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Human amniotic mesenchymal cells (HAM cells) are known to contain somatic stem cells possessing the characteristics of pluripotency. However, little is known about the biology of these somatic cells because isolated HAM cells from amniotic membrane have a limited lifespan. To overcome this problem, we attempted to prolong the lifespan of HAM cells by infecting retrovirus encoding human papillomavirus type16E6 and E7 (HPV16E6E7), bmi-1, and/or human telomerase reverse transcriptase (hTERT) genes and investigated their characteristics as stem cells. We confirmed the immortalization of the four lines of cultured HAM cells for about 1 year. Immortalized human amnion mesenchymal cells (iHAM cells) have continued to proliferate over 200 population doublings (PDs). iHAM cells were positive for CD73, CD90, CD105, and CD44 and negative for CD34, CD14, CD45, and HLA-DR. They expressed stem cell markers such as Oct3/4, Sox2, Nanog, Klf4, SSEA4, c-myc, vimentin, and nestin. They showed adipogenic, osteogenic, and chondrogenic differentiation abilities after induction. These results suggested that immortalized cell lines with characteristics of stem cells can be established. iHAM cells with an extended lifespan can be used to produce good experimental models both in vitro and in vivo.

Nebulized drugs for asthma treatment are often mixed together in order to simplify inhalation regimens, although not recommended. We therefore evaluated aerosol characteristics and physicochemical stability of the admixture of an inhaled corticosteroid suspension with a beta2-agonist solution.

Background A relevant relationship exists between the upper and lower airway, indicating the concept of a unified airway. This study aimed to evaluate whether rhinitis has an association with asthma in children.Methods A cross-sectional nationwide survey was performed among children 6–7, 13–14, and 16–17 years old, using the International Study of Asthma and Allergies in Children (ISAAC) questionnaire in Japan. According to the responses to the ISAAC core questions, a child who had experienced nasal symptoms in the past 12 months in the absence of a cold was defined as having current rhinitis.Results After excluding 11,475 children because of incomplete data, 136,506 children were analyzed. Even after adjusting for demographics, sex, and obesity, children with current rhinitis were more likely to have asthma (adjusted odds ratio [OR], 3.10 [95% CI, 2.92–3.30] in children aged 6–7 years; OR, 3.76 [95% CI, 3.45–4.10] in children aged 13–14 years; and OR, 3.59 [95% CI, 3.33–3.88] in children aged 16–17 years). Children whose daily activities were more impaired by rhinitis symptoms had a significantly higher prevalence of severe asthma. The adjusted ORs for severe asthma among asthmatic children whose daily activities were severely impaired by rhinitis symptoms were 3.66 (95% CI, 2.29–5.85) in children aged 6–7 years, 2.55 (95% CI, 1.64–3.96) in children aged 13–14 years, and 1.87 (95% CI, 1.24–2.82) in children aged 16–17 years compared with asthmatic children whose daily activities were not impaired at all.Conclusion There was a close association between rhinitis and asthma in young children to adolescents. Asthma should be examined in children with rhinitis symptoms.

Recent studies have shown an association between excessive smartphone use and health problems. Along with such mounting concerns, health risk information on excessive smartphone use has been presented through various media. Such information is usually aimed at making smartphone users understand the associated health risks, thereby preventing excessive use or reducing current use. However, according to self-affirmation theory, such information may pose a threat to heavy smartphone users because it implies that they are engaging in maladaptive behavior. Therefore, to defend themselves, they may not accept the information and may not be highly motivated to reduce their usage. According to self-affirmation theory, such maladaptive defensive responses can be reduced through the affirmation of important values. We examined whether self-affirmation prior to reading health risk information increased heavy users’ motivation to reduce smartphone usage. Participants (142 undergraduate students aged 18–22 years) reported their mean daily smartphone use. They then completed a writing task that affirmed/did not affirm an important personal value. Next, they read an article on the health risks of smartphone overuse and reported their motivation to reduce smartphone use. As a result, when heavy users did not self-affirm, they were significantly less motivated to reduce use than light users. However, when heavy users self-affirmed, their motivation was significantly higher than when they did not self-affirm. This effect of self-affirmation was not shown in light users. These results suggest that health risk information combined with self-affirmation is effective in reducing smartphone usage by heavy users.

The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to associate with PKC in an isoform-specific manner. Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC. ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro. Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo. Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains. These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

LIM-kinase 1 (LIMK1) is a serine/threonine kinase containing two LIM motifs at the N-terminus. The functional role of LIMKI has remained unknown. In this study, we examined the role of LIMK1 in cell growth of fibroblasts. Induced expression of LIMK1 in NIH3T3 cells led to growth retardation. Transfection of LIMK1 sense cDNA into NIH3T3 and H-ras-transformed FYJ10 fibroblasts significantly suppressed colony formation of these cells. In contrast, transfection with LIMK1 antisense cDNA strongly stimulated colony formation of the NIH3T3 cells. These findings suggest that LIMK1 functions as a negative regulator of fibroblast cell growth, and may play a role in tumor suppression.

LIM‐kinase 1(LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N‐terminus. The LIM motif is thought to be involved in protein‐protein interactions. We report here evidence that LIMK1 self‐associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMK1 revealed that the self‐association of LIMK1 was caused by interaction between the N‐terminal LIM domain and the C‐terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

By transfection of the expression plasmid containing a human c- metcDNA into COS-7 cells, high-affinity binding sites specific for HGF with a Kdvalue of 30 pM were newly detected. Furthermore, only in the c- mettransfected COS-7 cells, but not in the control COS-7 cells, DNA synthesis was markedly induced in response to HGF. Thus, transient expression of exogenous c- metcDNA resulted in the appearance of high-affinity receptor for HGF and conversion of the normally non-responsive COS-7 cells into the HGF-responsive cells. These results provide evidence for identifying the c- metproduct as a signal transducing high-affinity receptor for HGF.

ABSTRACTTo investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R’s deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

ABSTRACTTo investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R’s deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations inPEX5were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

LIM‐kinase 1 (LIMK1) is a serine/threonine kinase containing two LIM motifs at the N‐terminus. The functional role of LIMKI has remained unknown. In this study, we examined the role of LIMK1 in cell growth of fibroblasts. Induced expression of LIMK1 in NIH3T3 cells led to growth retardation. Transfection of LIMK1 sense cDNA into NIH3T3 and H‐ras‐transformed FYJ10 fibroblasts significantly suppressed colony formation of these cells. In contrast, transfection with LIMK1 antisense cDNA strongly stimulated colony formation of the NIH3T3 cells. These findings suggest that LIMK1 functions as a negative regulator of fibroblast cell growth, and may play a role in tumor suppression.

By transfection of the expression plasmid containing a human c‐metcDNA into COS‐7 cells, high‐affinity binding sites specific for HGF with a Kdvalue of 30 pM were newly detected. Furthermore, only in the c‐mettransfected COS‐7 cells, but not in the control COS‐7 cells, DNA synthesis was markedly induced in response to HGF. Thus, transient expression of exogenous c‐metcDNA resulted in the appearance of high‐affinity receptor for HGF and conversion of the normally non‐responsive COS‐7 cells into the HGF‐responsive cells. These results provide evidence for identifying the c‐metproduct as a signal transducing high‐affinity receptor for HGF.

LIM-containing protein kinase 1 (LIMK1) is a serine/threonine kinase with a structure composed of two LIM domains, a PDZ domain, and a protein kinase domain. We examined the subcellular localization of LIMK1 and its variously deleted mutants in HeLa cells by transfection with these cDNAs. Immunofluorescence analysis revealed that the full-length LIMK1 and its mutants deleted with LIM domain or protein kinase domain preferentially localized in the cytoplasm, while the mutants deleted with the PDZ domain or a 52 amino acid region (B region) within the PDZ domain localized mainly in the nucleus. When the normally nuclear cyclin A was fused with the PDZ domain or the B region of LIMK1, it was localized in the cytoplasm of transfected cells. The corresponding region of the PDZ domain of postsynaptic density protein (PSD)-95 had no such function. Additionally, the PDZ domain of LIMK1 had no potential to bind to the C-terminal S/TXV peptides, to which the PSD-95 PDZ domain can bind. Taken together these results suggest that the PDZ domain, particularly the B region, of LIMK1 has a specific function to localize the protein in the cytoplasm. When glutathioneS-transferase (GST) fused with the PDZ domain of LIMK1 (GST–PDZ) or GST–PDZ deleted with the B region (GST–PDZΔB) was microinjected into the nucleus of COS cells, GST–PDZ was almost completely excluded from the nucleus within 30 min, whereas GST–PDZΔB remained in the nucleus. These findings suggest that the B region of LIMK1 probably has nuclear export signal activity.

LIM-kinase 1(LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMK1 revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

The chemical shifts of methoxyl protons of a series of methoxybenzenes and the related compounds in cyclohexane, carbon tetrachloride, and benzene solutions have been investigated. The benzene-induced high-field shifts for methoxyl protons are large if the substituent at the benzene ring is a highly electron-withdrawing group. In carbon tetrachloride, the methoxyl proton signals appear at lower field than those in cyclohexane solutions. The low-field shifts also increase with the electronegativities of the substituents. An attempt has been made to interpret the solvent effects by the use of a model where benzene molecules are assumed to cluster around the methoxyl group and the benzene ring of the solute molecule. The attraction between solute and solvent molecules has been suggested to be due to the van der Waals interactions and partly to the charge-transfer interaction.

Background: Patients with systemic sclerosis (SSc) complicated by gastrointestinal dysmotility are difficult to treat and have high mortality. To clarify the pathogenesis of gastrointestinal manifestations, we aimed to demonstrate the association among the clinical features of SSc, the serological markers, the autoantibodies against nicotinic acetylcholine receptor at autonomic ganglia (gAChR). Methods: Fifty patients were enrolled and divided into two groups according to the presence or absence of gastrointestinal manifestations, and the characteristics were analyzed between these two groups. We measured biomarkers and the autoantibodies against two gAChRα3 and β4 subunits to test sera samples. Furthermore, patients were classified based on the presence or absence of anti-gAChR autoantibodies, and their clinical features were compared. Results: In patients with SSc and gastrointestinal manifestations, digital ulcers were more frequent (p= 0.050) and VEGF expression was significantly higher (p= 0.038). Seven subjects with SSc were seropositive for α3 subunit, whereas one patient was seropositive for β4 subunit. The mean level of anti-gAChRα3 autoantibodies in SSc patients with gastrointestinal manifestations was significantly higher than that in SSc patients without gastrointestinal manifestations (p= 0.001). The group of patients with SSc and gAChR autoantibodies had significantly higher endostatin levels (p= 0.046). Conclusions: This study is the first to demonstrate that clinical characteristics of SSc patients with seropositivity for gAChR autoantibodies. Patients with SSc have circulating autoantibodies against gAChR, which may contribute to gastrointestinal manifestations associated with this disease, suggesting that gAChR-mediated autonomic neurotransmission may provide a pathomechanism for gastrointestinal dysmotility in SSc.

Summary: A 31-year-old Japanese woman was referred to our hospital after experiencing a convulsion. Upon radiological examination, a heterogeneously enhanced tumor was found on the anterior skull base. The tumor was surgically removed. On light microscopy, the tumor cells appeared spindle-shaped, forming an interwoven pattern. The nuclei were arranged partially parallel mimicking a palisading pattern. At first, the tumor was thought to be schwannoma. However, it was positive for S-100 and negative for both epithelial membrane antigen (EMA) and Leu7. The final diagnosis was olfactory ensheathing cell (OEC) tumor. OECs are similar to Schwann cells in microscopic appearance and upon immunohistochemical staining. However, the OECs are negative for CD57 (Leu7), while the Schwann cells are positive for it. Our patient’s tumor had immunological characteristics identical to those of OEC. In the English and Japanese literature, 21 cases of solitary schwannoma on the anterior skull base have been reported. Although several theories have been suggested, the pathogenesis of subfrontal schwannoma has not been clarified. Also, OECs have never been considered as their origin. However, as in our case, OECs, rather than Schwann cells, are suspected as the origin in some of the cases.

A case of aneurysm of the middle cerebral artery with congenital porencephaly in the same region is reported. The cyst was opened and the aneurysm was wrapped with a muscle strip. The resected specimen of the cyst wall contained a parenchymal layer with hemosiderinladen macrophages. It is speculated that bleeding from the aneurysm during the perinatal period caused the porencephaly.

The cytoplasmic protein Dok-7 is required for the full activation of the receptor tyrosine kinase MuSK.