Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system

To establish a strategy for the comprehensive identification of human N‐myristoylated proteins, the susceptibility of human cDNA clones to protein N‐myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell‐free protein synthesis system. One‐hundred‐and‐forty‐one cDNA clones with N‐terminal Met‐Gly motifs were selected as potential candidates from ～2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N‐myristoylation was evaluated using fusion proteins, in which the N‐terminal ten amino acid residues were fused to an epitope‐tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N‐myristoylated. The metabolic labeling experiments both in an insect cell‐free protein synthesis system and in the transfected COS‐1 cells using full‐length cDNA revealed that 27 out of 29 proteins were in fact N‐myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N‐myristoylated proteins that have not been reported previously to be N‐myristoylated, indicating that this strategy is useful for the comprehensive identification of human N‐myristoylated proteins from human cDNA resources.     

Augmentation of fatality of influenza in mice by inhibition of phagocytosis

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages, and this leads to the inhibition of virus propagation in vitro. To assess if this were also true in vivo, mice infected with influenza A/WSN (H1N1) virus were administered with phagocytosis inhibitors and examined for the progress of influenza. Administration of the inhibitors caused a decrease in the level of phagocytosis observed with bronchoalveolar lavage cells. We found that both the lethality in mice and the extent of inflammation in the lung were augmented in those mice. These results suggest that phagocytosis of virus-infected cells helps suppress the progress of influenza in mice.     

Role of follicular dendritic cells in the early HIV-1 infection: in vitro model without specific antibody

About 90% of HIV-1 RNA in the lymph nodes is reported to localize in follicular dendritic cellsnetwork (FDC-NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV-1 infection, FDC-like cell strains (FDCLC) were established and they were characterized in the co-culture system with T cells for their effect on HIV-1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA-42, S-100alpha and intercellular desmosome-like junctions. L-SIGN and DC-SIGN were also detected in FDCLC. Alu-HIV-1 PCR analysis showed no HIV-1 integration in FDCLC. FDCLC trapped HIV-1 and transferred them to uninfected MOLT-4 T cells (MOLT-4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV-1 replication in HIV-1-pre-exposed MOLT-4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up-regulated CD4 expression in MOLT-4 and helped T cells escape from apoptosis in the co-culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV-1 in the absence of specific antibody.     

Role of afferent input in muscle atrophy.

It is well known that soleus muscle of rat atrophies following spaceflight or hindlimb suspension (Ohira et al., 1992). It is, further, reported that the electromyogram (EMG) of soleus muscle disappears immediately in response to unloading by exposure to actual micro-g environment (Kawano et al., 2002; Leterme and Falempin, 1998) and by hindlimb suspension of rats (Alford et al., 1987; Ohira et al., 2000). However, the EMG level is increased gradually to the control level following 7-10 days of continuous hindlimb suspension (Alford et al., 1987; Ohira, 2000), while muscle atrophy is progressing (Winiarski et al., 1987). We previously reported that reduction of the EMG level of rat soleus in response to actual micro-g environment, created by a parabolic flight of a jet airplane, was closely associated with a decrease of the afferent input recorded at the L5 segmental level of spinal cord (Kawano et al., 2002). However, it is still unclear how the EMG level of soleus muscle adapts to unloading condition. The current study was performed to investigate the responses of soleus EMG and both afferent and efferent neurogram at the L5 segmental level of spinal cord to acute (20 seconds) and chronic (14 days) unloading.     

Development and evaluation of intermediate frequency magnetic field exposure system for studies of in vitro biological effects

We have developed an intermediate frequency (IF) magnetic field exposure system for in vitro studies. Since there are no previous studies on exposure to heating-frequency magnetic fields generated from an induction heating (IH) cook top, there is a strong need for such an exposure system and for biological studies of IF magnetic fields. This system mainly consists of a magnetic-field-generating coil housed inside an incubator, inside which cultured cells can be exposed to magnetic field. Two systems were prepared to allow the experiment to be conducted in a double-blind manner. The level of the generated magnetic field was set to 532 microT rms in the exposure space, 23 kHz, 80 times the value in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines, with a spatial field uniformity better than 3.8%. The waveforms were nearly sinusoidal. It was also confirmed that the parasitic electric field was 157 V/m rms and the induced electric field was 1.9 V/m rms. The temperature was maintained at 36.5 +/- 0.5 degrees C for 2 h. Furthermore, leaked magnetic flux density was 0.7 microT rms or lower at extremely low frequency (ELF) and IF in the stopped system when the other system was being operated, and the environmental magnetic flux density was 0.1 microT rms or lower at the center of the coils. As a result, it was confirmed that this system could be successfully used to evaluate the biological effects of exposure to IF magnetic fields.     

Action of neltenexine on anion secretion in human airway epithelia

Neltenexine has been applied to human lung diseases such as chronic obstructive pulmonary disease (COPD) as a mucolytic agent. However, we have no information on the neltenexine action in bronchial epithelial cells. We studied the neltenexine action on the ion transport in human submucosal serous Calu-3 cells. Under a hyper-secreting condition caused by terbutaline (a beta2-adrenergic agonist), neltenexine diminished anion secretion by inhibiting the Cl- and HCO3- uptake via Na+/K+/2Cl- cotransporter and Na+/HCO3- cotransporter without blockade of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, and also diminished anion secretion via stimulation of Cl-/HCO3- exchanger, which facilitates the extrusion of more CFTR-permeant anion, Cl-, with the uptake of less CFTR-permeant anion, HCO3-. Thus, neltenexine reduced the hyper-secretion to keep an appropriate fluid level in the airway, providing a possibility that neltenexine can be an effective drug in airway obstructive diseases by decreasing the airway resistance under a hyper-secreting condition.     

Cyclodextrin-linked alginate beads as supporting materials for Sphingomonas cloacae, a nonylphenol degrading bacteria

Calcium alginate beads covalently linked with alpha-cyclodextrin (alpha-CD-alginate beads) were prepared and examined for their ability to serve as a supporting matrix for bacterial degradation of nonylphenol, an endocrine disruptor. Column chromatographic experiment using alpha-CD-alginate beads with diameter of 657+/-82 microm and with degree of CD substitution of 0.16 showed a strong affinity for nonylphenol adsorption. Although addition of alpha-CD (2.7-27 mM) to the culture broth of Sphingomonas cloacae retarded nonylphenol degradation, the immobilized bacteria on the CD-alginate beads were effective for the degradation. Batch degradation tests using the immobilized bacteria on alpha-CD-alginate-beads showed 46% nonylphenol recovery after 10-day incubation at 25+/-2 degrees C, and the recovery reached to about 17% when wide and shallow incubation tubes were used to facilitate uptake of the viscous liquid of nonylphenol on the surface of the medium. Scanning electron microscopic photographs revealed that multiplicated bacteria was present both on the surface and inside the beads and the matrix of CD-alginate was stable and suitable during 10-day incubation.     

Effects of functional overloading on the regenerative potential of injured skeletal muscles in mice

The purpose of this study was to investigate the effects of functional overload on the regeneration of injured skeletal muscles of male C57BL/6J mice. To activate a necrosis-regeneration cycle, cardiotoxin (CTX) was injected into soleus muscles both control and functionally overloaded groups. The recovery of muscle protein content, which was decreased by CTX injection, was significantly stimulated by application of functional overloading. The CTX-injection-related increment of satellite cell number in the overloaded groups was also greater than that in the group without overloading. Evidences suggest that the application of a mechanical stress on the injured skeletal muscles could activate satellite cells and facilitate the regeneration of the muscle.     

Nanobiotechnology: quantum dots in bioimaging

Many biological systems, including protein complexes, are natural nanostructures. To better understand these structures and to monitor them in real time, it is becoming increasingly important to develop nanometer-scale signaling markers. Single-molecule methods will play a major role in elucidating the role of all proteins and their mutual interactions in a given organism. Fluorescent semiconductor nanocrystals, known as quantum dots, have several advantages of optical and chemical features over the traditional fluorescent labels. These features make them desirable for long-term stability and simultaneous detection of multiple signals. Here, we review current approaches to developing a biological application for quantum dots.