The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ∼30 and ∼100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

Abstract. CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20–30–amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

No abstract

Asymptomatic community dwelling elderly individuals with postural hypotension as well as those with postural hypertension had poorer scores on neurobehavioral function tests and more advanced leukoaraiosis demonstrated on MRI than those without exaggerated postural changes in SBP.

No abstract

We have evaluated the association between trunk deformities of the sagittal plane and functional impairment of daily living in community-dwelling elderly subjects. The analysis involved a detailed assessment of indoor and outdoor activities of daily living, satisfaction with life, and mental status. The participants in this study were 236 community-dwelling older adults, aged 65 years and older, living in Kahoku district of Kochi in Japan. The participants were classified based on their posture, which was assessed using photographs of the subjects, and interviewed to assess their basic activities of daily living (BADL), instrumental ADL (IADL), and cognitive well-being in the cross-sectional study. The statistical analysis was performed using the Mann-Whitney U-test. The lumbar kyphosis group received significantly lower BADL and IADL scores than the normal group. The trunk deformity group which were defined as kyphosis, flat back, and lumbar lordosis groups exhibited decreases in activities that included going out, shopping, depositing and withdrawing money, and visiting friends in the hospital. These activities require going outdoors; thus, this study showed that the trunk deformity group had limitations in outdoor activities. There was no significant difference between the geriatric depression score (GDS) and the pattern of posture. The abnormal trunk deformity groups tended to score lower than the normal group with regard to subjective healthiness and life satisfaction measures, including subjective health condition, everyday feeling, satisfaction with human relationships, satisfaction with economic condition, and satisfaction with present life.

CARP abnormalities may be involved in the pathogenesis of HCM.