[Studies on Helicobacter pylori].
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Bacterial Adhesion↗
Biomedical subjects
T Nakazawa
Publications and source records attributed to T Nakazawa.
Transgenic mice overexpressing type 2 nitric-oxide synthase in pancreatic beta cells develop insulin-dependent diabetes without insulitis.
We generated transgenic mice carrying the mouse type 2 nitric-oxide synthase (NOS2) cDNA under the control of the insulin promoter. Western and immunohistochemical analyses revealed that NOS2 was expressed abundantly in transgenic islets but not in control islets. When islets were isolated and cultured, high levels of nitrite were released from the transgenic islets. In transgenic mice, the beta cell mass was markedly reduced without the infiltration of macrophages or lymphocytes, and extensive DNA strand breaks were detected in the islets by in situ nick translation. All the transgenic mice developed hypoinsulinemic diabetes by 4 weeks of age, and treatment with an inhibitor of NOS2, aminoguanidine (200 mg/kg body weight every 12 h), prevented or delayed the development of diabetes. The present study shows that the production of nitric oxide by beta cell NOS2 plays an essential role in the beta cell degeneration.
Animals↗
Generation of plasma cells from peripheral blood memory B cells: synergistic effect of interleukin-10 and CD27/CD70 interaction.
B cells can differentiate into the antibody-secreting cells, plasma cells, whereas the crucial signals that positively control the entry into the pathway to plasma cells have been unclear. Triggering via CD27 by CD27 ligand (CD70) on purified peripheral blood B cells yielded an increase in the number of plasma cells in the presence of interleukin-10 (IL-10). Differentiation into plasma cells by a combination of IL-10 and CD70 transfectants occurred in CD27+ B cells but not in CD27- B cells. Moreover, addition of IL-2 to the IL-10 and CD70-transfect activation system greatly induced differentiation into plasma cells. In the presence of only IL-2, IL-4, or IL-6, CD70 transfectants did not promote differentiation into plasma cells. On the other hand, CD40 signaling increased the expansion of a B-cell pool from peripheral blood B cells primarily activated by IL-2, IL-10, and anti-CD40 monoclonal antibody (MoAb). Finally, CD27 signaling also rescued B cells from IL-10-mediated apoptosis. These data demonstrate that CD27 ligand (CD70) is a key molecule to prevent the IL-10-mediated promotion of apoptosis and to direct the differentiation of CD27+ memory B cells toward plasma cells in cooperation with IL-10.
Adult↗
Arrangement of actin filaments and cytoplasmic granules in the sea urchin egg after TPA treatment.
Elongation of microvilli and formation of actin filaments after treatment with a phorbol ester, TPA, were investigated in unfertilized eggs of Hemicentrotus pulcherrimus. Microvilli on the egg surface were examined by scanning electron microscopy. Actin filaments in the cortical layer of the eggs were observed by fluorescence microscopy using rhodamine-labeled phalloidin. The actin molecules were polymerized and bundled to form long filaments inside the cortical layer of eggs after TPA treatment. Arrangement of the actin filaments was followed by spiral elongation of microvilli. Transmission electron microscopic studies showed that the cortical granules under the cell membrane of sea urchin eggs were transferred after TPA treatment from the surface to the interior of the cell [Ciapa et al., 1988: Dev. Biol. 128:142-149]. This movement of the cortical granules was inhibited by cytochalasin B, but not by nocodazole. Furthermore, the distribution of clear granules was changed following TPA treatment. From these results we conclude that intracellular actin filaments may cause the transport of cortical granules and clear granules into the central area of the egg by the activation of protein kinase C. The possible involvement of actin in the inward displacement of granules might be the result of the rearrangement of actin filaments in the cortical layer.
Actins↗
A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides.
We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.
Amino Acid Sequence↗
Human lung mast cells are enriched in the capacity to produce granulocyte-macrophage colony-stimulating factor in response to IgE-dependent stimulation.
By using reverse transcription-PCR, in situ hybridization, enzyme-linked immunosorbent assay and immunocytochemistry, we have studied the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lung mast cells induced by cross-linkage of high-affinity Fc epsilon receptors (Fc epsilonRI). We have also confirmed the bioactivity of GM-CSF released from lung mast cells by investigating the effect of the supernatant from lung mast cells activated with anti-IgE on the release of eosinophil cationic protein (ECP) from eosinophils. Mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 (93-99% pure). Purified mast cells were precultured with IgE for 16 h before challenge with 1 microg/ml anti-IgE with or without stem cell factor (SCF). Eosinophils were purified by immunomagnetic negative selection (> 98% pure). The activation of mast cells via Fc epsilonRI enhanced the intensity of the GM-CSF signal within 2 h and the cells produced GM-CSF protein 4 h after the activation. In the absence of recombinant human (rh) SCF, anti-IgE induced a median GM-CSF response of 202 (< 15 to approximately 681) pg/10(6) mast cells/24 h, whereas in the presence of rhSCF the median IgE-dependent GM-CSF release was 356 (152 to approximately 1216) pg/10(6) mast cells/24 h. This difference was statistically significant (p = 0.0029, n = 12). In contrast, mast cells produced only a small amount of GM-CSF in the absence of anti-IgE. The mast cell supernatant induced ECP release from eosinophils and the release was significantly inhibited by blocking mAb against GM-CSF. These findings indicate that human mast cells are an important cellular source of GM-CSF and as such may contribute to chronic eosinophil-mediated inflammation.
Blood Proteins↗
Differential expression of trypsin in human ovarian carcinomas and low-malignant-potential tumors.
It is widely recognized that matrix metalloproteinases and serine proteinases play an important role in cancer invasion and metastasis. We have reported that trypsin is synthesized in ovarian carcinomas as well as in some other types of cancers. In general, ovarian cancers easily tend to invade, metastasize, and spread widely into the peritoneal cavity. However, low-malignant-potential (LMP, borderline tumor) ovarian tumors are known to have limited malignant potential for progression, although microinvasion and distant metastasis have been reported among them. To analyze the relationship between varied degrees of trypsin expression and malignant behavior of ovarian tumors, immunohistochemical studies with monoclonal antibodies to human trypsin and clinicopathologic analysis were performed in human ovarian carcinomas, low-malignant-potential tumors, and benign cystadenomas. Thirteen (44.8%) cases of 29 ovarian carcinomas showed prominent trypsin expression, while only 2 (18.2%) cases of 11 LMP ovarian tumors demonstrated low levels of expression. Benign tumors and normal ovaries did not show any positivity for trypsin. These data suggest that tumor-derived heterotropic trypsin may be associated with ovarian tumors in parallel with malignant potential or behavior such as invasiveness or metastasis. At least in some ovarian carcinomas, prominent stromal invasion or metastasis might require the acquisition of or association with tumor-derived trypsin production.
Female↗
Alteration of bcl-2 and bax expression in embolized meningiomas.
We examined the expression of the p53 and bcl-2 family in embolized meningiomas and investigated the interaction between them. p53 and its downstream effector p21 accumulated mainly in perinecrotic areas, where apoptosis was also observed. bcl-2 was often expressed in the areas distant from necrosis, whereas Bax was immunostained more intensely in the perinecrotic areas. Double staining for both p53 and Bcl-2 showed a reciprocal pattern. This appeared to be due to the down-regulation of Bcl-2 by p53. However, regulation of Bax by p53 was not distinct. The expression of p21 was not apparent 13 days after embolization, and apoptosis was observed until 6 days after embolization. The effect of embolization was most likely temporary, although growth arrest has been reported to persist for a long time in a limited number of embolized cases.
Adult↗
Calcitonin-producing pancreatic somatostatinoma: report of a case.
A 59-year-old woman was hospitalized due to a 1-year history of diarrhea and weight loss. Echography and computed tomography of the abdomen revealed a 10 x 7 cm solid mass in the tail of the pancreas and gallstones, while selective celiac angiography revealed the presence of a hypervascular mass. High levels of somatostatin and calcitonin were detected in the plasma, 70 pg/ml (normal range <28 pg/ml) and 5550 pg/ml (normal range 37 +/- 8 pg/ml), respectively. This tumor was thus removed by means of a distal pancreatectomy and a splenectomy. After the pancreatic tumor was removed, the elevated levels of plasma somatostatin and calcitonin returned to the normal ranges, and the persistent diarrhea also dramatically disappeared. A postoperative immunohistochemical study showed the tumor cells to be diffusely positive for somatostatin and calcitonin. These results clearly indicate this patient to be a case of calcitonin-producing pancreatic somatostatinoma.
Calcitonin↗
Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells.
It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.
B-Lymphocytes↗
Metabolism of rosmarinic acid in rats.
The urine of rats administered rosmarinic acid (7) orally contained seven metabolites, which were identified as trans-caffeic acid 4-O-sulfate (1), trans-m-coumaric acid 3-O-sulfate (2), trans-ferulic acid 4-O-sulfate (3), trans-caffeic acid (4), m-hydroxyphenylpropionic acid (5), trans-m-coumaric acid (6), and unchanged rosmarinic acid (7) by spectroscopic and chemical data. The total cumulative amount of 1-7 excreted in the urine 48 h after the oral administration of rosmarinic acid was approximately 31.8% of the dose administered. On the other hand, the metabolites attributed to rosmarinic acid could not be found in the bile. Orally administered rosmarinic acid may thus be concluded to be excreted in the urine rather than in the bile, with cleavage of ester bonds, selective para-dehydroxylation, methylation, and sulfate-conjugation. Metabolites 2, 3, 5, and 6 were also detected in the plasma.
Animals↗
Post-translational modification and intracellular localization of a splice product of CD46 cloned from human testis: role of the intracellular domains in O-glycosylation.
We obtained a unique CD46 cDNA, STc/CY4, from the human testis, the predicted amino acid sequence of which suggested the presence of a novel isoform of CD46. This message was present predominantly in the testis, and the predicted isoform possessed a short (11 amino acids) transmembrane section (TM) and an unidentified cytoplasmic tail (CY). When expressed in Chinese hamster ovary (CHO) cells, this CD46 isoform underwent no O-glycosylation and was mostly retained in the endoplasmic reticulum. This unusual behaviour of the new isoform was due in part to the short TM and the unusual sequences of the CY. The molecular mass of this isoform was 42,000, approximately 20,000 smaller than conventional CD46. These properties of the STc/CY4 isoform were similar to those of sperm CD46. The only difference between sperm CD46 and the STc/CY4 isoform expressed on CHO cells was that only the latter possessed N-linked sugars of high mannose types. Since the STc/CY4 isoform may behave like sperm CD46 in cellular localization and post-translational modification, studies of sperm-egg interassociation were performed using hamster eggs and CHO cell clones expressing various isoforms including the STc/CY4. Rosette formation was seen most effectively between hamster eggs and STc/CY4-expressing CHO cells. These results infer that O-glycosylation perturbs CD46-mediated sperm-binding to eggs and thus sperm CD46 lacking O-linked sugars can serve as an adhesion molecule. The possible role of CD46 in fertilization and the structural differences between sperm and conventional CD46 are discussed.
Alternative Splicing↗
Persistent infection by Helicobacter pylori down-modulates virus-specific CD8+ cytotoxic T cell response and prolongs viral infection.
To determine whether Helicobacter pylori infection affects clearance of a concomitant viral infection and cytotoxic T lymphocyte (CTL) and cytokine response to that infection, H. pylori-infected BALB/c mice were challenged with a recombinant vaccinia virus expressing human immunodeficiency virus type 1 gp160. Two H. pylori strains, a colonizing clinical isolate (KS612) and an established standard noncolonizing strain (NCTC11637), were compared. Clearance of recombinant vaccinia virus was reduced in KS612-infected mice compared with NCTC11637-infected and control mice. As a potential mechanism, in contrast to control or NCTC11637-infected mice, the H. pylori clinical isolate KS612 diminished gp160-specific and vaccinia virus-specific CTL activity, even in the presence of exogenous interleukin-2. Furthermore, KS612-infected mice had reduced Th1 cytokine responses to gp120 in vitro compared with control or NCTC11637-infected mice. These results have implications for possible effects of prevalent H. pylori infection on other human diseases.
Animals↗
Local recurrence of renal cell carcinoma and acquired cysts 10 years after tumour nephrectomy.
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Adult↗
Chlamydia pneumoniae in coronary and iliac arteries of Japanese patients with atherosclerotic cardiovascular diseases.
Recent studies suggest the association of atherosclerotic cardiovascular diseases with Chlamydia pneumoniae infection in western populations. It is of great interest whether such an association exists in Asians with their distinct genetic background. Symptomatic patients with coronary heart disease (29) or arteriosclerosis obliterans (10) who underwent directional endo-atherectomy were studied. Atherectomy specimens of coronary and iliac arteries were examined for C. pneumoniae by culture, nested PCR and immunohistochemical stain (IHC) with one Chlamydia genus-specific, two C. pneumoniae species-specific, and two C. trachomatis species-specific monoclonal antibodies. Among the 29 patients with coronary artery disease, C. pneumoniae was detected in the coronary arteries of 13 by IHC, 16 by PCR and 20 by IHC or PCR, or both. C. pneumoniae was also found in the iliac arteries of four patients by IHC, three by PCR and five by IHC or PCR, or both, of the 10 patients with arteriosclerosis obliterans. Attempts to isolate C. pneumoniae by culture were unsuccessful. The re-stenotic rate after atherectomy was higher in the C. pneumoniae-positive group than in the negative group, but not significantly so. These findings support the high incidence of C. pneumoniae in atherosclerotic lesions of symptomatic patients with coronary heart disease and arteriosclerosis obliterans in Asians.
Aged↗
Urease plays an important role in the chemotactic motility of Helicobacter pylori in a viscous environment.
Helicobacter pylori exhibits chemotactic responses to urea, flurofamide, acetohydroxamic acid, and sodium bicarbonate. In buffer, the chemotactic activities of a urease-positive strain were higher than those of the isogenic urease-negative strain. Moreover, the chemotactic activities of the urease-positive strain were increased in a viscous solution containing 3% polyvinylpyrrolidone, whereas those of the urease-negative mutant were not. These results are in accordance with the fact that the mutant strain did not show swarming in motility agar regardless of having flagella. Incubation of the wild-type strain with flurofamide resulted in partial inhibition of the chemotactic activities in the viscous solution. In addition, incubation with acetohydroxamic acid, a low-molecular-weight, diffusible urease inhibitor, resulted in complete loss of chemotactic activity in the viscous solution. The inhibition of the chemotactic activity by urease inhibitors paralleled the inhibition of urease. The chemotactic activity of H. pylori was also inhibited by the proton carrier carbonyl cyanide m-chlorophenylhydrazone, showing that H. pylori utilizes proton motive force for motility. These results indicate that cytoplasmic urease plays an important role in the chemotactic motility of H. pylori under a condition that mimics the ecological niche of the bacterium, the gastric mucous layer.
Benzamides↗
Nucleotide sequence and characterization of cdrA, a cell division-related gene of Helicobacter pylori.
We identified cell division-related gene cdrA in Helicobacter pylori HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of H. pylori 26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a cdrA-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained cdrA mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a cdrA-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the cdrA-disrupted mutant remained mostly as short rods. Furthermore, the cdrA-disrupted mutant regained the filamentation phenotype when the intact cdrA gene was introduced by allelic exchange. Taken together, these observations show that the cdrA gene plays an important role in the cell growth of H. pylori.
Amino Acid Sequence↗
Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa.
Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast, Candida maltosa (T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030-5036, 1997). To understand the mechanism of this transition, we screened the gene library of C. maltosa for sequences which could suppress this morphological change. As a result, we isolated the 5' end of a new gene, EPD1 (for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epdlp was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae, and Phr1p and Phr2p of Candida albicans. The expression of EPD1 was moderately regulated by environmental pH. A homozygous EPD1 null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble beta-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by n-hexadecane at pH 4 or pH 7, suggesting that EPD1 is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the GAL promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length EPD1 did not. This result and the initial isolation of the 5' end of EPD1 as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.
Amino Acid Sequence↗