Enniatin has a new function as an inhibitor of Pdr5p, one of the ABC transporters in Saccharomyces cerevisiae

Pdr5p is one of the major multidrug efflux pumps whose overexpression confers multidrug resistance (MDR) in Saccharomyces cerevisiae. By using our original assay system, a fungal strain producing inhibitors for Pdr5p was obtained and classified as Fusarium sp. Y-53. The purified inhibitors were identified as ionophore antibiotics, enniatin B, B1, and D, respectively. A non-toxic concentration of each enniatin (5 microg/ml, approximately 7.8 microM) strongly inhibited a Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. The enniatins accumulated a fluorescent dye rhodamine 123, a substrate of Pdr5p, into yeast cells. The mode of Pdr5p inhibition of enniatin was competitive against FK506, and its inhibitory activity was more potent with less toxicity than that of FK506. The enniatins showed similar inhibitory profile as FK506 against S1360 mutants (S1360A and S1360F) of Pdr5p. The enniatins did not inhibit the function of Snq2p, a homologue of Pdr5p. Thus, it was found that enniatins are potent and specific inhibitors for Pdr5p, with less toxicities than that of FK506.     

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.     

Role of the RuvAB protein in avoiding spontaneous formation of deletion mutations in the Escherichia coli K-12 endogenous tonB gene

The endogenous tonB gene of Escherichia coli was used as a target for spontaneous deletion mutations which were isolated from ruvAB-, recG-, and ruvC- cells. The rates of tonB mutation were essentially the same in ruv+, ruvAB-, recG-, and ruvC- cells. We analyzed tonB mutants by sequencing. In the ruv+, recG-, and ruvC- strains, the spectra were different from those obtained from the ruvAB- cells, where deletions dominated followed by IS insertions, base substitutions, and frameshifts, in that order. We then analyzed the tonB-trp large deletion, due to simultaneous mutations of the trp operon, and found that the frequency in ruvAB- was higher than those in ruv+, recG-, and ruvC- cells. To characterize deletion formation further, we analyzed all the tonB mutants from one colicin plate. Seven deletions were identified at five sites from the 45 tonB mutants of ruv+ cells and 24 deletions at 11 sites from the 43 tonB mutants of ruvAB- cells. Thus, the ruvAB- strain is a deletion mutator. We discuss the role of RuvAB in avoiding deletions.     

Role of charged residues of pharaonis phoborhodopsin (sensory rhodopsin II) in its interaction with the transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppR(O)(-)(like)) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppR(O)(-)(like) decay rate and K(D) value for binding, implying that the carboxyl group in question is from Asp-193. The pK(a) of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193(p)()(pR) is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pK(a). In addition, we discuss the importance of Arg-162(p)()(pR) with respect to the binding activity.     

Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment

Comparative proteomic analysis was used to search for characteristic alterations in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment. Serum samples collected from eight patients before and after treatment were subjected to 2-DE. Eighty-eight protein spots differentially expressed with the treatment were selected by clustering analysis, and the proteins were identified by MS based on MALDI-TOF/TOF analysis and public database searches. The statistical analysis suggested that four proteins decreased after treatment (pro-apolipoprotein, alpha2-HS glycoprotein, apolipoprotein A-IV precursor, and PRO1708/PRO2044, which is the carboxy terminal fragment of albumin) and that seven proteins were increased after treatment, including leucine-rich alpha2-glycoprotein and alpha1-antitrypsin. These data facilitate the identification of differentially expressed proteins that are involved in HCC carcinogenesis and provide candidate biomarkers for the development of diagnostic and therapeutic tools.     

Synthesis and insulinomimetic activities of novel mono- and tetranuclear oxovanadium(IV) complexes with 3-hydroxypyridine-2-carboxylic acid

Two chargeless VO(IV) complexes with 3-hydroxypyridine-2-carboxylic acid (H2hpic), [VO(Hhpic-O,O)(Hhpic-O,N)(H2O)].3H2O (1) and the cyclic tetramer [(VO)4(mu-(hpic-O,O',N))4(H2O)4].8H3O (2), have been synthesized and characterized by elemental analysis, mass, infrared, electronic absorption, electron spin resonance (ESR) spectroscopies, and X-ray crystallography. Their coordination structures are similar to each other (and 1 is readily transformed into 2), but are quite different from that of bis(pyridine-2-carboxylato)oxovanadium(IV). The magnetic susceptibility of 2 indicates the presence of a weak ferromagnetic intramolecular interaction between the V atoms at low temperature, in addition to a weak antiferromagnetic intermolecular interaction. The ESR signal of 2 was broad, while 1 showed an eight-line hyperfine splitting pattern due to coupling of the unpaired electron with the 51V nucleus (I=7/2). The ESR spectrum and cyclic voltammogram of 2 clearly show that the cyclic tetramer remains intact in solution. The insulinomimetic activity of 1 and 2 was evaluated by means of in vitro measurements of the inhibition of free fatty acid release from epinephrine-treated isolated rat adipocytes. While 1 exerted higher insulinomimetic activity than VOSO4, the activity of 2 was significantly lower than that of VOSO4. Hence 2 appears to retain its cyclic structure during the in vitro test. These results indicate that the rational ligand design for VO complexes might be a promising approach to obtain superior insulinomimetic activity.     

Activity regulation of tyrosinase by using photoisomerizable inhibitors

Enzymatic activity of tyrosinase was controlled on the basis of cis-trans photoisomerization of inhibitors, 4-azobenzene carboxylic acid (ACA) and 4,4'-azobenzene dicarboxylic acid (ADCA). In the case of ACA, the cis form inhibited tyrosinase-catalyzed oxidation of L-tyrosine more strongly than the trans form. On the contrary, in the case of ADCA, the cis form was less inhibitory. The oxidation rate was controlled reversibly by light irradiation in the course of the reaction. In the presence of ACA, UV light irradiation, which isomerized trans to cis form, decelerated the tyrosine oxidation, while visible light irradiation, which isomerized backward, accelerated the reaction. In contrast, in the presence of ADCA, UV light accelerated and visible light decelerated the reaction.     

Diet mixing and its effect on polyphagous grasshopper nymphs

We examined the effects of diet mixture on the nymphal performance of a polyphagous grasshopper Parapodisma subastris Huang by developing the first-stadium nymphs with either single or various combinations of plants occurring in their natural habitat. Intrinsic quality in terms of nymphal survival differed largely across the 16 plant species. However, even combinations of the six worst quality plants (survival 10% or less in each) greatly improved nymphal survival when compared to that of superior quality plants (more than 70% survival). In contrast, the addition of either of two inferior plants (more than 10% and less than 40% survival) to the superior plant affected neither the survival nor the adult mass. Thus, diet mixture can be particularly important when only low quality plants are available. The adaptive significance of diet mixture was discussed in relation to the habitat flora and foraging habits of the grasshopper in the present study.