Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

Further characterization of a conserved actin-binding 27-kDa fragment of actinogelin and alpha-actinins and mapping of their binding sites on the actin molecule by chemical cross-linking

A conserved actin-binding domain (Mr = 27,000) of rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins (Mimura, N., and Asano, A. (1986) J. Biol. Chem. 261, 10680-10687) was separated into two components having different isoelectric points (peptides A and B) by chromatofocusing. Thermolysin digestion of peptide A generated peptide B with concomitant loss of peptide A. Amino acid compositions and tryptic maps of peptides A and B also demonstrated that peptide A is a precursor of peptide B upon thermolysin digestion. All of peptides A and B retained the activity to bind with F-actin competitively to each other. By the gel-filtration method, it was also shown that the native actin-binding 27-kDa fragments are monomeric and globular. The non-actin-binding 50- or 53.5-kDa fragment of actinogelin/alpha-actinins was, however, found to be asymmetric and dimeric in the native state. Chemical cross-linking of the 27-kDa fragment with F-actin with a water-soluble carbodiimide produced at least four different complexes (I-IV). Chemical cleaving analysis of the cross-linked products (complexes I and II) indicated that the 27-kDa fragment possesses two possible binding sites on actin at the NH2-terminal residues 1-12 (for complex I) and at residues spanning 86-119 or 123 (for complex II).     

A Modification of Mayer's Tannic Acid-Ferric Chloride Staining Method for Demonstrating Cellular Membranous Systems for Light Microscopy

To observe cellular membranous systems under a light microscope, we modified Mayer's tannic acid-ferric chloride stain method by adding a treatment with hematoxylin after the original procedure. We used the modified tannic acid-ferric chloride (MTA-Fe) stain method to examine kidneys, liver, heart, trachea, epididymides and other organs of rats and dogs. The MTA-Fe stain clearly demonstrated the basement membrane, brush border, basolateral invaginations and cell processes in the kidneys which enabled easy differentiation of the S1 and S3 segments of proximal convoluted tubules. Our technique also demonstrated hepatic cell membranes and bile canaliculi in the liver, cross striations and longitudinal traveling of myofibrils in the heart, cilia of the epithelial cells in the trachea, and stereocilia and terminal bars in the epididymis. The MTA-Fe stain is a convenient method to visualize cellular membranous systems even for light microscopy. The stain has the advantages of using no toxic materials, simple and easy technique, little variation of staining results, and little fading for several months after staining.     

Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.     

A transmembrane glycoprotein, gp38, is a novel marker for immature hepatic progenitor cells in fetal mouse livers

Previously, we clarified the surface antigen profiles of hepatic progenitor cells (HPCs) in fetal liver tissue as the CD49f(+)CD45(-)Thy1(-) cell fraction. However, these cells were a heterogeneous cell population containing various stages of differentiation. This study aimed to detect more immature HPCs, using a novel surface antigen, gp38. After the collagenase digestion of fetal livers harvested from E13.5 to E18.5 fetal mice, HPCs were obtained and divided into two subpopulations using flow cytometry: gp38-positive HPCs, and gp38-negative HPCs. Both types of HPCs were characterized by immunocytochemistry and RT-PCR. The proliferative activity was compared by BrdU incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay. Furthermore, the comprehensive gene expression was investigated by DNA microarray. Both types of HPCs expressed alpha-fetoprotein. However, the gp38-positive HPCs derived from E13.5 fetal livers did not express albumin or cytokeratin 19, while the gp38-negative HPCs did. DNA microarray revealed that some genes related to the Wnt signal pathway were up-regulated in the gp38-positive HPCs. Furthermore, Wnt3a had a proliferative effect on the gp38-positive HPCs. In conclusion, the gp38-positive HPCs derived from fetal liver tissue until E13.5 could therefore be candidates for hepatic stem cells in the fetal liver.     

Predicting the success of an invader: Niche shift versus niche conservatism

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.     

A bitter melon extract inhibits the P-glycoprotein activity in intestinal Caco-2 cells: monoglyceride as an active compound

P-glycoprotein (P-gp) is a 170 kDa membrane protein that belongs to the ATP-binding cassette (ABC) transporter superfamily. In normal tissues, P-gp functions as an ATP-dependent efflux pump that excretes highly hydrophobic xenobiotic compounds, playing an important role in protecting the cells/tissues from xenobiotics. In the present study, chemical substances that could directly modulate the intestinal P-gp activity were searched in vegetables and fruits. By using human intestinal epithelial Caco-2 cells as a model of the small intestinal cells, we observed that a bitter melon fraction extracted from 40% methanol showed the greatest increase of the rhodamine-123 accumulation by Caco-2 cells. Inhibitory compounds in the bitter melon fraction were then isolated by HPLC using Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by (1)H-NMR and FAB-MS, and the active compound was identified as 1-monopalmitin. It is interesting that certain types of monoglyceride might be involved in the drug bioavailability by specifically inhibiting the efflux mediated by P-gp.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.