Rapid Tryptophan Assay as a Screening Procedure for Quality Protein Maize.

Tryptophan is an essential amino acid deficient in cereals, especially maize. However, maize (Zea mays L.) is the main source of protein in some developing countries in Africa and Latin America. In general, the nutritional profile of cereals is poor, because they are deficient in essential amino acids such as tryptophan and lysine due to a relatively higher proportion of alcohol-soluble proteins. Quality protein maize (QPM) has been developed through genetic manipulation for the nutritional enrichment of maize to address these problems. Nevertheless, methods for protein, lysine and tryptophan are time-consuming and require relatively large amounts of samples. Therefore, we have advanced here a simple, cheap, fast, reliable and robust procedure for the determination of protein and tryptophan in the same biuret supernatant, which can also be used for chemical characterization of other cereals. Samples of 50 mg maize ground to pass through a 0.1 mm screen were sonicated for 5 min. in eppendorf vials with 1.5 mL of a biuret reagent each. After centrifugation and protein determination by biuret, 0.2 mL of supernatant was treated with 0.8 mL of a tryptophan reagent. Both total protein and tryptophan can be determined in microplates at 560 nm to speed up the measurements. The main advantage of the new micro-method is the rapid estimation of the nutrient quality of maize samples by a single weighing of a small amount of valuable plant materials.

Exploring Pyrrolo-Fused Heterocycles as Promising Anticancer Agents: An Integrated Synthetic, Biological, and Computational Approach.

Five new series of pyrrolo-fused heterocycles were designed through a scaffold hybridization strategy as analogs of the well-known microtubule inhibitor phenstatin. Compounds were synthesized using the 1,3-dipolar cycloaddition of cycloimmonium N-ylides to ethyl propiolate as a key step. Selected compounds were then evaluated for anticancer activity and ability to inhibit tubulin polymerization in vitro. Notably, pyrrolo[1,2-a]quinoline 10a was active on most tested cell lines, performing better than control phenstatin in several cases, most notably on renal cancer cell line A498 (GI50 27 nM), while inhibiting tubulin polymerization in vitro. In addition, this compound was predicted to have a promising ADMET profile. The molecular details of the interaction between compound 10a and tubulin were investigated through in silico docking experiments, followed by molecular dynamics simulations and configurational entropy calculations. Of note, we found that some of the initially predicted interactions from docking experiments were not stable during molecular dynamics simulations, but that configurational entropy loss was similar in all three cases. Our results suggest that for compound 10a, docking experiments alone are not sufficient for the adequate description of interaction details in terms of target binding, which makes subsequent scaffold optimization more difficult and ultimately hinders drug design. Taken together, these results could help shape novel potent antiproliferative compounds with pyrrolo-fused heterocyclic cores, especially from an in silico methodological perspective.

Ultrasound-Assisted Synthesis of Fluorescent Azatetracyclic Derivatives: An Energy-Efficient Approach.

We report here an energy-efficient and straight synthesis of two new classes of derivatized fluorescent azatetracycles under ultrasound (US) irradiation. A first class of azatetracyclic compounds was synthesized by heterogeneous catalytic bromination of the α-keto substituent attached to the pyrrole moiety of the tetracyclic cycloadducts, while for the second, one class was synthesized by nucleophilic substitution of the bromide with the azide group. Comparative with conventional thermal heating (TH) under US irradiation, both types of reactions occur with substantially higher yields, shortened reaction time (from days to hours), lesser energy consumed, easier workup of the reaction, and smaller amounts of solvent required (at least three to five-fold less compared to TH), which make these reactions to be considered as energy efficient. The derivatized azatetracycle are blue emitters with λmax of fluorescence around 430-445 nm. A certain influence of the azatetracycle substituents concerning absorption and fluorescent properties was observed. Compounds anchored with a bulky azide group have shown decreased fluorescence intensity compared with corresponding bromides.

Zinc Binding to NAP-Type Neuroprotective Peptides: Nuclear Magnetic Resonance Studies and Molecular Modeling.

Aggregation of amyloid-ß peptides (Aß) is a hallmark of Alzheimer's disease (AD), which is affecting an increasing number of people. Hence, there is an urgent need to develop new pharmaceutical treatments which could be used to prevent the AD symptomatology. Activity-dependent neuroprotective protein (ADNP) was found to be deficient in AD, whereas NAP, an 8-amino-acid peptide (1NAPVSIPQ8) derived from ADNP, was shown to enhance cognitive function. The higher tendency of zinc ion to induce Aß aggregation and formation of amorphous aggregates is also well-known in the scientific literature. Although zinc binding to Aß peptides was extensively investigated, there is a shortage of knowledge regarding the relationship between NAP peptide and zinc ions. Therefore, here, we investigated the binding of zinc ions to the native NAP peptide and its analog obtained by replacing the serine residue in the NAP sequence with tyrosine (1NAPVYIPQ8) at various molar ratios and pH values by mass spectrometry (MS) and nuclear magnetic resonancespectroscopy (NMR). Matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry confirmed the binding of zinc ions to NAP peptides, while the chemical shift of Asp1, observed in 1H-NMR spectra, provided direct evidence for the coordinating role of zinc in the N-terminal region. In addition, molecular modeling has also contributed largely to our understanding of Zn binding to NAP peptides.

A Simple Synthesis Route for Selectively Methylated ß-Cyclodextrin Using a Copper Complex Sandwich Protecting Strategy.

This communication reports a novel synthesis route for the preparation of monofunctionalized ß-cyclodextrin in a single stage. The approach involves only the in-situ protection of secondary hydroxyl groups as an excellent alternative to the classical procedure involving a series of five steps of protection and deprotection of hydroxyl groups (both primary and secondary ones) belonging to ß-cyclodextrin.

Cytotoxic substituted indolizines as new colchicine site tubulin polymerisation inhibitors.

A potential microtubule destabilising series of new indolizine derivatives was synthesised and tested for their anticancer activity against a panel of 60 human cancer cell lines. Compounds 11a, 11b, 15a, and 15j showed a broad spectrum of growth inhibitory activity against cancer cell lines representing leukaemia, melanoma and cancer of lung, colon, central nervous system, ovary, kidney, breast, and prostate. Among them, compound 11a was distinguishable by its excellent cytostatic activity, showing GI50 values in the range of 10-100 nM on 43 cell lines. The less potent compounds 15a and 15j in terms of GI50 values showed a high cytotoxic effect against tested colon cancer, CNS cancer, renal cancer and melanoma cell lines and only on few cell lines from other types of cancer. In vitro assaying revealed tubulin polymerisation inhibition by all active compounds. Molecular docking showed good complementarity of active compounds with the colchicine binding site of tubulin.

Direct evidence for binding of aluminum to NAP anti-amyloid peptide and its analogs.

NAP (NAPVSIPQ) is a small peptide derived from the activity-dependent neuroprotective protein (ADNP), which provides neuroprotection against amyloid-ß peptide toxicity associated with Alzheimer disease. Several metal ions are able to promote the formation of amyloid-ß peptide oligomers and protofibrils in human brain tissue. Although the relationship between metal ions and amyloid-ß peptide peptides is extensively investigated, that with the NAP peptide is less understood. Nevertheless, our previous research revealed unexpected iron binding to NAP peptide and its analogs. However, a link between aluminum ions, Alzheimer disease and amyloid-ß peptide or NAP peptides still remains controversial. Therefore, we have investigated the possible binding of aluminum ions to NAP peptide and its four analogs. Indeed, MALDI-ToF mass spectrometry (MS), including MS/MS study, and Fourier transform infrared (FT-IR) spectroscopy revealed an unexpected pattern of aluminum ion binding to both NAP peptide and its analogs. Our results have been discussed with respect to NAP protection against Alzheimer disease-related neurotoxicity.

Letter: Mass spectrometric evidence for iron binding to the neuroprotective peptide NAP and its Cys5 mutant.

The NAP peptide (H(2)N-(1)NAPVSIPQ(8)-CONH(2)) is a truncated version of the activity-dependent neuroprotective protein. Its neuroprotective activities consist of the inhibition of Aß(25-35) and Aß(1-40) fibrillogenesis as well as protection against Aß-induced neurotoxicity and prevention of microtubule disruption associated with Alzheimer's disease. Therefore, we synthesized NAP and its mutant peptide with the sequence: H(2)N-(1)NAPVCIPQ(8)-COOH (NAPCOH), by replacing serine S(5) with cysteine C(5). Both native and mutant peptides were further used to study their interaction with iron ions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Fourier transform infrared spectroscopy and also atomic force microscopy were used to probe Fe(3+) binding to both peptides. Contrary to the expected results, the investigated peptides underwent different oxidation processes, with resultant reduced Fe(2+) ions. These ions, and not the original Fe(3+) ions, were found to bind to each of non-oxidized peptides.

Ultrasound-based protein determination in maize seeds.

The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.