Prediction of intracellular targets of a small compound by analyzing peptides presented on MHC class I

In chemical biology, the elucidation of chemical target is crucial for successful drug development. Because MHC class I molecules present peptides from intracellular damaged proteins, it might be possible to identify targets of a chemical by analyzing peptide sequences on MHC class I. Therefore, we treated cells with the autophagy-inducing chemical TMD-457 and identified the peptides presented on MHC class I. Many of the peptides were derived from molecules involved in ER trafficking and ER stress, which were confirmed by morphological and biochemical analyses. Therefore, our results demonstrate that analyzing MHC class I peptides is useful for the detection of chemical targets.     

Evidence that C4b-binding protein (proline-rich protein) is synthesized by hepatocytes

C4b-binding protein (C4bp), a glycoprotein involved in regulating the classical pathway of the complement system, binds the activated form of C4b and accelerates the decay rate of the C4b, C2a complex. Recently, sequence analysis of the cDNA for proline-rich protein (PRP) demonstrated that PRP is identical with C4bp. We measured the concentration of C4bp in serum by single radial immunodiffusion in patients with various liver diseases. Concentration of C4bp was significantly lower in hepatic cirrhosis (P = 0.001) and higher in fatty liver (P = 0.0002) than the control values, after adjusting for age, sex, and concentration of total cholesterol, triglyceride, and C-reactive protein. Significant positive correlations were observed between the concentration of C4bp in serum and total protein, albumin, cholinesterase level, and lecithin-cholesterol acyltransferase activity. Immunohistochemical analysis of human liver with specific antiserum to human C4bp demonstrated reaction endproducts in the hepatocytes around the central veins. These observations provide evidence that C4bp is synthesized by hepatocytes.     

Human ABCA1 contains a large amino-terminal extracellular domain homologous to an epitope of Sjögren's Syndrome

ABCA1 has been suggested to play a key role in cellular lipid release from peripheral cells. In order to study structure-function relationship of this protein, the protein product of a full-length human ABCA1 cDNA was examined for its functions and topological orientation. The electrophoretic mobilities of human ABCA1 expressed in transfected cells increased when treated with N-glycosidase F, suggesting that ABCA1 is highly glycosylated. The ABCA1 was photoaffinity-labeled with ATP and mediated the apoA-I-dependent-release of cholesterol and phospholipid. The influenza hemagglutinin (HA) epitope was introduced into the amino-terminus (N-HA) or between the residues 207 and 208 (207-HA) of the protein. While an antibody against the C-terminus peptide of ABCA1 detected both fusion proteins, an anti-HA antibody did not react with the N-HA fusion protein. Confocal microscopy demonstrated strong cell surface signal with the anti-HA antibody of nonpermeabilized HEK293 cells expressing the 207-HA fusion protein. The results suggested that the signal peptide in the amino-terminal region is cleaved off in its mature form and that the following large hydrophilic region is exposed to outside of cells unlike previously proposed models. We found that this amino-terminal extracellular domain contains a segment homologous to the autoantigen SS-N, an epitope of Sj?gren's syndrome, and further identified that ABCA7 codes for the autoantigen SS-N.     

The distribution of sugar chains on the vomeronasal epithelium observed with an atomic force microscope

The distribution of sugar chains on tissue sections of the rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined with an atomic force microscope (AFM). AFM tips were modified with a lectin, Vicia villosa agglutinin, which recognizes terminal N-acetyl-D-galactosamine (GalNAc). When a modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample surface were observed. The final rupture force was calculated to be approximately 50 pN based on the spring constant of the AFM cantilever. Distribution patterns of sugar chains obtained from the force mapping image were very similar to those observed using fluorescence-labeled lectin staining. AFM also revealed distribution patterns of sugar chains at a higher resolution than those obtained with fluorescence microscopy. Most of the adhesive interactions disappeared when the scanning solution contained 1 mM GaINAc. The adhesive interactions were restored by removing the sugar from the solution. Findings suggest that the adhesion force observed are related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium.     

Onset of amnesia is delayed with a decrease in inhibition of protein synthesis during odor-taste associative learning in the terrestrial slug Limax valentianus

Slugs can retain odor-taste associative memory for several weeks, and this requires protein synthesis. We examined the dose-dependency of the onset time of amnesia caused by the protein synthesis inhibitor anisomycin, and showed that with reduced dose, the onset is shifted from 2 days to 3 days after conditioning; we could not shift the onset delay later than 3 days. Our results suggest that the mechanism underling memory retention is different in the period up to 3 days, versus the period later than 3 days. Our results also suggest that sustained inhibition of protein synthesis in the period from zero to 3 hr after conditioning is necessary to cause amnesia.     

Molecular evidence of the existence of two sibling species within the echinothurioid echinoid Asthenosoma ijimai from Japanese waters

The echinoid, Asthenosoma ijimai belonging to the order Echinothurioida from Japanese waters shows the geographical variation in morphological and ecological characters. The echinothurioid from Ryukyu Islands in southern Japan is cleary different from that of Sagami Bay and Suruga Bay in the middle part of Japan at non-molecular level.Their phylogenetic and taxonomic relationships were studied at the molecular level by allozyme analysis. The results demonstrated that the two echinothurioids from Ryukyu Islands and Sagami Bay do not share gene pools with each other, and they were fixed for different alleles at five genetic loci (Mdh, G6pd, Po, Alk-3 and Est-7) in a total of 23 enzyme genetic loci scored. This indicates no gene flow between the two echinothurioids, and is a molecular evidence for that they are reproductively isolated and genetically distinct species. The Nei's genetic distance (D=0.181) between the two were significantly higher than those between conspecific local populations, and comparable to those between closely related species in many other animals containing echinoderms. The present molecular data are well consistent with the non-molecular evidence from morphology, developmental biology and ecology. Putting these data together, we propose that the two echinothurioids should be classified as two sibling species of the genus Asthenosoma and would like to give the following scientific names: the echinothurioid species from Sagami Bay is Asthenosoma ijimai and that from Ryukyu Islands is A. ijimai R.     

Chironomid Egg Masses as a Natural Reservoir of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Non-O1 and Non-O139 in Freshwater Habitats

Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year. The natural reservoir of the bacterium is environmental. A recent report indicated an association between V. cholerae and chironomid egg masses. Chironomids, the non-biting midges (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater. Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the waters edge where bacteria are abundant and may encounter the nutrient-rich substrate. Here we report the isolation of non-O1 and non-O139 V. cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa. In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region. The bacterial population rose as water temperature surpassed 25°C. Thirty-five different serogroups of V. cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity. Two strains of V. cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive. Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V. cholerae.