Cellular hydrophobicity of Listeria monocytogenes involves initial attachment and biofilm formation on the surface of polyvinyl chloride

Aims: To clarify the cellular properties of Listeria monocytogenes involved in adhesion to and biofilm formation on polyvinyl chloride, a widely used material in the food manufacturing process. Methods and Results: A significant correlation between the ability of initial adherence to and biofilm formation on PVC was observed for 24 L. monocytogenes strains (Spearman rank‐correlation coefficient, r_s = 0·89). The swimming motility assay revealed no relationship between initial adherence and motility of L. monocytogenes. The microbial adhesion to solvent assay revealed an interaction of L. monocytogenes cells with nonpolar solvents, and a significant correlation was also observed between the degree of interaction with nonpolar solvents and initial adherence to PVC (r_s = 0·87 and r_s = 0·84, between initial adherence and affinities to decane and hexadecane, respectively). Conclusions: Results indicate that cellular hydrophobicity of L. monocytogenes is an important property involved in the initial adherence to and biofilm formation on PVC. Significance and Impact of Study: This study clarified the factors involved in the adherence to and biofilm formation ability of L. monocytogenes strains with PVC.     

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.     

PAL31 expression in rat trophoblast giant cells

PAL31 is a proliferation-related acidic nuclear protein that belongs to the leucine-rich protein family and is expressed cell-cycle-dependently. Trophoblasts differentiate into the trophoblast giant cells (TGCs) through the unusual type of cell cycle, namely endoreduplication. In the present study, we investigated the spatiotemporal pattern of PAL31 expression in rat placenta and Rcho-1 cell line. The PAL31 mRNA concentration varied in different areas of the placenta, and was barely detectable in the TGC layer. In Rcho-1 cells, although the level of PAL31 mRNA decreased dramatically during differentiation, PAL31 was detected even after differentiation. The site of intranuclear localization of PAL31 mostly overlapped with that of PCNA in the undifferentiated Rcho-1 cells, while they were not overlapped in differentiated cells. Thus, the subcellular localization of PAL31 in Rcho-1 cells significantly changed, and loss of cell cycle dependency after differentiation was noted. PAL31 is suggested to play a role in the endoreduplication distinct from the usual DNA duplication.     

Ricin A-chain inhibitors resembling the oxacarbenium ion transition state

Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a D(N)*A(N) mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609-1617]. On the basis of this information, stem-loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem-loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds four times better (0.57 microM) than the 10-base RNA stem-loop with adenosine at the depurination site (2.2 microM). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 microM and tightens to 0.75 microM in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds with a K(d) of 1.3 microM and improves to 0.65 micro;M with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem-loop, the Kd was 0.48 microM. Addition of 9-deazaadenine tightens the binding to 0.10 microM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative D(N)*A(N) mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.     

Combinatorial evaluation of the chiral discrimination of permethylated carbohydrates using fast-atom bombardment mass spectrometry

The chiral discrimination abilities of several variously permethylated carbohydrates toward various amino acid 2-propyl esters were combinatorially evaluated from the relative peak intensity of the 1:1 diastereomeric complex ions with the deuterium-labeled L-amino acid 2-propyl ester protonated ion and with the unlabeled D-amino acid 2-propyl ester protonated ions in FAB mass spectrometry. The chiral discrimination abilities evaluated using FAB mass spectrometry approximately corresponded to the ratio of the association constants (K(R)/K(S)) toward each enantiomer in the solution. Therefore, this evaluation method is very useful for the screening of the chiral discrimination abilities of carbohydrates and their derivatives.     

Endothelin-1 stimulates leptin production in adipocytes

Leptin is an adipocyte-derived hormone that regulates body fat stores and feeding behavior. In an effort to identify endogenous diffusible modulators of leptin production, we found that endothelin-1 (ET-1) up-regulates leptin expression in adipocytes. ET-1 is as potent and efficacious as insulin in stimulating leptin production in two different adipocyte cell lines. Endothelins stimulate leptin production via the endothelin-A receptor (ET(A)), as judged by a potency rank order of ET-1 ET-3. We detected expression of ET(A) but not ET(B) in both cell lines by Northern blot analysis. In addition, the ET(A)-selective antagonist FR139317 inhibited ET-1-induced leptin expression more potently than did the ET(B)-selective antagonist BQ788. ET-1 and insulin positively interact with each other in increasing leptin production in adipocytes. In primary mouse white fat cells, we detected expression of both ET(A) and ET(B) by Northern blot and in situ hybridization analyses. We conclude that ET-1 stimulates leptin production via the ET(A) receptor in cultured adipocytes.     

Serine proteinase inhibitor 3 and murinoglobulin I are potent inhibitors of neuropsin in adult mouse brain

Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 +/- 0.22 x 10(6) M(-1) s(-1), showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively.     

Rearrangements of the DNA in carbon ion-induced mutants of Arabidopsis thaliana

To elucidate the nature of structural alterations in plants, three carbon ion-induced mutations in Arabidopsis thaliana, gl1-3, tt4(C1), and ttg1-21, were analyzed. The gl1-3 mutation was found to be generated by an inversion of a fragment that contained GL1 and Atpk7 loci on chromosome 3. The size of the inverted fragment was a few hundred kilobase pairs. The inversion was found to accompany an insertion of a 107-bp fragment derived from chromosome 2. The tt4(C1) mutation was also found to be due to an inversion. The size of the intervening region between the breakpoints was also estimated to be a few hundred kilobase pairs. In the case of ttg1-21, it was found that a break occurred at the TTG1 locus on chromosome 5, and reciprocal translocation took place between it and chromosome 3. From the sequences flanking the breakpoints, the DNA strand breaks induced by carbon ions were found to be rejoined using, if present, only short homologous sequences. Small deletions were also observed around the breakpoints. These results suggest that the nonhomologous end-joining (NHEJ) pathway operates after plant cells are exposed to ion particles.     

Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.