pancreatic PLA2 in the serum IP-PLA2 were determined by radioimmunoassay with an antibody directed against active human PLA2. The subjects of this study included eight patients who underwent endoscopic retrograde pancreatography (ERP), nine patients with acute pancreatitis, and six healthy controls. The serum IP-PLA2 levels were elevated after ERP and during acute pancreatitis. The amount of active pancreatic PLA2 in the serum was determined after its separation from pancreatic prophospholipase A2 using reverse-phase high-performance liquid chromatography (HPLC). The serum IP-PLA2 was separated into two peaks on reverse-phase HPLC. The one which eluted faster contained the PLA2 activity; the other peak did not. The latter IP-PLA2 peak consisted of pancreatic prophospholipase A2 as judged by HPLC analysis and PLA2 activity determination of the products after treatment with trypsin. The proportion of active pancreatic PLA2 in the serum IP-PLA2 of patients after ERP (13.9 +/- 0.5%) increased slightly compared with that in fasting, healthy controls (8.0 +/- 1.1%). For those with acute pancreatitis, the proportion of active pancreatic PLA2 within 48 hours of hospital admission increased more markedly (44.0 +/- 5.7%) than that after ERP. These findings demonstrate that the proportion of active pancreatic PLA2 in the serum IP-PLA2 markedly increases during the early stage of acute pancreatitis, and that an ERP-induced rise in the intraductal pressure leads to the leakage of pancreatic PLA2 into the circulation, but not to a marked activation of the leaked enzyme.

Acute Disease↗
PubMed1993

[Adrenocortical zonation in human growth hormone gene (hGH) transgenic mice].

Adrenocortical zonation was histologically examined in 2 infertile female transgenic (Tg) mice which carried human growth hormone (hGH) gene and had a high circulating level of hGH. The adrenal cortices of Tg mice were characterized by the appearance of hypertrophied zona fasciculata cells with the cytoplasm filled with many lipid droplets even in size, the absence of the distinctive z. reticularis and the persistence of the X zone with nodules in one and without them in the other. The last finding might be explained by the reflection of the strain differences used in Tg mouse construction. It was, however, suggested that the X zone degeneration might not be associated with the adenohypophysis. The changes observed in z. fasciculata cells were interpreted as the effects of ACTH-like function of circulating hGH in addition to the known PRL-like function. The absence of the z. reticularis may be explained by its morphological similarity to z. fasciculata under the effects of the circulating hGH at a high level. In addition to genetic analysis of various inbred strains, Tg mice with ectopic hGH production provide a new methodology in analyzing the mechanism of adrenocortical zone formation.

Adrenal Cortex↗
PubMed1993

[Immunocytochemical observation of adenohypophysis in a human growth hormone (hGH) gene transgenic mouse].

Adenohypophysis was immunocytochemically examined in an infertile female transgenic (Tg) mouse which carried human growth hormone (hGH) gene and had a high circulating level of hGH. No GH positive cells were detected. This confirmed the extrahypophyseal (ectopic) production of hGH and was coincident with the disappearance of parenchymal cells showing affinity to azocarmine in Azan staining. The normal frequency of ACTH positive cells was in accordance with the previous suggestion based on the changes found in zona fasciculata cells of the adrenal cortex. Most interesting findings were the detection of many PRL positive cells and the ovarian histology with nearly normal characteristics except for the presence of thick capsule and interstitial gland-like structure composed of large and light cells. Ovarian histology was also clearly different among individual Tg mice, even though they stemmed from the same line or progenitors, and had a similar phenotype. The current immunocytochemical observation well documented the changes revealed with Azan staining in the adenohypophysis about GH but not about PRL or ACTH. Thus, the immunocytochemical analysis of the adenohypophysis will provide useful methodology in assessment of endocrinological circumstances of Tg mice.

Adrenocorticotropic Hormone↗
PubMed1993

Elevation of phospholipase A2 protein in sera of patients with Crohn's disease and ulcerative colitis.

The protein mass of group II phospholipase A2 (PLA2) levels were immunochemically measured in sera of 43 patients with Crohn's disease (CD), 43 patients with ulcerative colitis (UC), and 35 healthy volunteers, and compared with PLA2 enzymatic activity. Serum immunoreactive group II PLA2 (IR-PLA2 II) levels of active CD patients were significantly higher than those of the controls and inactive CD patients. Serum IR-PLA2 II levels were also significantly greater in patients with severe UC and moderate UC than in the controls and patients with mild UC. Serum IR-PLA2 II levels were found to be closely correlated with serum PLA2 activity in patients with CD and UC. Serum IR-PLA2 II levels and PLA2 activity decreased by antiinflammatory treatment in six patients with CD and two with UC. These results suggest that serum IR-PLA2 II levels can be a useful parameter in the evaluation of disease activity of CD and UC, and that elevated PLA2 enzymatic activity in sera of patients with CD and UC is attributable to increased protein concentration of group II PLA2, in agreement with our previous results.

Adolescent↗
PubMed1993

[A case of eosinophilic pneumonia with histologically proven mediastinal lymph node involvement].

Intrathoracic lymphadenopathy in eosinophilic pneumonia is very rare and few cases have been confirmed histologically. This is the first case of eosinophilic pneumonia with mediastinoscopic lymph node biopsy reported in Japan. The case is a 42-year-old man who was admitted to our hospital complaining of cough, general fatigue and dyspnea of one month's duration. Chest X-ray demonstrated bilateral hilar and mediastinal lymph node enlargement with interlobar pleural thickening and infiltrative shadows in right lower lung field. Computed tomography revealed infiltrative shadows at right S4, S5, S8 and S9 and S10 segments and #2, #3, #5, #6 lymph node enlargement. Biopsy of the lymph node via mediastinoscopy demonstrated that the architecture was preserved, sinusoids were filled with histiocytes and eosinophils, and lymphoid follicles were compressed. Immunohistochemical technique using monoclonal antibody EG2 which reacts with the secreted form of Eosinophilic Cationic Protein (ECP), demonstrated that eosinophils infiltrating the mediastinal lymph nodes were activated. The pathogenesis of mediastinal lymphadenopathy in eosinophilic pneumonia remains to be determined, but our case suggests that mediastinal lymph nodes may play a role in eosinophilic pneumonia.

Adult↗
PubMed1993

[Flow cytometric analysis of the DNA content in resected thymomas].

Nuclear DNA contents were determined by flow cytometry in 31 resected thymomas. Ten (32%) of the 31 thymomas showed DNA aneuploid, and the presence of aneuploidy was well correlated with advanced clinical stage. Patients with an aneuploid tumor had a poor prognosis than those with a diploid tumor, demonstrating a survival rate of 50% at 7 years in aneuploidy compared to 100% at the same period in diploid patients (p < 0.05). In addition, patients with a high DNA index (DI > or = 1.5) tended to have a poor prognosis than those with a low DI. The analysis of DNA content can be an important prognostic index in patients with a thymoma.

Adult↗
PubMed1993

Reverse-phase high-performance liquid chromatographic assay of phospholipases: application of spectrophotometric detection to rat phospholipase A2 isozymes.

This paper appraises an HPLC method for assaying phospholipase A2 (PLA2). The procedure is based on heptane-isopropanol-H2SO4 extraction of fatty acids released by the enzyme in the presence of margaric acid as an internal standard, and precolumn derivatization with 9-anthryldiazomethane. The derivatives of naturally occurring long-chain fatty acids were accurately determined by reverse-phase HPLC with ultraviolet detection at 254 nm; the fatty acids were identical with margaric acid in terms of their extraction efficiency in the presence or absence of a bile salt, reactivity with the labeling reagent, and molar extinction coefficients of their derivatives. HPLC conditions were optimized so as to separate the derivatives of palmitic and oleic acids completely within 7 min. The use of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/cholate system as substrate proved useful for the sensitive detection of PLA2 activities in rat tissue homogenates. Distribution of immunoreactive pancreatic and group II phospholipases A2 was estimated from the degree of inhibition of enzyme activities by specific antibodies raised against either forms of phospholipase A2 isozymes. The results were consistent with those of immunoblot analyses.

Animals↗
PubMed1992

Group II phospholipase A2 induced by interleukin-1 beta in cultured rat gingival fibroblasts.

Previously, we reported the presence of group II-like phospholipase A2 activity in the soluble fraction of rat gingiva. In the present study, we found that treatment of rat gingival cells with human recombinant interleukin-1 beta resulted in dose-dependent stimulation of intracellular and extracellular phospholipase A2 activity. Antisera against group II phospholipase A2 totally blocked the interleukin-1 beta-induced phospholipase A2 activity, but antisera against group I phospholipase A2 did not. Moreover, immunoblot analysis showed that the induced phospholipase A2 was group II phospholipase A2. These findings suggest that the induced enzyme belongs to the group II phospholipase A2 family of proinflammatory enzymes.

Animals↗
PubMed1992

Raised serum activity of phospholipase A2 immunochemically related to group II enzyme in inflammatory bowel disease: its correlation with disease activity of Crohn's disease and ulcerative colitis.

Calcium dependent phospholipase A2 activity in the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was measured in sera of 39 patients with Crohn's disease, 40 patients with ulcerative colitis, and 40 healthy controls. The phospholipase A2 activity was significantly raised in those sera of the patients with active Crohn's disease and those with moderate and severe ulcerative colitis. The major phospholipase A2 activity derived from the sera was separated into two peaks by reverse phase high performance liquid chromatography. The phospholipase A2 active fractions were immunochemically characterised using specific antibody directed against human group II phospholipase A2 purified from rheumatoid synovial fluid. The results suggest that raised serum phospholipase A2 activity in patients with Crohn's disease and ulcerative colitis was mainly attributed to the two forms of phospholipase A2 immunochemically related to group II enzyme. In patients with Crohn's disease, serum phospholipase A2 activity decreased in parallel with clinical improvement, and correlated with serum C-reactive protein and erythrocyte sedimentation rate. The results suggest that serum phospholipase A2 activity may serve as an additional indicator of disease activity. Serum phospholipase A2 activity in patients with ulcerative colitis tends to increase in relation with endoscopic severity, and may be a more sensitive laboratory index than serum C-reactive protein and erythrocyte sedimentation rate to evaluate disease activity.

Acute Disease↗
PubMed1992

Effect of the new quinolone antibacterial agent levofloxacin on multiple organ carcinogenesis initiated with wide-spectrum carcinogens in rats.

A multiple organ carcinogenesis model was used in male F344 rats to test the carcinogenic potential of (-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo- 7H-pyrido[1,2,3-de][1,4] benzoxazine-6-carboxylic acid hemihydrate (levofloxacin, DR-3355, CAS 10086-85-4). After sequential treatment with diethylnitrosamine (DEN: carcinogen for the liver), N-methylnitrosourea (MNU: carcinogen for the esophagus, forestomach and thyroid) and dihydroxy-di-N-propylnitrosamine (DHPN: carcinogen for the lungs, kidney and urinary bladder), rats were treated with DR-3355, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), catechol (CC) or phenobarbital (PB) to examine whether these compounds modified the carcinogenesis in multiple organs. As a result of histopathological examinations at study week 20, DR-3355 did not induce neoplastic lesions, nor did it enhance the occurrences of proliferative preneoplastic lesions. In contrast, BBN increased the incidences of hyperplasias and papillomas of the urinary bladder. CC enhanced the occurrences of hyperplasias and papillomas of the forestomach as well as submucosal glandular growth for the glandular stomach. PB increased the number of altered cell foci in the liver and the incidence of follicular cysts and hyperplasias of the thyroid. These results indicate that DR-3355 is not capable of promoting the development of tumors in rat multiple organs.

Animals↗
PubMed1991

Preferential distribution of group-II-like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver.

The localization of calcium-dependent phospholipase A2, (PLA2) immunochemically closely related to the enzyme of the viperid and crotalid type (group II), in cells isolated from rat spleen and liver was examined using a polyclonal antibody directed against rat spleen group II, PLA2 (PLA2M). In isolated spleen cells, the monocyte/macrophage fraction had the highest PLA2 activity (1.28 +/- 0.35.min-1.10(6) cells-1) which was almost completely inhibited by the anti-PLA2M antibody. An immunoblot analysis confirmed the presence of the enzyme in this fraction. An immunocytochemical study revealed that the PLA2 was present in spleen macrophages. In the isolated liver cells, Kupffer cells (0.92 +/- 0.22 nmol.min-1.10(6) cells-1) contained higher anti-PLA2M-antibody-inhibitable PLA2 activity than parenchymal cells (0.26 +/- 0.06.min-1.10(6) cells-1). The immunocytochemical study showed that cells immunopositive with anti PLA2M antibody were Kupffer cells. These results suggest that the mononuclear phagocytic cells in rat spleen and liver have relatively high activity of group-II-like PLA2. Subcellular distribution patterns of the anti-PLA2M-antibody-inhibitable phospholipase A2 activity in different cell populations from spleen and liver were compared. A mode of the distribution of the enzyme in the spleen macrophages was essentially similar to that in the spleen lymphocytes. The distribution in Kupffer cells was similar to that in parenchymal cells.

Animals↗
PubMed1991

Induction of group II-like phospholipase A2 by lipopolysaccharide in the liver of BCG-primed rat.

The specific activity of phospholipase A2 (PLA2) in the liver homogenate was elevated 1.7-fold in bacillus Calmette-Guerin (BCG)-treated rats, 1.6-fold in lipopolysaccharide (LPS)-treated rats, and 2.4-fold in BCG-infected rats treated with LPS, compared with that of control rats. These increased activities were almost completely inhibited by the antibody directed against rat splenic group II PLA2 (PLA2M) but not by anti-pancreatic PLA2 antibody. The results of immunoblot analysis confirmed that the PLA2 immunochemically related to the group II enzyme was induced by treatment with BCG and/or LPS. The anti-PLA2M antibody-inhibitable PLA2 activity per a single cell was elevated not only in nonparenchymal cell fraction but also in hepatocyte fraction, as in the case of whole liver. On the contrary, the PLA2 concentration and its specific activity did not change by the same treatment both in spleen homogenate and in isolated spleen cell fractions although a 3-fold increase in spleen mass occurred by BCG treatment. These results suggested that a tissue-specific mechanism of the PLA2 induction by these inflammatory mediators may operate in liver.

Animals↗