Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

Evidence for a single essential thiol in the yeast hexokinase molecule.

In yeast hexokinase B, two thiols per monomer appeared to be essential when enzymic inactivation was produced by the concurrent alkylation of both of them, by several reagents including the affinity reagent N-bromoacetyl-2-D-galactosamine. However, it is shown that only one of these thiols is actually essential. Three of the four thiols present can be blocked by alkylation in the presence of a substrate in appropriate conditions, without loss of enzymic activity. Subsequently, in the absence of substrate, the affinity reagent reacts at the one remaining thiol, with complete inactivation. The same behavior can be obtained by reaction with iodoacetamide or by the formation of the -SCN group. The affinity reagent inactivates hexokinase B faster than does the isomeric glycosidic compound (glycosides being nonsubstrates), although the latter has twice the reactivity of the former toward glutathione. The reactions with alkylating agents, with or without substrate present, are used to classify the four thiols in the monomer. The temperature dependence of the alkylation of the essential thiol provides evidence for a transition in the molecule at about 31 degrees C. The inactive monomer containing the -SCN group can regenerate, by thiolysis, active enzyme with the thiol free. It can also perform an intramolecular cleavage of the chain. The latter reaction was used to locate the essential cysteine residue in the chain, at 80% of the length from the N terminus.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

ESR as a valuable tool for the investigation of the dynamics of EPC and EPC/cholesterol liposomes containing a carboranyl-nucleoside intended for BNCT

Electron Spin Resonance (ESR) spectroscopy of long-chain nitroxides (5-, 7-, and 16-doxyl stearic acid) has been used to evaluate the perturbations induced by β-5-o-carboranyl-2′-deoxyuridine (CDU) on the structure and dynamics of egg phosphatidylcholine (EPC) and EPC/cholesterol liposomes. Loaded liposomes are intended for the use in Boron Neutron Capture Therapy (BNCT). From a detailed analysis of the motional and order parameters determining the ESR line shape as a function of temperature and of CDU content in liposomes, an increased order and a hindered motion of the phospholipid membranes resulted in the presence of increasing CDU concentration. This occurred particularly at the liposome surface level. Both higher ordering and increased rigidity of the membrane lipids were the result of the constraints exerted by the embedded carboranyl-nucleoside.     

ESR as a valuable tool for the investigation of the dynamics of EPC and EPC/cholesterol liposomes containing a carboranyl-nucleoside intended for BNCT

Electron Spin Resonance (ESR) spectroscopy of long-chain nitroxides (5-, 7-, and 16-doxyl stearic acid) has been used to evaluate the perturbations induced by beta-5-o-carboranyl-2'-deoxyuridine (CDU) on the structure and dynamics of egg phosphatidylcholine (EPC) and EPC/cholesterol liposomes. Loaded liposomes are intended for the use in Boron Neutron Capture Therapy (BNCT). From a detailed analysis of the motional and order parameters determining the ESR line shape as a function of temperature and of CDU content in liposomes, an increased order and a hindered motion of the phospholipid membranes resulted in the presence of increasing CDU concentration. This occurred particularly at the liposome surface level. Both higher ordering and increased rigidity of the membrane lipids were the result of the constraints exerted by the embedded carboranyl-nucleoside.     

Stochasticity of the step size in the force generating process due to a single myosin molecule

We formulate a motional model on the basis of an experimental optical trapping system which reveals the single molecular events of the force generation due to actin and myosin, and we discuss the origin of the dispersion of the displacements of the beads bound to the actin filament. In some experimental data sets, this dispersion is larger than that presumed from the model under the condition that the thermal agitation is the only source of the fluctuation. Thus, other fluctuating elements besides that due to thermal agitation are supposed to cause the dispersion. A possible fluctuating element is the step size itself. We discuss this possibility and estimate the fluctuation in step size using two reported experimental data sets.     

Evidence for a single catalytic and two binding sites in the almond emulsin β-D-glucosidase molecule

An isoenzyme of glucosidase- isolated from sweet almond emulsin - and composed of a β-D-glucosidase, a β-D-galactosidase and a β-D-fucosidase, has been shown to possess β-D-xylosidase activity, as well. On the basis of the following results it has been concluded that the β-D-glucosidase and β-D-galactosidase activities reside in one catalytic site, but there are two kinetically distinst binding sites in the active center: 1./D-Glucono-1,5-lactone is shown to excert competitive inhibition on the hydrolysis of β-D-glucopyranoside and non-competitive inhibition on the hydrolysis of β-D-galactopyranoside. 2./ D-galactono-1,5-lactone competitively inhibits the hydrolysis of β-D-galactopyranoside, but possesses non-competitive inhibition on the hydrolysis of β-D-glucopyranoside. 3./ When the enzyme is incubated with two p-nitrophenyl glycoside substrates at or above their respective K_m values, the rate of p-nitrophenol formation is not additive but rather it is equal to the value calculated from the individual K_m values and relative maximum rates.     

Construction of the control system of target molecule expression in Escherichia coli: application to a validation platform for bactericidal and bacteriostatic profiles due to suppression of a target molecule

Validation of bactericidal profiles owing to a deficiency of target bacterial molecule provides opportunities to discover antimicrobial drug candidates. In this study, we constructed genetic-engineered Escherichia coli strains, in which the target gene expression is conditionally regulated by a tryptophan promoter, while the target protein expression is regulated by N-end rule-based proteolysis. Among 10 genes, whose correspondent proteins are target candidates of antibiotics for community acquired respiratory tract infection, it was clearly demonstrated that the suppression of DnaB, GlmU, or DnaX results in a bactericidal profile, while the suppression of FabB, PyrG, DnaG, Der, PyrH, Era, or IspA leads to a bacteriostatic profile. This study is the first to predict the antibacterial inhibition profiles of Der, DnaG, DnaX, Era, GlmU, IspA, PyrG, and PyrH, and confirms previous findings for DnaB and FabB. The results suggested that the system constructed in this study is a novel and useful tool to validate whether the target bacterial molecule has appropriate properties as a target of antimicrobial agents.     

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

"Sequential" boron neutron capture therapy (BNCT): a novel approach to BNCT for the treatment of oral cancer in the hamster cheek pouch model

In the present study the therapeutic effect and potential toxicity of the novel "Sequential" boron neutron capture therapy (Seq-BNCT) for the treatment of oral cancer was evaluated in the hamster cheek pouch model at the RA-3 Nuclear Reactor. Two groups of animals were treated with "Sequential" BNCT, i.e., BNCT mediated by boronophenylalanine (BPA) followed by BNCT mediated by sodium decahydrodecaborate (GB-10) either 24 h (Seq-24h-BNCT) or 48 h (Seq-48h-BNCT) later. In an additional group of animals, BPA and GB-10 were administered concomitantly [(BPA + GB-10)-BNCT]. The single-application BNCT was to the same total physical tumor dose as the "Sequential" BNCT treatments. At 28 days post-treatment, Seq-24h-BNCT and Seq-48h-BNCT induced, respectively, overall tumor responses of 95 ± 2% and 91 ± 3%, with no statistically significant differences between protocols. Overall response for the single treatment with (BPA + GB-10)-BNCT was 75 ± 5%, significantly lower than for Seq-BNCT. Both Seq-BNCT protocols and (BPA + GB-10)-BNCT induced reversible mucositis in the dose-limiting precancerous tissue around treated tumors, reaching Grade 3/4 mucositis in 47 ± 12% and 60 ± 22% of the animals, respectively. No normal tissue toxicity was associated with tumor response for any of the protocols. "Sequential" BNCT enhanced tumor response without an increase in mucositis in dose-limiting precancerous tissue.     

Human aldo-keto reductases 1B1 and 1B10: a comparative study on their enzyme activity toward electrophilic carbonyl compounds

Aldo-keto reductase family 1 member B1 (AKR1B1, 1B1 in brief) and aldo-keto reductase family 1 member B10 (AKR1B10, 1B10 in brief) are two proteins with high similarities in their amino acid sequences, stereo structures, and substrate specificity. However, these two proteins exhibit distinct tissue distributions; 1B10 is primarily expressed in the gastrointestinal tract and adrenal gland, whereas 1B1 is ubiquitously present in all tissues/organs, suggesting their difference in biological functions. This study evaluated in parallel the enzyme activity of 1B1 and 1B10 toward alpha, beta-unsaturated carbonyl compounds with cellular and dietary origins, including acrolein, crotonaldehyde, 4-hydroxynonenal, trans-2-hexenal, and trans-2,4-hexadienal. Our results showed that 1B10 had much better enzyme activity and turnover rates toward these chemicals than 1B1. By detecting the enzymatic products using high-performance liquid chromatography, we measured their activity to carbonyl compounds at low concentrations. Our data showed that 1B10 efficiently reduced the tested carbonyl compounds at physiological levels, but 1B1 was less effective. Ectopically expressed 1B10 in 293T cells effectively eliminated 4-hydroxynonenal at 5 μM by reducing to 1,4-dihydroxynonene, whereas endogenously expressed 1B1 did not. The 1B1 and 1B10 both showed enzyme activity to glutathione-conjugated carbonyl compounds, but 1B1 appeared more active in general. Together our data suggests that 1B10 is more effectual in eliminating free electrophilic carbonyl compounds, but 1B1 seems more important in the further detoxification of glutathione-conjugated carbonyl compounds.     

Comparative mapping of a region on chromosome 10 containing QTL for reproduction in swine

Several quantitative trait loci (QTL) for important reproductive traits (age of puberty, ovulation rate, nipple number and plasma FSH) have been identified on the long arm of porcine chromosome 10. Bi-directional chromosome painting has shown that this region is homologous to human chromosome 10p. Because few microsatellite or type I markers have been placed on SSC10, we wanted to increase the density of known ESTs mapped in this region of the porcine genome. Genes were chosen for their position on human chromosome 10, sequence availability from the TIGR pig gene indices, and their potential as a candidate gene. The PCR primers were designed to amplify across introns or 3'-UTR to maximize single nucleotide polymorphism (SNP) discovery. Parents of the mapping population (one sire and seven dams) were amplified and sequenced to find informative markers. The SNPs were genotyped using primer extension and mass spectrometry. These amplification products were also used to probe a BAC library (RPCI-44, Roswell Park Cancer Institute) for positive clones and screened for microsatellites. Six genes from human chromosome 10p (AKR1C2, PRKCQ, ITIH2, ATP5C1, PIP5K2A and GAD2) were mapped in the MARC swine mapping population. Gene order was conserved within these markers from centromere to telomere of porcine chromosome 10q, as compared with human chromosome 10p. Four of these genes (PIP5K2A, ITIH2, GAD2 and AKR1C2), which map under QTL, are potential candidate genes. Identification of porcine homologues near important QTL and development of a comparative map for this chromosome will allow further fine- mapping and positional cloning of candidate genes affecting reproductive traits.     

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 μg/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173–175) for tyrosine hydroxylase.     

NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo

Natural killer (NK) cells have been shown critical in reducing tumor lung metastasis in various murine cancer models. Effector molecules such as perforin and IFN-γ may play important roles in inhibition of metastasis. However, most of these conclusions were based on experiments that involved quantitation of metastatic colonies several weeks after tumor challenge. The roles of NK cells and their effector molecules (perforin and IFN-γ) in the initial immune responses against tumor metastasis in lungs are still unknown. By using the B16F10 melanoma tumor model combined with confocal microscopy, we observed an increase in numbers of B16F10 cells in NK-depleted mice at 60 min post tumor inoculation, but this effect was independent of perforin or IFN-γ. In addition, NK cell numbers in lungs after tumor injection rapidly increased suggesting a redistribution of NK cells in the lungs. However, NK cells were not found in contact with tumor cells until day 6 or later. Our data indicate that during early responses against B16F10 cells, NK cells use another mechanism(s) besides perforin and IFN-γ to prevent tumor metastasis.     

Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds

Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid hydroxylase exhibiting selectivity for long-chain substrates (12–20 carbons). Replacement of Phe87 in P450 BM-3 by Val (F87V) greatly increased its activity towards a variety of aromatic and phenolic compounds. The apparent initial reaction rates of F87V as to benzothiophene, indan, 2,6-dichlorophenol, and 2-(benzyloxy)phenol were 227, 204, 129, and 385 nmol min^–1 nmol^–1 P450, which are 220-, 66-, 99-, and 963-fold those of the wild type, respectively. These results indicate that Phe87 plays a critical role in the control of the substrate specificity of P450 BM-3. Furthermore, F87V catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, F87V showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone. With F87V as the catalyst, 0.71 mg ml^–1 2-(benzyloxy)hydroquinone was produced from 1.0 mg ml^–1 2-(benzyloxy)phenol in 4 h, with a molar yield of 66%.