Estimation of the hydrophobicity of 2,4-diphenyl-1,3-oxazoline analogs and QSAR analysis of their ovicidal activity against Tetranychus [corrected] urticae

Partition coefficients of six 2-phenyl-1,3-oxazoline congeners containing 2-I, 2-NO2, 2-CF3, 2,6-(CH3)2, 2,6-F2, and 2-F-6-Cl substitutions on the phenyl moiety were measured in a 1-octanol/water system using the flask-shaking method. The effect on the hydrophobicity (LogP) of substituents on the phenyl moiety of 2-phenyl-1,3-oxazolines linearly correlated with that of benzamide congeners. logP values of other 2-(substituted phenyl)-1,3-oxazoline analogs were empirically estimated from the corresponding substituted benzamides. The ovicidal activity of 2-(substituted phenyl)-4-phenyl-1,3-oxazoline analogs against the two-spotted spider mite Tetranychus [corrected] urticae was quantitatively analyzed using the classical QSAR (Hansch-Fujita) method. Results showed that ovicidal activity increases with hydrophobicity. The introduction of inductive electron-withdrawing groups at ortho-positions increased ovicidal activity, but addition of steric bulk was unfavorable. Substitution at either the meta- or para-position was detrimental to the acaricidal activity.     

Response of blanket bog vegetation to drain‐blocking

Question: Does blocking of moorland drains increase bog vegetation on blanket peat? Location: Two sites with blocked drains and two with unblocked drains on Forsinard Flows National Nature Reserve, Sutherland, UK. Methods: Vegetation cover was recorded from 70 locations, with 12 sampling points at different distances (0.5‐14.5 m) from moorland drains in each location. Gradients in the cover of species indicative of wet and dry conditions, as well as bog recovery and degradation in relation to distance from drain, were compared from a sample of drains at two sites with blocked drains and two with unblocked drains. Results: There was evidence for drain‐blocking having a negative effect on vegetation indicative of drier conditions and bog degradation. One of the blocked sites had the lowest values of these indices near to the drain and increasing at greater distances perpendicular from the drain. The two unblocked sites, and the other blocked site, had a contrasting pattern of highest values of these indices close to the drain declining with distance. Cover of species indicative of bog recovery was greater where the drains had been blocked for the longest time. Conclusions: In some cases drain‐blocking can improve the ecological functioning of blanket bogs by increasing cover of healthy bog vegetation. Further studies into the causes of such variability in restoring vegetation through drain‐blocking are needed to aid targeting of peatland restoration projects to areas or methods most likely to be effective.     

Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes

Quantitative structure toxicity relationship (QSTR) equations were obtained to predict and describe the cytotoxicity of 31 phenols using logLD(50) as a concentration to induce 50% cytotoxicity of isolated rat hepatocytes in 2 h and logP as octanol/water partitioning: logLD(50) (microM)=-0.588(+/-0.059)logP+4.652(+/-0.153) (n=27, r(2)=0.801, s=0.261, P<1 x 10(-9)). Hydroquinone, catechol, 4-nitrophenol, and 2,4-dinitrophenol were outliers for this equation. When the ionization constant pK(a) was considered as a contributing factor a two-parameter QSTR equation was derived: logLD(50) (microM)=-0.595(+/-0.051)logP+0.197(+/-0.029)pK(a)+2.665(+/-0.281) (n=28, r(2)=0.859, s=0.218, P<1 x 10(-6)). Using sigma+, the Brown variation of the Hammet electronic constant, as a contributing parameter, the cytotoxicity of phenols towards hepatocytes were defined by logLD(50) (microM)=-0.594(+/-0.052)logP-0.552(+/-0.085)sigma+ +4.540(+/-0.132) (n=28, r(2)=0.853, s=0.223, P<1 x 10(-6)). Replacing sigma+ with the homolytic bond dissociation energy (BDE) for (X-PhOH+PhO.-->X-PhO.+PhOH) led to logLD(50) (microM)=-0.601(+/-0.066)logP-0.040(+/-0.018)BDE+4.611(+/-0.166) (n=23, r(2)=0.827, s=0.223, P<0.05). Hydroquinone, catechol and 2-nitrophenol were outliers for the above equations. Using redox potential and logP led to a new correlation: logLD(50) (microM)=-0.529(+/-0.135)logP+2.077(+/-0.892)E(p/2)+2.806(+/-0.592) (n=15, r(2)=0.561, s=0.383, P<0.05) with 4-nitrophenol as an outlier. Our findings indicate that phenols with higher lipophilicity, BDE, or sigma+ values or with lower pK(a) and redox potential were more toxic towards hepatocytes. We also showed that a collapse of hepatocyte mitochondrial membrane potential preceded the cytotoxicity of most phenols. Our study indicates that one or a combination of mechanisms; i.e. mitochondrial uncoupling, phenoxy radicals, or phenol metabolism to quinone methides and quinones, contribute to phenol cytotoxicity towards hepatocytes depending on the phenol chemical structure.     

离体鼠肝细胞及P-450酶系与拟除虫菊酯类杀虫剂的相互作用

经研究表明顺式氯氰菊酯、甲氰菊酯、氟氰菊酯、氰戊菊酯在正辛醇——水相中的分配系数(logP)分别为6.13,5.50,5.55和4.02。离体培养的大白鼠原代肝细胞对上述杀虫剂(除氟氰菊酯外)的吸收系数(q值)分别为322.6,281.1,201.9。上述杀虫剂在离体培养的原代肝细胞中降解很快,温育30分钟后降解速率分别为90.0％(顺式氯氰菊酯)、86.2％(甲氰菊酯)、69.5％(氰戊菊酯)。同时有明显的减少离体培养的鼠肝细胞中P-450含量的作用,其中以溴氰菊酯和顺式氯氰菊酯作用较明显。在溴氰菊酯处理组中,随杀虫剂浓度的升高,这种减少P-450含量的作用明显增强。检查温育反应90分种后的细胞悬浮反应液,对照组仍维持高的细胞存活率(74.4％和91.2％),而杀虫剂处理组,除甲氰菊酯处理组仍有42.4％的存活率外,其余各杀虫剂(溴氰菊酯各浓度组和顺式氯氰菊酯处理组)均未检出存活的细胞。结果表明,试验所用的几种拟除虫菊酯类杀虫剂具有一定的细胞毒性。它们在鼠肝细胞中的降解代谢可能主要不是由于多功能氧化酶起作用。另外,杀虫剂的亲脂性与q值及在肝细胞中的降解速率三者间有一定的正相关。     

Synthesis and evaluation of fluorine-substituted 1H-pyrrolo[2,3-b]pyridine derivatives for dopamine D4 receptor imaging

Seven fluorine-substituted 1H-pyrrolo[2,3-b]pyridine derivatives were synthesized based on a lead ligand, 3-[[4-(4-iodophenyl)piperazin-1-yl]-methyl]-1H-pyrrolo[2,3-b]pyridine (L-750,667) and evaluated as potential dopamine D(4) receptor imaging agents by positron emission tomography (PET). Binding affinities of these ligands for the dopamine D(2), D(3), and D(4) receptor subtypes were measured in vitro. Most ligands showed high and selective binding for the D(4) receptor. Ligand 7 had high affinity for the D(4) receptor, whereas ligands 1, 2, and 6 showed high selectivity for the D(4) receptor. LogP values were calculated for the ligands in this series and ligand 6 had the lowest lipophilicity. (18)F-labeled ligand 7 demonstrated a uniform regional brain distribution and a rapid washout in mice, probably due to nonspecific binding. Based on their in vitro binding properties and calculated logP values, ligand 6 appears to have the most promise for dopamine D(4) receptor imaging.     

The prediction of human acute systemic toxicity by the EDIT/MEIC in vitro test battery: the importance of protein binding and of partitioning into lipids

The aim of the two studies presented in this paper was to further improve the predictability of the original Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vitro test battery for acute systemic toxicity. In the first study, whether a protein-free cytotoxicity assay could improve the prediction of human acute systemic toxicity was investigated. The cytotoxicity of 39 MEIC reference chemicals was measured by the neutral red uptake inhibition test after 30 minutes in phosphate-buffered saline (PBS), with hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Mercuric chloride and hexachlorophene were much more cytotoxic in PBS, as was the case, to a lesser extent, for seven other chemicals. Potassium cyanide and eight other chemicals were less cytotoxic in PBS than in complete culture medium, probably because of poor physiological conditions. The correlation between the cytotoxicity measured in PBS and human acute toxicity was rather low, but became of the same order as for other assays, when mercuric chloride and hexachlorophene were withdrawn from the comparison. In the second study, modelling of human lethal blood concentrations by using the results of the three cell line tests of the original MEIC test battery were complemented by logP (octanol-water partition coefficient) values. The introduction of logP into the modelling did not improve the correlations, but some improvement of both R(2) and Q(2) was obtained by expanding the logP values with logP(2) values. The highest R(2) (0.84) and Q(2) (0.80) values were obtained for a model in which both experimental and calculated (ambiguous) logP values were used. When only experimental logP values were used, the corresponding values were 0.80 and 0.78. These two studies showed that including protein binding and the partition of chemicals in the MEIC in vitro test battery is important, in order to improve the predictability of the results obtained.     

Computational methods for prediction of T-cell epitopes--a framework for modelling, testing, and applications

Computational models complement laboratory experimentation for efficient identification of MHC-binding peptides and T-cell epitopes. Methods for prediction of MHC-binding peptides include binding motifs, quantitative matrices, artificial neural networks, hidden Markov models, and molecular modelling. Models derived by these methods have been successfully used for prediction of T-cell epitopes in cancer, autoimmunity, infectious disease, and allergy. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures and performed according to strict standards. This requires careful selection of data for model building, and adequate testing and validation. A range of web-based databases and MHC-binding prediction programs are available. Although some available prediction programs for particular MHC alleles have reasonable accuracy, there is no guarantee that all models produce good quality predictions. In this article, we present and discuss a framework for modelling, testing, and applications of computational methods used in predictions of T-cell epitopes.     

HPLC columns partition by chemometric methods based on peptides retention

In recent years, multivariate techniques have been utilized to evaluate reversed-phase high-performance liquid chromatographic data. In the present study, 11 high-performance liquid chromatography (HPLC) columns were divided into several groups according to the retention factors of 12 peptides. Principal component analysis (PCA) and cluster analysis (CA) were used in column and peptides' comparison and grouping. CA results indicated that all stationary phases may be generally grouped into several clusters, due to stationary phase structure and properties. On the other hand, interesting results were obtained with the use of PC. There is almost linear relationship between classified HPLC columns in the space of new PCs, which is connected with meaning of the PC's reflected in their loading values. The first component describes non-polar properties of peptides, whereas the second component is loaded with polar peptides having much lower logP values. PCA and CA were also used in peptides comparison however, complete explanation of peptides grouping still remains unclear.     

Towards the in silico identification of class II restricted T-cell epitopes: a partial least squares iterative self-consistent algorithm for affinity prediction

MOTIVATION: The immunogenicity of peptides depends on their ability to bind to MHC molecules. MHC binding affinity prediction methods can save significant amounts of experimental work. The class II MHC binding site is open at both ends, making epitope prediction difficult because of the multiple binding ability of long peptides. RESULTS: An iterative self-consistent partial least squares (PLS)-based additive method was applied to a set of 66 peptides no longer than 16 amino acids, binding to DRB1*0401. A regression equation containing the quantitative contributions of the amino acids at each of the nine positions was generated. Its predictability was tested using two external test sets which gave r(pred) = 0.593 and r(pred) = 0.655, respectively. Furthermore, it was benchmarked using 25 known T-cell epitopes restricted by DRB1*0401 and we compared our results with four other online predictive methods. The additive method showed the best result finding 24 of the 25 T-cell epitopes. AVAILABILITY: Peptides used in the study are available from http://www.jenner.ac.uk/JenPep. The PLS method is available commercially in the SYBYL molecular modelling software package. The final model for affinity prediction of peptides binding to DRB1*0401 molecule is available at http://www.jenner.ac.uk/MHCPred. Models developed for DRB1*0101 and DRB1*0701 also are available in MHCPred.     

Cyclic, Linear, Cycloretro-Isomer, and Cycloretro-Inverso Peptides Derived from the C-Terminal Sequence of Bradykinin as Substrates or Inhibitors of Serine and Cysteine Proteases

We investigated the inhibition of trypsin, human tissue (hK1) and human plasma kallikrein (HuPK), papain, and cathepsin L, B, and X by synthetic cyclic, cycloretro-isomer, cycloretro-inverso, and linear peptides derived from the C-terminal sequence of bradykinin. c(FSPFRG) and Ac-FSPFRG-NH2 were taken as the references for cyclic and linear peptides, respectively. Longer and more flexible analogs of them with addition of 2, 3, or 4 Gly and cycloretro-isomer and cycloretro-inverso analogs of c(FSPFRG) and c(GGGFSPFRG) were obtained and assayed. The susceptibility to hydrolysis of the peptides to all proteases was also examined. The highest affinities were found for c(FSPFRG) with hK1, Ac-GGFSPFRG-NH2 with HuPK, and psi (NHCO) c(fspfrG) with cathepsin L. The Ki values for cathepsin B and X with cyclic peptides were lower than those of linear peptides. The serine proteases hydrolyzed all linear and cyclic peptides, except c(FSPFRG) and c(GFSPFRG). The cysteine proteases hydrolyzed only the linear peptides, which were poor substrates. Although the Ki values obtained in the current work were in the microM range, the cyclic and cycloretro-inverso peptides seem to be a promising approach to develop efficient and resistant to hydrolysis inhibitors for the kallikreins and lysosomal cysteine proteases.     

Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis,inconsequent of mitosis

Virgin mouse mammary gland in explant culture will differentiate and synthesize casein and α-lactalbumin when insulin, hydrocortisone, and prolactin (IFPRL) are present in the culture medium. Explants whose DNA synthesis has been blocked differentiate cytologically, mobilize lipid, synthesize RNA, and incorporate ³H-amino acids into proteins to the same extent as unblocked tissue. Nevertheless, casein synthesis as measured by immunoprecipitation with casein-specific antiserum remains at the zero-time level in blocked explants while unblocked explants produce casein at five- to eightfold greater levels. Electrophoretic analysis of immunoprecipitated radioactive proteins showed that the IFPRL-treated virgin tissue made all four size classes of mouse casein. Immunoperoxidase studies of explants revealed that the number of mammary epithelial cells positive for casein was 2–8% in blocked and 24–31% in unblocked, in good agreement with the radioimmunoprecipitation results. Immunoelectron microscopy demonstrated the accumulation of casein within the cisternae of the granular endoplasmic reticulum and in Golgi vacuoles in the unblocked epithelial cells. Similar accumulation did not occur in blocked cultures despite the secretory appearance of the cells. Autoradiographic analysis of blocked and unblocked explants, incubated in the presence of IFPRL and [³H]thymidine for 72 hr, showed that 53–57% of the epithelial cells synthesized DNA in unblocked explants, whereas only 2% incorporated the label in the presence of cytosine arabinoside. When explants were incubated with IFPRL and various concentrations of colchicine, only 5–6% of the epithelial cells were found to enter mitosis. Since cell duplication cannot account for the severalfold increase in casein-producing cells in the unblocked explants, the results suggest that the requirement for DNA synthesis in this system may involve either polyploid cells or the augmentation of specific sequences necessary for the facilitation of terminal differentiation. Similar requirements for DNA synthesis were not observed in mammary explants from pregnant and primiparous (but nonpregnant) mice.     

Characterization of oligopeptides that cross-react with carbohydrate-specific antibodies by real time kinetics, in-solution competition enzyme-linked immunosorbent assay, and immunological analyses

Phage displaying random cyclic 7-mer, and linear 7-mer and 12-mer peptides at the N terminus of the coat protein, pIII, were panned with the murine monoclonal antibody, 9-2-L379 specific for meningococcal lipo-oligosaccharide. Five cyclic peptides with two sequence motifs, six linear 7-mers, and five linear 12-mers with different sequence motifs were identified. Only phage displaying cyclic peptides were specifically captured by and were antigenic for 9-2-L379. Monoclonal antibody 9-2-L379 exhibited "apparent" binding affinities to the cyclic peptides between 11 and 184 nm, comparable with lipo-oligosaccharide. All cyclic peptides competed with the binding of 9-2-L379 to lipo-oligosaccharide with EC(50) values in the range 10-105 microm, which correlated with their apparent binding affinities. Structural modifications of the cyclic peptides eliminated their ability to bind and compete with monoclonal antibody 9-2-L379. Mice (C3H/HeN) immunized with the cyclic peptide with optimal apparent binding affinity and EC(50) of competition elicited cross-reactive antibodies to meningococcal lipo-oligosaccharide with end point dilution serum antibody titers of 3200. Cyclic peptides were converted to T-cell-dependent immunogens without disrupting these properties by C-terminal biotinylation and complexing with NeutrAvidin. The data indicate that constrained peptides can cross-react with a carbohydrate-specific antibody with greater specificity than linear peptides, and critical to this specificity is their structural conformation.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

On the nature of 5' termini in nuclear pre-mRNA of Ehrlich carcinoma cells.

5' terminal nucleosides of nuclear pre-mRNA of Ehrlich ascites carcinoma cells were analyzed by a combination of different chromatographic methods and phosphatase treatment. The heavy nuclear pre-mRNA contains mainly unblocked triphosphorylated nucleosides at the 5' end, although some capped 5' ends could also be found. In this respect, it differs from cytoplasmic poly(A)+ mRNA which contains blocked 5' termini and no triphosphorylated ends. The 5' terminal nucleotides in pre-mRNA are pppGp and pppAp (in a ratio of 3:2). The determination of pppNp content in poly (A)+, poly(U)+, and poly (A)-(U)- fragments of RNA has been used as an approach to establish the topography of pre-mRNA. We also established that the technique for isolation of triphosphorylated 5' terminal fragments of RNA based on hydroxyapatite chromatography (Bajszár, Samarina, and Georgiev, 1974) is still valid in the presence of blocked oligonucleotides. The latter do not interfere with fragments containing free triphosphate groups. Using this technique, we showed that a small but significant portion of triphosphorylated 5' end fragments of 100 nucleotides in length contain oligo(U) sequences reacting with poly(A)-Sepharose.     

QSAR studies in substituted 1,2,3,4,6,7,12,12a-octa-hydropyrazino[2',1':6,1]pyrido[3,4-b]indoles--a potent class of neuroleptics

A series of nineteen substituted 1,2,3,4,6,7,12,12a-octahydropyrazino[2',1':6,1]pyrido[3, 4-b]indoles analogues of neuroleptic drug, Centbutindole have been studied using quantitative structure-activity relationship analysis. The derived models display good fits to the experimental data (r>or=0.75) having good predictive power (r(cv)>or=0.688). The best model describes a high correlation between predicted and experimental activity data (r=0.967). Statistical analysis of the equation populations indicates that hydrophobicity (as measured by pi(R), logP(o/w) and SlogP_VSA8), dipole y and structural parameters in terms of indicator variable, (In(1)) and globularity are important variables in describing the variation in the neuroleptic activity in the series.