Biomedical subjects

Hiroko Saito

Publications and source records attributed to Hiroko Saito.

14 recordsLinked to original sources

PubMed2005

Haemopoietic mechanisms in murine allergic upper and lower airway inflammation.

Eosinophil recruitment to the airways, including involvement of haemopoietic eosinophil-basophil progenitors (Eo/B-CFU), is primarily regulated by interleukin-5 (IL-5) and eotaxin. In this study, we investigated the haemopoietic mechanisms in upper and lower airway eosinophilic inflammation. Ovalbumin (OVA) sensitized and challenged BALB/c mice were used to establish isolated upper (UAC), isolated lower (LAC), or combined upper and lower airway (ULAC) inflammation. Airway, blood and bone marrow responses were evaluated in each model. Numbers of airway eosinophils and CD4(+) cells were increased significantly in the nasal mucosa in UAC and ULAC mice, and in the lung tissue in LAC and ULAC groups. Levels of IL-5 and eotaxin were increased significantly in the nasal lavage fluid (NL) in UAC and ULAC mice, and in the bronchoalveolar lavage fluid (BAL) in LAC and ULAC groups. The proportion of IL-5-responsive bone marrow Eo/B-CFU was significantly higher than the control in all treatment groups, but peaked much earlier in the ULAC group. Kinetic studies revealed that IL-5 and eotaxin in NL, BAL and serum peaked between 2 and 12 hr after OVA challenge in ULAC mice, and at 24 hr in UAC mice, related to the timing of maximal progenitor responses. These data support the concept that the systemic mechanisms linking rhinitis to asthma depend on the location and extent of airway allergen exposure.

Animals↗

PubMed2004

Reduced expression of class II histone deacetylase genes is associated with poor prognosis in lung cancer patients.

HDAC genes are thought to be involved in gene expression through the regulation of chromatin structure, alterations of which may cause abnormal gene silencing in cancers. To clarify the possible role of HDAC genes during tumor development and progression, we studied their expression and influence on clinical features. Expression levels of HDAC class I and class II genes in cancer tissues resected from 72 patients with NSCLC were measured with real-time RT-PCR. Their association with clinicopathologic features was statistically investigated. Reduced expression of each class II HDAC gene was significantly associated with poor prognosis and an independent predictor of poor prognosis. Of all the genes, HDAC10 was the strongest predictor of poor prognosis. Hierarchical clustering analysis showed that lung cancer tissues could be divided into 3 groups based on the expression level of class I and class II HDAC genes. The group with reduced expression of class II HDACs showed poor prognosis. These results suggest that class II HDACs may repress critical genes and that low expression of these genes may play a role in lung cancer progression. Results of clustering analyses imply that class II HDAC genes may be regulated by a similar mechanism and deregulated during cancer development.

Adenocarcinoma↗

PubMed2004

Effects of a cysteinyl leukotriene receptor antagonist on eosinophil recruitment in experimental allergic rhinitis.

The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting through a receptor (cysLT1-R) which can be targeted in rhinitis and asthma. We investigated the effects of cysLT1-R antagonism in experimental allergic rhinitis, focusing on bone marrow eosinophil progenitor responses. BALB/c mice were sensitized, then given daily intranasal ovalbumin for 2 weeks, with montelukast sodium (5 mg/kg or 2.5 mg/kg) or placebo by gavage. Bone marrow eosinophil/basophil colonies were enumerated, and colony cells were morphologically assessed as indices of eosinophil differentiation and maturation. Montelukast treatment resulted in a significant decrease of eosinophils in the nasal mucosa, and in either bone marrow interleukin (IL)-5-, but not IL-3-, or granulocyte-macrophage colony-stimulating factor-responsive eosinophil/basophil colony-forming units, and IL-5-stimulated eosinophil maturation. These results indicate that cysLT1-R antagonism in vivo limits both IL-5-responsive eosinophilopoiesis, acting at several stages of eosinophil differentiation and maturation. The anti-allergic effects of cysLT1-R antagonists are consistent with the concept that cysLTs and IL-5 act together in the recruitment of eosinophils and eosinophil progenitors from the marrow during upper airway allergic inflammation.

Acetates↗

PubMed2004

Identification of another surface protein antigen I/II gene, paaB, and a putative transcriptional regulator gene, par, from Streptococcus cricetus.

The flanking region of the antigen I/II gene, paaA, in Streptococcus cricetus was examined using the gene-walking technique. In the region downstream of the paaA gene, another antigen I/II gene designated as paaB was found. The paaB gene was disrupted at the alanine-rich region (A region) by a novel insertion sequence element, ISScr1. ISScr1 is a member of the IS982 family and is composed of a 962-bp sequence and duplicated target DNA (the sequence 5'-TAGCTAAAT-3') resulting from its insertion. To clarify the structural divergence of the two antigen I/II proteins (PAaA and PAaB), computational analysis of the paaB gene was performed and the two structures were compared. The amino acid sequence homology indicated that PAaB resembled PAaA, but the middle region showed little similarity to that of PAaA. Phylogenetic analysis showed that PAaB was better classified in a major group with S. mutans PAc and S. gordonii SspA and SspB than with PAaA. The transcriptional expression of paaA and paaB was demonstrated by reverse transcription (RT)-PCR. In the region upstream of the paaA gene, three genes homologous to the genes located in the region upstream of the S. sobrinus antigen I/II gene (pag) were found. Of the three genes, ORF3 showed homology to the par gene encoding a transcriptional repressor for the pag gene in S. sobrinus. Therefore, ORF3 was designated the par gene of S. cricetus. Southern hybridization revealed that the par gene of S. cricetus was not found in other oral streptococci examined in this study.

Amino Acid Sequence↗

PubMed2003

Detailed characterization of a homozygously deleted region corresponding to a candidate tumor suppressor locus at distal 17p13.3 in human lung cancer.

17p13.3 is one of the chromosomal regions most frequently affected by allelic loss in a variety of human neoplasms including lung cancer. A number of loss of heterozygosity (LOH) analyses have suggested the existence of a tumor suppressor gene at 17p13.3, distal to the p53 locus at 17p13.1. In the present study, we characterized a homozygous deletion at 17p13.3 in a small cell lung cancer cell line by constructing a bacterial artificial chromosome (BAC) contig and a restriction map surrounding the region, as well as by utilizing publicly available draft sequences. We defined the breakpoint, assigned and analysed two known genes, 14-3-3 epsilon and CRK, and a novel gene LOST1 within or at the end of the homozygous deletion of about 170 kb in size. Marked reduction of LOST1 expression was detected in 69% (11/16) of lung cancer specimens by quantitative real-time RT-PCR, while significant DNA hypermethylation was observed at the 5' end of the LOST1 gene in four of six lung cancer cell lines with negligible LOST1 expression. We also show here that a polymorphic marker D17S1174, which resides within the homozygous deletion, was apparently located in the middle of the minimum LOH region, providing further supportive evidence for the presence of a tumor suppressor gene(s) in this region.

Amino Acid Sequence↗

PubMed2003

Association between mitotic spindle checkpoint impairment and susceptibility to the induction of apoptosis by anti-microtubule agents in human lung cancers.

Anti-microtubule agents such as vinorelbine and paclitaxel, which are extensively used in the treatment of lung cancers, activate mitotic spindle checkpoint. Although defects of the mitotic spindle checkpoint are thought to play a role in the genesis of chromosome instability, we previously reported its frequent impairment in human lung cancer cell lines. In this study, we examined a panel of 13 human cancer cell lines comprising 11 lung and 2 other cancers and found a significant difference in the resistance to apoptosis induced by anti-microtubule agents between mitotic spindle checkpoint-impaired and -proficient cancer cell lines. This finding was in marked contrast to a lack of such correlation with a DNA damaging agent, cis-platin. Interestingly, anti-microtubule agent-induced apoptosis in mitotic spindle checkpoint-proficient cell lines, NCI-H460 and A549, was shown to be markedly reduced by staurosporine treatment in association with the shortened mitotic arrest, whereas various inhibitors of caspases seemed to have very modest effects. Taken together, these findings suggest the potential involvement of mitotic spindle checkpoint in the induction of apoptosis by anti-microtubule agents in human lung cancers, warranting further studies on the underlying mechanisms.

Antineoplastic Agents↗

PubMed2002

Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency.

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5(-/-)) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5(+/+) and IL-5(-/-) BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5(-/-) OVA-sensitized mice had significantly higher nasal mucosal CD4(+) cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Ralpha on bone marrow CD34(+)CD45(+) progenitor cells in IL-5(-/-) mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.

Animals↗

PubMed2002

Identification of frequent G(2) checkpoint impairment and a homozygous deletion of 14-3-3epsilon at 17p13.3 in small cell lung cancers.

Accumulating evidence suggests that a coordinately controlled G(2) checkpoint prevents cells with damaged DNA from entering mitosis, thus playing an important role in the maintenance of chromosomal integrity. In the study presented here, we identified a homozygous deletion of the 14-3-3epsilon gene, which resides within a previously identified, commonly deleted region at 17p13.3 in lung cancers, in two small cell lung cancer cell lines that originate from distinct metastatic sites of the same patients. The introduction of 14-3-3epsilon induced significantly restored G(2) checkpoint responses, which resulted in the reduction of mitotic cells as well as of aberrant mitotic figures in the X-ray-irradiated 14-3-3epsilon-null small cell lung cancer cell line. Interestingly, we also found that the G(2) checkpoint response is frequently impaired to various degrees in a large fraction of small cell lung cancer cell lines. These findings suggest the possible involvement of the perturbed G(2) checkpoint in the pathogenesis of this aggressive form of human lung cancers.

14-3-3 Proteins↗

PubMed2002

Identification and characterization of the human protein kinase-like gene NTKL: mitosis-specific centrosomal localization of an alternatively spliced isoform.

Although the centrosome has an essential role in mitosis, its molecular components have not been fully elucidated. Here, we describe the molecular cloning and characterization of the human gene NTKL, which encodes an evolutionarily conserved kinase-like protein. NTKL mRNA is found ubiquitously in human tissues. NTKL is located on 11q13 and is mapped around chromosomal breakpoints found in several carcinomas, suggesting that NTKL dysfunction may be involved in carcinogenesis. Alternative splicing generates two variant forms of NTKL mRNA that encode protein isoforms with internal deletions. When fused to green fluorescent protein, the full-length product and one of the variant proteins are found in cytoplasm. The other variant product also exists in the cytoplasm during interphase, but is found in the centrosomes during mitosis. Endogenous NTKL protein is also localized to the centrosomes during mitosis. This cell-cycle-dependent centrosomal localization suggests that NTKL is involved in centrosome-related cellular functions.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Mutation analysis of the mel-18 gene that shows decreased expression in human breast cancer cell lines.

BACKGROUND: Mammalian mel-18 is a member of the polycomb group, and it acts as a transcriptional repressor with DNA binding activity. Murine mel-18 negatively regulates the cell cycle through the c-myc/cdc25 cascade, and mice haploinsufficient for mel-18 develop mammary gland tumors. In addition, the human homolog of mel-18 is located at 17q, on which candidate tumor suppressor genes for breast cancer have been suggested for a long time. These observations indicate that the mel-18 gene may be a tumor suppressor gene for breast cancer. To investigate this possibility, we examined the expression of mel-18 mRNA in human breast cancer cell lines and searched for mel-18 gene mutations in sporadic and familial breast cancers. METHODS: The expression of mel-18 mRNA was examined in five breast cancer cell lines by RT-PCR, and somatic and germline mutations of the mel-18 gene were analyzed by the PCR-SSCP and sequence methods in 48 sporadic breast cancers, including 16 cases with loss of heterozygosity (LOH) at the mel-18 locus, and in 23 cases from 18 breast cancer families, respectively. RESULTS: We found that most cell lines examined here showed decreased expression of mel-18 mRNA, however, no alteration other than a single nucleotide change that did not lead to amino acid alteration in one patient was identified. CONCLUSION: Our results reveal that mel-18 gene mutations are exceedingly rare in human breast cancers, and a reduction of mel-18 expression in human breast cancer cell lines would support a role for mel-18 haploinsufficiency in breast carcinogenesis.

Adult↗

PubMed2002

Anti-allergic therapies: effects on eosinophil progenitors.

Marked eosinophilic infiltration is the typical inflammatory response associated with allergic inflammation. Previous research involving animal and human models has established a role for the eosinophil/basophil hematopoietic progenitor in a systemic process of allergic inflammation. In this article, we will review the evidence implicating eosinophil/basophil progenitors in this systemic response and will discuss the rationale for targeting this cell in the treatment of allergic disease. In this context, we discuss corticosteroid treatment of allergic diseases, such as asthma and its effects on hematopoietic mechanisms, the effects of therapies that inhibit the actions of cysteinyl leukotrienes, the effects of in vivo blockade of the eosinophil-active cytokine interleukin-5, and the effects of antihistamines on hematopoiesis. It is suggested that several anti-allergic therapies exert their beneficial effects on allergic inflammation by influencing eosinophil production systemically. Therefore, targeting the systemic hematopoietic response may provide additional, more beneficial, therapeutic effects.

Animals↗

PubMed2001

Interaction between Erbin and a Catenin-related protein in epithelial cells.

Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Human regulatory factor X 4 (RFX4) is a testis-specific dimeric DNA-binding protein that cooperates with other human RFX members.

Regulatory factor X (RFX) members are evolutionarily conserved transcription factors that share a highly conserved winged helix DNA-binding domain. Human RFX4 has been isolated from breast cancer as a partial cDNA encoding a short RFX-type DNA-binding domain fused to the estrogen receptor, but the entire structure of RFX4 has been unknown. Here, we report the molecular cloning and characterization of human RFX4. RFX4 contains evolutionarily conserved regions, including a RFX-type DNA-binding domain, a dimerization domain, and other conserved regions, and is closely related to RFX1, RFX2, and RFX3 in structure. The expression of RFX4 is restricted to testis. In vitro synthesized RFX4 protein bound to typical RFX binding sites in a sequence-dependent manner. Immunoprecipitation analyses showed that RFX4 interacts physically with RFX2, RFX3, and RFX4 itself but not with RFX1. In contrast to other mammalian RFX members that form dimers, RFX4 is revealed to have no distinct transcriptional activation domains. By using a chimeric protein of RFX1 and RFX4, the C-terminal domain of RFX4 was shown to be a possible transcriptional repression domain. Taken together, these results indicate that RFX4 is the first mammalian member of RFX family without transcriptional activation capacity and might function through selective interactions with other RFX members in transcriptional regulation.

Amino Acid Sequence↗

PubMedDate not supplied

An improved method for assaying phosphatidylcholine in mouse tissue.

INTRODUCTION: To measure levels of phosphatidylcholine (PtdCh) in various mouse tissues, we developed a rapid and precise method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. To generate an example data set, the effect of methoxamine (an alpha1-adrenergic agonist) on the PtdCh levels was examined by this method in the artery and the submandibular gland of the mouse in vivo. METHODS: Under our modifications of the method of Zapata et al. [J. Neurosci. 18 (1998) 3597], the mixture of lipophilic choline metabolites (PtdCh, lyso-PtdCh, and sphingomyelin) extracted by chloroform from the tissue homogenate was dried without prior separation and hydrolyzed with free choline by a 1-N perchloric acid solution containing ethylhomocholine (an internal standard for choline assay) at 90 degrees C for 1 h. Subsequently, the hydrolyzed mixture was injected directly into the HPLC system for PtdCh assay. RESULTS: The present method permitted PtdCh assay within 5 min in one chromatographic run. Recovery of an authentic PtdCh sample was 99% (n = 10). The within-run coefficients of variation for choline derived from PtdCh in the same tissue samples were 0.6% (n = 10) and 1.3% (n = 30). Under the present method, the lowest and highest PtdCh values in tissue samples were about 2 micromol/g (eye ball) and 29 micromol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland. DISCUSSION: Under the present sample processing procedure, the choline values originating from lyso-PtdCh and sphingomyelin were much less than those originating from PtdCh hydrolysis. Thus, it was possible to inject the hydrolyzed mixture directly into the HPLC system for PtdCh assay. Since the present method provides simple, rapid, and highly reliable PtdCh determination, it is suitable for routine assay of PtdCh in a large number of samples.

Adrenergic alpha-Agonists↗