Biomedical subjects

K Nagata

Publications and source records attributed to K Nagata.

At least 145 records · Page 8Linked to original sources

PubMed2000

Immunoglobulin beta signaling regulates locus accessibility for ordered immunoglobulin gene rearrangements.

The antigen receptor gene rearrangement at a given locus is tightly regulated with respect to cell lineage and developmental stage by an ill-defined mechanism. To study the possible role of precursor B cell antigen receptor (pre-BCR) signaling in the regulation of the ordered immunoglobulin (Ig) gene rearrangement during B cell differentiation, a newly developed system using mu heavy (H) chain membrane exon (microm)-deficient mice was employed. In this system, the antibody-mediated cross-linking of Igbeta on developmentally arrested progenitor B (pro-B) cells mimicked pre-BCR signaling to induce early B cell differentiation in vivo. Analyses with ligation-mediated polymerase chain reaction revealed that the Igbeta cross-linking induced the redirection of Ig gene rearrangements, namely, the suppression of ongoing rearrangements at the H chain locus and the activation of rearrangements at the light (L) chain locus. Upon the cross-linking, the kappaL chain germline transcription was found to be upregulated whereas the V(H) germline transcription was promptly downregulated. Notably, this alteration of the accessibility at the H and L chain loci was detected even before the induction of cellular differentiation became detectable by the change of surface phenotype. Thus, the pre-BCR signaling through Igbeta appears to regulate the ordered Ig gene rearrangement by altering the Ig locus accessibility.

Animals↗

PubMed2000

Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets.

Several effector molecules, including cytokines, are differentially produced by Th1 and Th2 cells. We used a gene expression screen method to identify a gene encoding hematopoietic PG D synthase (hPGDS) which was preferentially expressed in human Th2 but not Th1 clones. Studies with anti-hPGDS mAbs confirmed the Th2-dominated expression of hPGDS protein. Upon stimulation with anti-CD3 plus anti-CD28 mAbs, coordinated cyclooxygenase-2 expression and PGD2 production were induced in Th2 lines. hPGDS expression was also observed in a small population (<1.0%) of peripheral blood CD4+ lymphocytes from healthy adults. Most hPGDS-expressing CD4+ lymphocytes showed a typical Th2-type cytokine pattern. Our results suggest that, at the sites of Ag presentation, at least part of the Th2 cell population produces PGD2, which may be involved in various aspects of Th2-related immune responses similar to mast cells.

Adult↗

PubMed2000

Deficiency of a STE20/PAK family kinase LOK leads to the acceleration of LFA-1 clustering and cell adhesion of activated lymphocytes.

Lymphocyte-oriented kinase (LOK) is a member of the STE20/p21-activated kinase (PAK) family and expressed predominantly in lymphoid organs. Generation of LOK-deficient mice revealed that the leukocyte-function-associated antigen (LFA-1)/intercellular adhesion molecules (ICAM)-mediated aggregation of mitogen-stimulated T cells was greatly enhanced in the absence of LOK. Though levels of total LFA-1 and ICAMs as well as the active form of LFA-1 on T cell blasts were comparable in the presence and absence of LOK, clustering of active LFA-1 detected by binding of soluble ICAM-1 was accelerated in the absence of LOK. These results suggest that LOK is potentially involved in the regulation of LFA-1-mediated lymphocyte adhesion.

Animals↗

PubMed2000

Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop.

LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.

Animals↗

PubMed2000

Isolation, characterization, and mapping of the mouse Fgd3 gene, a new Faciogenital Dysplasia (FGD1; Aarskog Syndrome) gene homologue.

FGD1 gene mutations result in faciogenital dysplasia (FGDY, Aarskog syndrome), an X-linked developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase Cdc42. By way of Cdc42, FGD1 regulates the actin cytoskeleton and activates the c-Jun N-terminal kinase signaling cascade to regulate cell growth and differentiation. Previous work shows that FGD1 is the founding member of a family of related genes including the mouse Fgd2 gene and the rat Frabin gene. Here, we report on the isolation, characterization, and mapping of the mouse Fgd3 gene, a new and novel member of the FGD1 gene family. Fgd3 cDNA encodes a 733-amino-acid protein with a predicted mass of 81 kDa. Fgd3 and FGD1 share a high degree of sequence identity that spans >560 contiguous amino acid residues. Like FGD1, Fgd3 contains adjacent RhoGEF and pleckstrin homology (PH) domains, a second carboxy-terminal PH domain, and a distinctive FYVE domain. Together, these domains appear to form a canonical core structure for FGD1 family members. In addition, compared to other FGD1 family members, Fgd3 contains different structural regions that may be involved in distinct signaling interactions. Microinjection studies show that Fgd3 stimulates fibroblasts to form filopodia, actin microspikes formed upon the stimulation of Cdc42. Fgd3 transcripts are present in several diverse tissues and during mouse embryogenesis, suggesting a developmentally regulated pattern of expression and a potential role in embryonic development. Genetic linkage and radiation hybrid mapping data show that Fgd3 and the human FGD3 ortholog map to syntenic regions of murine chromosome 13 and human chromosome 9q22, respectively. We conclude that Fgd3 is a new and novel member of the FGD1 family of RhoGEF proteins.

3T3 Cells↗

PubMed2000

Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate.

In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis.

Animals↗

PubMed2000

Bax and Bcl-2 protein expression following radiation therapy versus radiation plus thermoradiotherapy in stage IIIB cervical carcinoma.

BACKGROUND: The relative amounts of Bcl-2 and Bax proteins determine cell survival or death following an apoptotic stimulus. To clarify the molecular mechanism of cell death after radiotherapy or thermoradiotherapy and its relation to the response of AJCC/UICC Stage IIIB cervical carcinomas, the expression of Bax and Bcl-2 proteins was investigated both before and in the course of treatment given during this study. METHODS: Thirty-seven patients with Stage IIIB carcinoma of the uterine cervix were treated with external beam irradiation to the pelvis combined with iridium-192 high-dose-rate intracavitary brachytherapy. All patients were randomized to one of the following two groups: the radiotherapy (RT) group of 19 patients who were given radiotherapy alone, and the thermoradiotherapy (TRT) group of 18 patients who were given 3 sessions of hyperthermia in addition to RT. Specimens of the cervical tumors were obtained by punch biopsy both before and in the course of the treatment (after a total dose of 10.8 grays ¿Gy for the RT group or after 10.8 Gy plus 1 session of hyperthermia for the TRT group). The tumor sections were stained with anti-Bax and anti-Bcl-2 monoclonal antibody. On the basis of the percentage of immunopositive cells, both pretreatment and posttreatment samples were scored. Furthermore, relative changes in protein expression were determined by comparing the pretreatment scores with those in the course of treatment. In addition, treatment response was evaluated. RESULTS: A complete response was achieved in 52.6% (10 of 19) of the RT group versus 83. 3% (15 of 18) of the TRT group (P = 0.049). Better tumor control was accompanied by increased Bax expression, i.e., 10.5% (2 of 19) of the RT group versus 44.4% (8 of 18) of the TRT group (P = 0.02). The respective number of patients who partially responded (PR) or did not respond to treatment (NC) was 26.3% (5 of 19) and 21.1% (4 of 19) of the RT group versus 11.1% (2 of 18) and 5.6% (1 of 18) of the TRT group (P = 0.2 for both the PR and NC subgroups). CONCLUSIONS: TRT was found to result in better treatment responses than RT for patients with Stage IIIB cervical carcinoma. An additive or synergistic antitumor effect of TRT is likely to occur through induction of apoptosis involving one of the bax pathways.

Aged↗

PubMed2000

Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence.

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis-trans isomerase (N-PPIase) and double-labeled (13C- and 15N-) protein (DL-PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPIase and four bands for DL-PPIase. Since the N-terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), were hydrolyzed and their amino acid composition was analyzed using a highly sensitive 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (sigma(T%-E%)2) for similarity of proteins was calculated by summation of the square difference between the theoretical (T%) and the experimental (E%) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins were 16812 and 17694 by electrospray ionization (ESI)-mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3721 for N-PPIase and 5285 for DL-PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for characterization of a recombinant protein.

Amino Acids↗

PubMed2000

CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.

Cells expressing the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the chemokine C receptor (CCR)4 were consistently detected in the circulation of healthy subjects, whereas numbers of cells expressing CCR3 were much lower. While all CCR4+ cells were T cells, a small proportion of CRTH2+, and about a half of the few CCR3+ cells were basophils. Only CRTH2+ T cells contained Th2 or Tc2 cells, but neither Th0 or Tc0, nor Th1 or Tc1 cells, although not all of them produced Th2-type cytokines. By contrast, CCR4+ T cells contained both Th2 or Tc2 and Th0 or Tc0 cells and even Th1 or Tc1 cells, whereas the few CCR3+ T cells were not clearly classifiable for their cytokine profile. CRTH2+ T lymphocytes were virtually devoid of chemokine CX receptor (CXCR)3+ and CCR5+ cells, but enriched in CCR3+ and CCR4+ cells. By contrast, CCR3+ or CCR4+ T cells did not show a similar clear-cut dichotomy in the expression of CCR5/CXCR3 or CCR3/ CCR4. Subjects with atopic dermatitis or HIV infection with low levels of circulating CD4+ T cells revealed a significant increase of CRTH2+ cells within both the CD4+ and the CD8+ T cell subset. These data support the concept that at present CRTH2 is the more reliable marker for detection of both human Th2 and Tc2 cells in health and disease.

Antiretroviral Therapy, Highly Active↗

PubMed2000

Degeneration of the cerebellar dentate nucleus in corticobasal degeneration: neuropathological and morphometric investigations.

To resolve the controversy regarding the involvement of the dentate nucleus in corticobasal degeneration (CBD), an entire profile of the dentate nucleus was exposed in a sagittal plane and divided into four fields, and the number of neurons in each filed was counted separately using a computer-assisted analyzer in five cases of CBD and compared to those from seven age-matched controls. The size of the nucleus and the number of neurons were significantly reduced in all five cases of CBD. The neuronal loss had a definite regional predilection, more severe in the caudolateral neodentatum than in the rostromedial palaeodentatum, and was accompanied by grumose degeneration and astrogliosis which paralleled the severity of the neuronal loss. Thus, the dentate nucleus appears to be a cardinal target in CBD.

Aged↗

PubMed2000

Immunohistochemical study of NGF and its receptors in the synovial membrane of the ankle joint of adjuvant-induced arthritic rats.

To study the role of nerve growth factor (NGF) in local inflammation, we investigated the expression of NGF and its receptors, trkA and p75, in the ankle joints of adjuvant-induced arthritic rats. Infiltrated mononuclear cells revealed a positive immunoreactivity for NGF and trkA; they were also positive for immunostaining for W3/25 and ED1, which mainly stain T cells and macrophages, respectively. Changes in the ratios of NGF-positive cells to mononuclear cells showed a relatively similar pattern for trkA-positive cells, which peaked at weeks 2 to 3 after the adjuvant injection. In double-immunofluorescence staining, 80% and 65% of NGF-positive cells stained for W3/25 and ED1, respectively. Similarly, 67% and 80% of trkA-positive cells also corresponded to W3/25- and ED1-positive cells, respectively. However, p75 immunoreactivity localized on the nerve fibers but not on the cells of the ankle joints. Dense meshworks of p75-positive nerve fibers with numerous terminal varicosities were observed at weeks 2 to 4. The present findings suggest that infiltrated mononuclear cells may secrete NGF in an autocrine or paracrine manner in the inflamed synovium. An upregulation of NGF in these mononuclear cells and an increase in density of the synovial nerve fibers may be involved in the development of adjuvant arthritis in rats.

Animals↗

PubMed2000

Intradiscal pressure after intradiscal injection of hypertonic saline: an experimental study.

Although chemonucleolysis with chymopapain is a long-established treatment for lumbar intervertebral disc herniation, serious complications have been reported. Accordingly, alternative substances for chemonucleolysis have been sought. The main beneficial effect of chemonucleolysis derives from the decrease in intradiscal pressure. Several previous studies have investigated the relationship between physiological saline injection and disc mechanics in cadaveric specimens [2, 5, 16]. However, no previous study has assessed the intradiscal pressure after intradiscal injection of "hypertonic saline" in living animals. The present study compared the changes in intradiscal pressure after intradiscal injection of hypertonic saline with those after chymopapain injection. The lumbar intervertebral discs of 26 living rabbits were examined: 10% hypertonic saline was injected in ten rabbits, and chymopapain (10 pikokatal units) was injected intradiscally in another ten, with the remaining six being used as controls. The intradiscal pressure was measured at 1, 4, and 12 weeks after injection. The intradiscal pressure of the hypertonic saline-injected group at 4 weeks was significantly lower than that of the control group, but by 12 weeks it had recovered. On the other hand, that of the chymopapain-injected group remained significantly lower than that of the control group at 12 weeks. The results of this study found that hypertonic saline injected into the intervertebral discs temporarily decreased the intradiscal pressure.

Animals↗

PubMed2000

Fibrillin gene (FBN1) mutations in Japanese patients with Marfan syndrome.

Marfan syndrome (MFS; MIM #154700) is a connective tissue disorder characterized by cardiovascular, skeletal, and ocular abnormalities. The fibrillin-1 gene (FBN1; MIM no. 134797) on chromosome 15 was revealed to be the cause of Marfan syndrome. To date over 137 types of FBN1 mutations have been reported. In this study, two novel mutations and a recurrent de-novo mutation were identified in patients with MFS by means of single-strand conformational polymorphism (SSCP) analysis. The two novel mutations are a 4-bp deletion at nucleotide 2820-2823 and a G-to-T transversion at nucleotide 1421 (C474F), located on exon 23 and exon 11, respectively. A previously reported mutation at the splicing donor site of intron 2 (IVS2 G + 1A), which is predicted to cause exon skipping, was identified in a sporadic patient with classical MFS.

Amino Acid Substitution↗

PubMed2000

A novel interstitial deletion of KAL1 in a Japanese family with Kallmann syndrome.

We identified a novel interstitial deletion that spanned from exons 5 to 10 of KAL1 in two Japanese brothers with X-linked Kallmann syndrome (KS; MIM no. 308700). Both brothers had hypogonadism, unilateral renal agenesis, and disturbance of the sense of smell, but they had no other neurological manifestations, including mental disturbance. Their mother was confirmed to be an asymptomatic carrier, by use of a comparative multiplex polymerase chain reaction (PCR) analysis. The present patients are further examples of patients with KS without mental disturbance caused by a mutation confined to KAL1.

Adult↗

PubMed2000

Evaluation of immunological rapid urease testing for detection of Helicobacter pylori.

The aim of this study was to evaluate in clinical specimens the immunological rapid urease test (IRUT), a new diagnostic system for detection of Helicobacter pylori which employs a monoclonal antibody against Helicobacter pylori urease. Helicobacter pylori urease adsorbed on a solid-phase tip coated with a monoclonal antibody against Helicobacter pylori urease after 15 min of incubation with a gastric mucus sample solution was measured by the pH change of the urea solution inside the tip. The detection limit of Helicobacter pylori urease using this system was determined and compared with that of a commercially available rapid urease test. Clinical evaluation of the system was performed in 155 patients. The IRUT could detect 0.25 milli-international units (mIU) of Helicobacter pylori urease per milliliter in less than 20 min. If a patient with at least one positive result in a standard test for Helicobacter pylori was considered to be Helicobacter pylori positive, the sensitivity, specificity, positive and negative predictive values of the system were calculated as 95.2%, 98.9%, 98.4% and 96.8%, respectively. However, 10 of 19 Helicobacter pylori-positive patients in whom the pH change was less than 0.1 had negative results in at least one of the standard tests, whereas the IRUT correctly detected Helicobacter pylori in all but 3 of these 19 patients. The IRUT accurately determined the Helicobacter pylori status of 75 (98.7%) of 76 patients who had completed treatment. This system has high sensitivity for the detection of Helicobacter pylori, especially in patients with low urease activity.

Adult↗

PubMed2000

Effect of n-3 polyunsaturated fatty acid supplementation on lipid peroxidation of rat organs.

The present study was undertaken in order to reexamine the effect of n-3 polyunsaturated fatty acid (PUFA)-rich diet supplementation on lipid peroxidation and vitamin E status of rat organs. Male Wistar rats were fed a diet containing safflower or fish oil at 50 g/kg diet and an equal amount of vitamin E at 59 mg/kg diet (1.18 g/kg oil; and 1.5 g/kg PUFA in safflower oil diet, and 4.3 g/kg PUFA in fish oil diet) for 6 wk. Fatty acid composition of total lipids of brain, liver, heart, and lung of rats fed fish oil was rich in n-3 PUFA, whereas that of each organ of rats fed safflower oil was rich in n-6 PUFA. The vitamin E levels in liver, stomach, and testis of the fish oil diet group were slightly lower than those of the safflower oil diet group, but the levels in brain, heart, lung, kidney, and spleen were not different between the two diet groups. The levels of phospholipid hydroperoxides were determined by the high-performance liquid chromatography-chemiluminescence method and the levels of thiobarbituric acid-reactive substances (TBARS) were determined at pH 3.5 in the presence of butylated hydroxytoluene with or without EDTA. Levels of phospholipid hydroperoxides and TBARS in the brain, liver, heart, lung, kidney, spleen, stomach and testis of the fish oil diet group were similar to those of the safflower oil diet group. The results indicate that high fish oil intake does not induce increased levels of phospholipid hydroperoxides and TBARS in rat organs.

Animals↗

PubMed2000

A novel translocation t(3;22)(q21;q11) involving 3q21 in myelodysplastic syndrome-derived overt leukemia with thrombocytosis.

We report here a novel translocation t(3;22)(q21;q11) in myelodysplastic syndrome (MDS)-derived overt leukemia with thrombocytosis. A 44-year-old female was initially diagnosed as MDS with a low platelet count and normal karyotype. After 4 months, blood leukemic cells and platelets rapidly increased concomitantly and a diagnosis of acute myeloblastic leukemia (AML M1) was made. Chromosome analysis showed 46, XX, t(3;22)(q21;q11) in 14 of 20 metaphases. Fluorescence in situ hybridization analysis confirmed both the der(3)t(3;22) and the der(22)t(3;22). Our results suggest that unidentified gene(s) at 3q21 breakpoint may be implicated in the pathogenesis of abnormal thrombopoiesis as observed in the 3q21q26 syndrome.

Adult↗

PubMed2000

Interstitial deletion of the short arm of chromosome 12 during clonal evolution in myelodysplastic syndrome with t(5;12)(q13;p13) involving the ETV6 gene.

We report here a 65-year-old man with a myelodysplastic syndrome (MDS), refractory anemia with excess of blasts. He had received chemotherapy with tegafur for renal carcinoma. Chromosome analysis of bone marrow cells revealed complex karyotypes; del(5)(q13) was observed in all 20 metaphase spreads, and two related aberrations, add(12)(p11) and add(12)(p13), were detected in 13 and 7 cells, respectively. Fluorescence in situ hybridization (FISH) analysis with chromosome-specific DNAs revealed that these alterations originated from a reciprocal translocation (5;12)(q13;p13). Therefore, del(5)(q13), add(12)(p11), and add(12)(p13) were revised as der(5)t(5;12)(q13;p13), der(12)del(12)(p11p13)t(5;12)(q13;p13), and der(12)t(5;12)(q13;p13), respectively. Fluorescence in situ hybridization with a series of cosmid probes spanning the ETV6 gene showed that the 12p13 breakpoint on the der(12)t(5;12)(q13;p13) was located in intron 1, but the exon 1 signal was deleted. Our results suggest that a fusion gene was generated between the 5'-end of an unidentified partner at 5q13 and the 3'-end of ETV6 by t(5;12)(q13;p13), and that the interstitial deletion (12)(p11p13) occurred following t(5;12) during clonal evolution. del(12)(p11p13), including the rearranged ETV6 gene, may be implicated in the progression of MDS.

Aged↗