The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. The receptor for MAG or signals that elicit morphological changes in neurons remained to be established. Here we show that the neurotrophin receptor p75 (p75(NTR)) is the signal transducing element for MAG. Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice carrying a mutation in the p75(NTR) gene are insensitive to MAG with regard to neurite outgrowth. MAG activates small GTPase RhoA, leading to retarded outgrowth when p75(NTR)) is present. Colocalization of p75(NTR) and MAG binding is seen in neurons. Ganglioside GT1b, which is one of the binding partners of MAG, specifically associates with p75(NTR). Thus, p75(NTR) and GT1b may form a receptor complex for MAG to transmit the inhibitory signals in neurons.     

Immune tolerance to combined organ and bone marrow transplants after fractionated lymphoid irradiation involves regulatory NK T cells and clonal deletion

Immune tolerance to organ transplants has been reported in laboratory animals and in humans after nonmyeloablative conditioning of the host and infusion of donor bone marrow cells. We examined the mechanisms of immune tolerance to mouse cardiac allografts in MHC-mismatched hosts that developed mixed chimerism after posttransplant conditioning with a 2-wk course of multiple doses of lymphoid tissue irradiation, depletive anti-T cell Abs, and an infusion of donor bone marrow cells. When CD1(-/-) or J(alpha)281(-/-) hosts with markedly reduced NK T cells were used instead of wild-type hosts, then the conditioning regimen failed to induce tolerance to the heart allografts despite the development of mixed chimerism. Tolerance could be restored to the CD1(-/-) hosts by infusing enriched T cells from the bone marrow of wild-type mice containing CD1-reactive T cells but not from CD1(-/-) host-type mice. Tolerance could not be induced in either IL-4(-/-) or IL-10(-/-) hosts given the regimen despite the development of chimerism and clonal deletion of host T cells to donor MHC-Ags in the IL-10(-/-) hosts. We conclude that immune tolerance to bone marrow transplants involves clonal deletion, and tolerance to heart allografts in this model also involves regulatory CD1-reactive NK T cells.     

Csx/Nkx2-5 is required for homeostasis and survival of cardiac myocytes in the adult heart

Csx/Nkx2-5, which is essential for cardiac development of the embryo, is abundantly expressed in the adult heart. We here examined the role of Csx/Nkx2-5 in the adult heart using two kinds of transgenic mice. Transgenic mice that overexpress a dominant negative mutant of Csx/Nkx2-5 (DN-TG mice) showed degeneration of cardiac myocytes and impairment of cardiac function. Doxorubicin induced more marked cardiac dysfunction in DN-TG mice and less in transgenic mice that overexpress wild type Csx/Nkx2-5 (WT-TG mice) compared with non-transgenic mice. Doxorubicin induced cardiomyocyte apoptosis, and the number of apoptotic cardiomyocytes was high in the order of DN-TG mice, non-transgenic mice, and WT-TG mice. Overexpression of the dominant negative mutant of Csx/Nkx2-5 induced apoptosis in cultured cardiomyocytes, while expression of wild type Csx/Nkx2-5 protected cardiomyocytes from doxorubicin-induced apoptotic death. These results suggest that Csx/Nkx2-5 plays a critical role in maintaining highly differentiated cardiac phenotype and in protecting the heart from stresses including doxorubicin.     

A novel lantibiotic, nukacin ISK-1, of Staphylococcus warneri ISK-1: cloning of the structural gene and identification of the structure

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.     

Production of interferon-β in a culture of fibroblast cells on some polymeric films

Normal human skin (NB1-RGB) cells were cultured in the presenceof polyinosinic and polycytidylic acids, diethylaminoethyldextran, cycloheximide and actinomycin D, which induced humaninterferon-. The simplest induction method, that requiredonly polyinosinic and polycytidylic acids and diethylaminoethyldextran was found to give the highest production ofinterferon- by the cells. The cell growth and productionof interferon- were investigated for NB1-RGB cellscultured on silk fibroin, poly(-methyl-L-glutamate),poly(-benzyl-L-glutamate) and collagen films prepared bythe Langmuir-Blodgett (LB) and casting methods. The cell densityof NB1-RGB cells cultured on the LB films was found to be higherthan that on the cast films made of the same polymer. Thisindicates that not only the chemical structure of the polymersused for the preparation of the films but the preparationmethods of the films, i.e., casting and LB methods, are also astrong factor affecting the cell growth. The production ofinterferon- per unit number of cells was found to behigher on the cast films than that on the LB films made of thesame polymer. This is explained by the fact that the optimalsuppressed growth of NB1-RGB cells on the cast films leads tothe enhanced production of interferon- on the cast filmscompared to those on the LB films prepared by the same polymer.     

Diurnal variation in the P300 component of human cognitive event-related potential

Diurnal variation in the P300 component of the human cognitive event-related potential (ERP) was examined. The P300 component is considered to be a measure of neuroelectric activity related to cognitive functions such as attention allocation and information processing. Nine diurnally active healthy male subjects whose sleep-wake rhythms were synchronized prior to the experiment were studied. The P300 components oral temperature, heart rate, left- and right-hand grip strength, reaction time, subjectively rated sleepiness, attention level, and fatigue were measured at 08:00, 11:00, 14:00, 17:00, and 20:00. Significant diurnal variations in P300 latency, P300 amplitude, oral temperature, heart rate, left- and right-hand grip strength, subjectively rated sleepiness, and attention level were observed. The P300 latency at 08:00 was significantly longer than at 11:00, 17:00, and 20:00, while the P300 amplitude at 08:00 was significantly greater than at 17:00 and 20:00. The P300 latency was correlated positively with subjectively rated sleepiness and negatively correlated with subjectively rated attention level. These results suggest the existence of diurnal variation in human cognitive functions.     

Reliability and validity of the questionnaire to determine the biosocial rhythms of daily living in the disabled elderly

The questionnaire to determine the biosocial rhythms of daily living in the disabled elderly was newly developed. This questionnaire was aimed to evaluate a state of synchronization of biological rhythms in the disabled elderly. Eighteen items of the questionnaire relating to the synchronization of biological rhythms were finally selected by the test-retest method that was conducted for 68 disabled elderly living in a community with a duration of one year. The factor analysis showed that the questionnaire consisted of five factors: outdoor activities, ultradian rhythms, subjective evaluation of health status, social support, and sleep habits. The cumulative contribution rate of five factors was 53.2%. Reliability of the questionnaire was confirmed by a calculation of the Equal-length Spearman-Brown coefficients ranging from 0.60 to 0.80. Regarding the construct validity of the questionnaire, results of factor analysis showed five factors that were consistent with the synchronizers known in chronobiology. The total score of the questionnaire was significantly correlated to Barthel Index score and the competence score, suggesting that it partly reflects the activities of daily living of the disabled elderly. We conclude that a new questionnaire to determine the biosocial rhythm of daily living in the disabled elderly is useful to evaluate the biosocial synchronization of the disabled elderly because of its high reliability and validity.     

Regulation of human extravillous trophoblast function by membrane-bound peptidases

During human placentation, the invasion of extravillous trophoblasts (EVTs) into maternal decidual tissues, especially toward maternal spiral arteries, is considered an essential process for subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVTs from further invasion have not been well understood. Recently, we found that two cell surface peptidases, dipeptidyl peptidase IV (DPPIV) and carboxypeptidase-M (CP-M,) are differentially expressed on EVTs. DPPIV expression was mainly observed on EVTs that had already ceased invasion. CP-M was detected on migrating EVTs including endovascular trophoblasts in the maternal arteries. The enzymatic inhibition of these peptidases affected the invasive property of choriocarcinoma-derived cell lines, BeWo and JEG3 cells. In addition, a chemokine, RANTES, that is one of the substrates for DPPIV, enhanced invasion of EVTs isolated from primary villous explant culture and its receptor, CCR1, was specifically expressed on migrating EVTs toward maternal arteries. Furthermore, a novel membrane-bound cell surface peptidase, named laeverin, was found to be specifically expressed on EVTs that had almost ceased invasion. These findings suggest that membrane-bound peptidases are important factors regulating EVT invasion during early placentation in humans.     

Sulindac activates nuclear translocation of AIF, DFF40 and endonuclease G but not induces oligonucleosomal DNA fragmentation in HT-29 cells

Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100-300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells.     

Different cation sensitivities and binding site domains of Na+-Ca2+-K+ and Na+-Ca2+ exchangers

We examined inhibitory effects of external multivalent cations Ni(2+), Co(2+), Cd(2+), La(3+), Mg(2+), and Mn(2+) on reverse-mode exchange of the K(+)-dependent Na(+)/Ca(2+) exchanger NCKX2 and the K(+)-independent exchanger NCX1 expressed in CCL-39 cells by measuring the rate of Ca(2+) uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca(2+) uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li(+) stimulated reverse-mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K(+). Thus, both exchangers exhibited differential sensitivities to not only K(+) but also many other external cations. We attempted to locate the putative binding sites within the alpha motifs for multivalent cations by site-directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the alpha-1 motif, but not by mutations in the alpha-2 motif. These results contrast with those for NCX1 where mutations in both alpha-1 and alpha-2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the alpha-1 and alpha-2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular-facing alpha-1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both alpha-1 and alpha-2 motifs together form the binding sites in NCX1.