Molecular monitoring of the developmental bacterial community in the gastrointestinal tract of Japanese infants

The dynamics of the developmental bacterial community in the Japanese neonatal gastrointestinal tract were examined by monitoring 16S ribosomal RNA gene (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE). The results showed a certain pattern common in infants without antibiotic treatment, in which aerobes, e.g., Pseudomonas, appeared first and were then immediately replaced by facultative anaerobe, Enterococcus, Streptococcus, and Enterobacteriaceae through the first month, and finally strictly anaerobic Bifidobactrerium appeared.     

Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phase

In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host-guest interactions might contribute to this enantiomer separation.     

Relationship between cerebral activity and movement frequency of maximal finger tapping

To examine the cerebral activity of the motor cortex during maximum movement, we measured regional cerebral blood flow (rCBF) in twelve normal volunteers, using near infrared spectroscopy (NIRS). Repetitive tapping of the right index finger was performed at 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 Hz, and during maximum effort (ME). The relative increase rate of rCBF during movement beginning with a resting condition was calculated for each movement condition. The left primary sensorimotor cortex showed significant activation during ME compared to the other frequencies. The rapid increase of rCBF was seen immediately after the initiation of finger tapping at all the tested frequencies but showed no increase following that. However, the rCBF during ME continued to increase until the end of the task.Change of the integrated electromyogram (iEMG) for the frequency and change of rCBF for the frequency at all the tested frequencies showed similar tendencies.     

Cytoplasmic transport signal is involved in phogrin targeting and localization to secretory granules

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic beta-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein (AP)-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules.     

Platelet activating factor induces cytoskeletal reorganization through Rho family pathway in THP-1 macrophages

In the process of atherosclerosis, platelet activating factor (PAF) promotes the infiltration of inflammatory cells into atherosclerotic plaque by modulating their cytoskeleton. Here, we examined whether Rho family proteins are involved in PAF-induced cytoskeletal reorganization in THP-1 macrophages. PAF stimulation rapidly induced cell elongation, accompanied by filopodia formation. The inhibition of Rho family proteins by the overexpression of Rho-GDI attenuated the PAF-mediated morphological changes. Both RhoA and Cdc42 were activated in response to PAF. Inhibition of RhoA or Cdc42 by dominant negative mutants abrogated morphological changes induced by PAF. Collectively, PAF regulates cytoarchitecture through Rho family proteins in macrophages.     

Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (delta5G). In delta5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and delta5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in delta5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in delta5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.     

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.     

S19 ribosomal protein dimer augments metal-induced apoptosis in a mouse fibroblastic cell line by ligation of the C5a receptor

To analyze the role of S19 ribosomal protein (RP S19) in apoptosis, murine NIH3T3 were transfected with either hemagglutinin peptide-tagged (HA) wild-type human RP S19 or a mutant (Gln137Asn) that is resistant to transglutaminase-catalyzed cross-linked-dimerization. Transfection with the mutant HA-RP S19 inhibited manganese (II) (Mn II)-induced apoptosis whereas the wild-type HA-RP S19 augmented apoptosis and a mock transfection had no effect. Release of the wild-type HA-RP S19 dimer but not the mutant HA-RP S19 was observed during the apoptosis. The reduced rate of apoptosis of the cells transfected with the mutant HA-RP S19 was overcome by addition of extracellular wild-type RP S19 dimer. The apoptosis rates in cells transfected with either form of human HA-RP S19 and in mock transfectants were reduced to about 40% by the presence of anti-RP S19 antibody in the culture medium. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analysis showed that the cell surface expression of the receptor for cross-linked RP S19 dimer, C5a receptor, increased during apoptosis, concomitant with phosphatidylserine exposure. The expression of the C5a receptor gene also increased twofold. Apoptosis rates in the transfected and control cell lines were also reduced by the presence of an anti-mouse C5a receptor monoclonal antibody or of a peptide C5a receptor antagonist. These results indicated the presence of an RP S19 dimer- and C5a receptor-mediated autocrine-type augmentation mechanism during Mn II-induced apoptosis in the mouse fibroblastic cell line. In contrast to the RP S19 dimer, C5a actually inhibited apoptosis, suggesting that signaling through the C5a receptor varies depending on the ligand bound.     

The influence of severe long-term exercise on the mouse hippocampus

To determine whether severe long-term exercise affects on the brain, we investigated the mice brain after 12-week treadmill exercise. The mice (ddN, male, 25-35 g in body weight) were divided into severe, mild, and non-exercise group. Mice in severe groups ran on a treadmill at a speed of 25 m/min for 12 weeks and mice in mild group ran on a treadmill at a speed of 10 m/min for 12 weeks. The mice were killed by transcardial perfusion with 0.1 M phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde in 0.1 M PB. The another sets of mice were fixed by 2.5% glutaraldehyde-2% osmium tetroxide for electromicroscope (EM). The brains were serially sectioned in the coronal plane at a thickness of 20-microns with a vibratome and then processed for histology, by means of hematoxylin-eosin (HE) staining and immunohistochemistry. Fifty % of mice in severe exercise showed hyperchromatic and shrunken nerve cells with nuclear pyknosis (dark neuron) in the hippocampus, but not in mild exercise and non-exercise groups. The immunoreactivity of microtuble associated protein-2 (MAP-2) decreased, while the heat-shock protein/cognate 70 (HSP/C 70) increased in the hippocampus of severe exercise group. Many destroyed mitochondria were observed in dark neurons by Electron micrograph. These findings suggested that severe long-term exercise might damage hippocampal neurons.