Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Summary Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)-α-hydroxymethyltyrosine in position 1 and (R)- and (S)-α-hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that α-hydroxymethyl substitution of amino acid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

A comparative study of cell classifiers for image-based high-throughput screening

Background

Millions of cells are present in thousands of images created in high-throughput screening (HTS). Biologists could classify each of these cells into a phenotype by visual inspection. But in the presence of millions of cells this visual classification task becomes infeasible. Biologists train classification models on a few thousand visually classified example cells and iteratively improve the training data by visual inspection of the important misclassified phenotypes. Classification methods differ in performance and performance evaluation time. We present a comparative study of computational performance of gentle boosting, joint boosting CellProfiler Analyst (CPA), support vector machines (linear and radial basis function) and linear discriminant analysis (LDA) on two data sets of HT29 and HeLa cancer cells.

Results

For the HT29 data set we find that gentle boosting, SVM (linear) and SVM (RBF) are close in performance but SVM (linear) is faster than gentle boosting and SVM (RBF). For the HT29 data set the average performance difference between SVM (RBF) and SVM (linear) is 0.42 %. For the HeLa data set we find that SVM (RBF) outperforms other classification methods and is on average 1.41 % better in performance than SVM (linear).

Conclusions

Our study proposes SVM (linear) for iterative improvement of the training data and SVM (RBF) for the final classifier to classify all unlabeled cells in the whole data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-342) contains supplementary material, which is available to authorized users.     

Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.     

Identification of Bacteria in Blood Culture Broths Using Matrix-Assisted Laser Desorption-Ionization Sepsityper? and Time of Flight Mass Spectrometry

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths. We evaluate for the first time, the performance of the MALDI Sepsityper™ Kit and MS for the identification of bacteria compared to standard phenotypic methods using the manufacturer''s specified bacterial identification criteria (spectral scores ≥1.700–1.999 and ≥2.000 indicated identification to genus and species level, respectively). Five hundred and seven positive blood culture broths were prospectively examined, of which 379 (74.8%; 358 monomicrobial, 21 polymicrobial) were identified by MALDI-TOF MS; 195 (100%) and 132 (67.7%) of 195 gram-positive; and 163 (100%) and 149 (91.4%) of 163 gram-negative organisms from monomicrobial blood cultures were correctly identified to genus and species level, respectively. Spectral scores <1.700 (no identification) were obtained in 128/507 (25.2%) positive blood culture broths, including 31.6% and 32.3% of gram-positive and polymicrobial blood cultures, respectively. Significantly more gram-negative organisms were identified compared to gram-positive organisms at species level (p<0.0001). Five blood cultures were misidentified, but at species level only; including four monomicrobial blood cultures with Streptococcus oralis/mitis that were misidentified as Streptococcus pneumoniae. Positive predictive values for the direct identification of both gram-positive and gram-negative bacteria from monomicrobial blood culture broths to genus level were 100%. A diagnostic algorithm for positive blood culture broths that incorporates gram staining and MALDI-TOF MS should identify the majority of pathogens, particularly to genus level.     

A convenient route to optically pure α-alkyl-β-(sec-amino)alanines

The cyclization of N-Boc-α-alkylserines to corresponding β-lactones under Mitsunobu reaction conditions and the ring opening with heterocyclic amines (pyrrolidine, piperidine, morpholine and thiomorpholine) produced N-Boc-α-alkyl-β-(sec-amino)alanines. The removal of the Boc group gives di-hydrochlorides of non-protein amino acids.     

Study of the mitotic and meiotic chromosomes of sotol (<i>Dasylirion cedrosanum</i> Trel.)

The genus Dasylirion is a group of plants typically present in the Chihuahuan Desert, perennial, with a dioecious sexual behavior and commonly called sotoles. This genus has been little studied from the biological point of view, and the bases of its reproductive response remain unknown. In this work we studied the chromosome number and meiotic response of Dasylirion cedrosanum in the county of Saltillo, Coahuila, located at the North East of Mexico. For the preparation of mitotic chromosomes, we used a technique based on enzymatic treatment with pectolyase and cellulase, as well as staining with acetocarmin dye. For the study of meiosis, male flower buds were collected, fixed and stained for analysis with the same dye. As a result, the gametic (n = x = 19) and somatic chromosome (2n = 38) numbers of D. cedrosanum are reported for the first time, being consistent with previous findings in other Dasylirion species, which points to a constant ploidy level across the genus. Variation was observed in the morphology and size of the somatic chromosomes, with types ranging from submetacentric to subtelocentric, and sizes oscillating in a range of 4.43 µm, with an average total length of 112.38 µm for the diploid chromosome complement. This shows that the chromosome complement of D. cedrosanom would belong to a 3B classification of Stebins, with a medium variation between chromosome lengths and low chromosome asymmetry. This variation indicates the feasibility of constructing a chromosome ideotype for this species. The meiotic chromosome pairing showed a chromosome behavior consistent with a disomic inheritance characteristic of a diploid species, with prevalence of ring and chain bivalents, typically without pairing abnormalities. Bivalent configurations in all cases were symmetrical.The normal and symmetrical meiotic pairing indicates a balanced production of gametes, and suggests the absence of heteromorphic sex determination.     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

Inducible cytochrome P450 activities in renal glomerular mesangial cells: biochemical basis for antagonistic interactions among nephrocarcinogenic polycyclic aromatic hydrocarbons

BACKGROUND: Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes in vitro and in vivo. While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons. METHODS: To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 - 30 microM) or in the presence of ANTH (3 microM) or CHRY (3 microM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC). RESULTS: Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism. CONCLUSION: These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential.     

Isolation and characterization of microsatellite loci in the two‐spotted spider mite,Tetranychus urticae (Acari: Tetranychidae)

Sixteen microsatellite loci are described for the two‐spotted spider mite Tetranychus urticae, which is an agricultural pest. The microsatellite loci were obtained through the construction of an enriched library; these loci exhibited polymorphisms (2–5 alleles per locus) and high levels of observed (0.033–0.667, average 0.415) and expected (0.033–0.602, average 0.336) heterozygosities. The isolated microsatellite markers are expected to be useful for the construction of a linkage map of this species.