AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria and are important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel TolC and the membrane fusion protein AcrA. Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer. Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.

Objective: Fascin, an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers, induces membrane protrusions and increases cell motility in various transformed cells. The expression of fascin in epithelial neoplasms has been described only recently, and its role in gastric cancer is still unknown. Methods: Paraffin sections of gastric carcinoma from 214 patients were immunohistochemically investigated using monoclonal antifascin antibody. Staining more than 5% of tumor cells was recorded as positive immunoreactivity. Results: Overall, fascin immunoreactivity was detected in 54 out of a total of 214 patients (25%). 26 patients were classified as 1+ (5–25% immunoreactive tumor cells) and 28 were 2+ (>25%). In these patients, 7 tumors showed high (>75%) fascin immunoreactivity. Increased immunoreactivity of fascin was sometimes seen at the edge of the tumor. Fascin immunoreactivity was increased according to the extent of primary tumor (p = 0.026). Fascin expression was correlated with age (p = 0.005), serosal invasion (p = 0.013), positive lymph node metastasis (p = 0.006), histopathological grading (p = 0.019), TNM stage (p = 0.003) and recurrence (p = 0.006); however, it was not correlated with distant metastasis (p = 0.108), Lauren’s type (p = 0.205), or R classification (p = 0.056). Among 166 patients with T1, T2, T3 or T4, those with fascin-positive tumors had a significantly poorer prognosis than those with fascin-negative tumors (p = 0.029). Multivariate analysis showed that fascin expression was not an independent poor prognostic factor. Conclusion: Our findings suggest that the immunohistochemical detection of fascin could provide useful information as one of the prognostic factors in gastric cancer patients.

Human melanoma cells can process the The discoveries of genes encoding a number of human melanoma-associated antigens (1-6) and of sequences of antigenic peptides serving as the cytolytic T-lymphocyte (CTL) epitopes on the appropriate major histocompatibility complex (MHC)class I molecules (7-11) have raised considerable interest in peptide-based specific melanoma vaccines. All the CTLdetermined melanoma-associated peptide epitopes that have so far been described are, however, "self' peptides (i.e., without any alteration in the sequence in the encoding gene found in the autologous normal cells). The question of whether these self peptide epitopes are immunogenic in humans has, therefore, become a crucial issue. To test the immunogenic potential of a peptide-based melanoma vaccine in vivo, we have taken an approach based on the physiologic principles underlying peptide presentation to T cells. This approach involves presenting a relevant CTL-determined peptide on the appropriate MHC-restricting elements of autologous professional antigen presenting cells (APCs). We chose to study the immunogenicity of the MAGE-1 nonapeptide EADPTGHSY (7) in vivo, since the MAGE-1 gene is not expressed in any normal tissues with the exception of certain cells in the testis (1).We have observed that a population of APCs, obtained by culturing autologous blood-derived monocyte/macrophages in granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit many of the essential features of authentic APCs (high levels of MHC class I and class II molecules, CD11b antigen, CD54 antigen, B7 molecules, and dendritic morphology expressed by a proportion of the cells). They are also capable of simultaneously providing an additional stimulatory signal(s) toward T-cell activation-analogous to some form of costimulatory, or just another stimulatory, second signal.These cultured APCs can efficiently present the MAGE-1 nonapeptide to the MAGE-1 antigen-specific CTLs (12). More importantly, they seem to be capable of overriding the negative influence of melanoma cells while activating autologous T cells in vitro (12). Based on these findings, this study was designed to examine whether the cultured APCs, pulsed with the synthetic peptide EADPTGHSY, could recruit a MAGE-1-specific CTL response in vivo at the immunization sites and at distant sites of melanoma deposits in patients with advanced stage IV melanoma, who are HLA-A1+, and whose tumor cells express the MAGE-1 gene.MATERIALS AND METHODS Patients. Patients with advanced metastatic melanoma were studied with informed consent.Cell Lines. The melanoma cell lines RM-M (HLA-A1, -3; B8, -44) and HS-M (HLA-A1, -28; B14, -57) were established from explants of metastatic melanoma in our laboratory from the two study cases according to procedures described earlier.The melanoma cell line MZ 3.1 (MAGE-1+/HLA-A1+) and the MAGE-1-/HLA-A1+ variant melanoma line MZ 2.2 and the MAGE-1 antigen-specific CTL line MZ 82/30 were gifts of Thierry Boon (Ludwig Institute, Brussels).APCs. Autologous perip...

AcrB is the major multidrug exporter in Escherichia coli. Although several substrate-entrances have been identified, the specificity of these various transport paths remains unclear. Here we present evidence for a substrate channel (channel 3)  from the central cavity of the AcrB trimer, which is connected directly to the deep pocket without first passing the switch-loop and the proximal pocket . Planar aromatic cations, such as ethidium, prefer channel 3 to channels 1 and 2. The efflux through channel 3 increases by targeted mutations and is not in competition with the export of drugs such as minocycline and erythromycin through channels 1 and 2. A switch-loop mutant, in which the pathway from the proximal to the deep pocket is hindered, can export only channel 3-utilizing drugs. The usage of multiple entrances thus contributes to the recognition and transport of a wide range of drugs with different physicochemical properties.

Sex determination in cucumber (Cucumis sativus L.) plants is genetically controlled by the F and M loci. These loci interact to produce three different sexual phenotypes: gynoecious (M-F-), monoecious (M-ff), and andromonoecious (mmff). Gynoecious cucumber plants produce more ethylene than do monoecious plants. We found that the levels of ethylene production and the accumulation of CS-ACS2 mRNA in andromonoecious cucumber plants did not differ from those in monoecious plants and were lower than the levels measured in gynoecious plants. Ethylene inhibited stamen development in gynoecious cucumbers but not in andromonoecious ones. Furthermore, ethylene caused substantial increases in the accumulation of CS-ETR2, CS-ERS, and CS-ACS2 mRNA in monoecious and gynoecious cucumber plants, but not in andromonoecious one. In addition, the inhibitory effect of ethylene on hypocotyl elongation in andromonoecious cucumber plants was less than that in monoecious and gynoecious plants. These results suggest that ethylene responses in andromonoecious cucumber plants are reduced from those in monoecious and gynoecious plants. This is the first evidence that ethylene signals may influence the product of the M locus and thus inhibit stamen development in cucumber. The andromonoecious line provides novel material for studying the function of the M locus during sex determination in flowering cucumbers.

It has been reported that ethylene production by cucumber plants is strongly related to the sex expression of their flowers. It has also been shown that both CS-ACS2 gene expression and ethylene evolution are much greater in gynoecious cucumber plants than monoecious ones. To investigate the action mechanism of ethylene in the induction of femaleness of cucumber flowers, we isolated three ethylene-receptor-related genes, CS-ETR1, CS-ETR2 and CS-ERS, from cucumber (Cucumis sativus L.) plants. Of these three genes, CS-ETR2 and CS-ERS mRNA accumulated more substantially in the shoot apices of the gynoecious cucumber than those of the monoecious one. Their expression patterns correlated with the expression of the CS-ACS2 gene and with ethylene evolution in the shoot apices of the two types of cucumber plants. Accumulation of CS-ETR2 and CS-ERS mRNA was significantly elevated by the application of Ethrel, an ethylene-releasing agent, to the shoot apices of monoecious cucumber plants. In contrast, the accumulation of their transcripts was lowered when aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene biosynthesis, was applied to the shoot apices of gynoecious cucumber plants. Thus, the expression of CS-ETR2 and CS-ERS is, at least in part, regulated by ethylene. The greater accumulation of CS-ETR2 and CS-ERS mRNA in gynoecious cucumber plants may be due to the higher level of endogenous ethylene, which plays a role in the development of female flowers.

1,3 : 2,4-Di-O-benzylidene-d-sorbitol (d-DBS) can cause gelation of organic solvents of a wide range of polarity into a gel state. The aggregated structures of d-DBS/organic solvent systems were studied, mainly by using IR, UV, and circular dichroism (CD) spectroscopy. In the case of the racemate Dl-DBS, no gel state was formed. In IR spectra of d-DBS and Dl-DBS in the solid state, the intensities of νOH and νCO absorption in d-DBS were found to be larger than those in Dl-DBS. It is assumed that the hydrogen bonding formed between an acetal oxygen and a hydroxyl group in the aggregation of d-DBS. From the changes in the IR spectra from solution into a gel state, we can conclude that the chirality and the hydrogen bonding of DBS molecules are essential for the formation of the gel. Comparing the gel-forming ability of d-DBS with its derivatives, 5- or 6-hydroxyl-group-blocked compounds by methyl group, the 6-hydroxyl group seems to be important in the formation of d-DBS aggregates. The results of UV spectra suggest that the benzene rings are ordered in a side by side arrangement. The molar ellipticities in CD spectra increased about 500 times during the change from the solution to the gel state. From these results, it is concluded that d-DBS forms a helical structure of thin fibrous crystals in the gel state.