N-Methylated Analogs of hIAPP Fragments 18-22, 23-27, 33-37 Inhibit Aggregation of the Amyloidogenic Core of the Hormone.

Amylin (hIAPP) aggregation leads to the formation of insoluble deposits and is one of the factors in the development of type II diabetes. The aim of this research was to find N-methylated analogs of the aggregating amylin fragments 18-22, 23-27, and 33-37, which would not themselves be susceptible to aggregation and would inhibit the aggregation of the amyloidogenic cores of the hormone. None of the analogs of fragment 18-22 containing one or two N-methylated amino acid residues showed any tendency to aggregate. Only the peptide H-F(N-Me)GA(N-Me) IL-OH (6) derived from the 23-27 hIAPP hot spot did not form fibrous structures. All analogs of the 33-37 amylin fragment were characterized by the ability to form aggregates, despite the presence of N-methylated amino acids in their structures. N-Methylated peptides 1-5 demonstrated inhibitory properties against the aggregation of fragment 18-22. Aggregation of the amyloidogenic core of 23-27 was significantly inhibited by N-methylated peptides 1-3 derived from the (18-22) H-HSSNN-OH fragment and by the H-F(N-Me)GA(N-Me)IL-OH (6) fragment derived from the 23-27 amylin hot spot. Fragment (33-37) H-GSNTY-NH2 was found to be inhibited in the presence of N-methylated peptides 1-3 derived from the 18-22 fragment and by the double methylated peptide H-F(N-Me)GA(N-Me)IL-OH (6). Research on the possibility of using N-methylated analogs of amyloidogenic amylin cores as inhibitors of hormone aggregation is ongoing, with a focus on finding the minimum concentration of N-methylated peptides capable of inhibiting the aggregation of hIAPP hot spots.

Non-Aggregating Amylin Fragments as an Inhibitors of the Aggregation Process of Susceptible to Aggregation Fragments 18-22, 23-27, and 33-37 of Hormone.

Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1-7, 8-12, 13-17 and 28-32 of amylin would inhibit the aggregation of the amyloidogenic cores 18-22, 23-27, 33-37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1-7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18-22, 23-27, 33-37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1-7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.

Synthesis and cellular effects of novel 1,3,5-triazine derivatives in DLD and Ht-29 human colon cancer cell lines.

This study provides new information on the cellular effects of 1,3,5-triazine nitrogen mustards with different peptide groups in DLD and Ht-29 human colon cancer cell lines. A novel series of 2,4,6-trisubstituted 1,3,5-triazine derivatives bearing 2-chloroethyl and oligopeptide moieties was designed and synthesized. The most cytotoxic derivative was triazine with an Ala-Ala-OMe substituent on the ring (compound 7b). This compound induced time- and dose-dependent cytotoxicity in the DLD-1 and HT-29 colon cancer cell lines. The triazine derivative furthermore induced apoptosis through intracellular signaling pathway attenuation. Compound 7b may be a candidate for further evaluation as a chemotherapeutic agent against colorectal cancer.

New Human Islet Amyloid Polypeptide Fragments Susceptible to Aggregation.

Human Islet Amyloid Polypeptide (hIAPP) plays a key role in the pathogenesis of type II diabetes. The aim of this research was to search for new amyloidogenic fragments of hIAPP. An initial attempt to predict the amyloidogenic cores of polypeptides/proteins using five different computer programs did not provide conclusive results. Therefore, we synthesized hIAPP fragments covering the entire hormone. The fragments were assessed for their aggregation ability, using recommended methods to search for the amyloidogenic fragments of the polypeptides/proteins. It was found that fragments (18-22) H-HSSNN-OH and (33-37) H-GSNTY-NH2 aggregate and form stable amyloid-like structures. Both of these fragments have a much higher antiproliferative activity relative to the RIN-5F cell compared to the (23-27) H-FGAIL-OH fragment widely regarded as the amyloidogenic core of amylin. The analog of (33-37) H-GSNTY-NH2 containing a free carboxy group on the C-terminal amino acid (H-GSNTY-OH) does not have amyloidogenic properties and can therefore be considered as a potential inhibitor of amylin aggregation. Research on the use of non-aggregating amylin fragments as potential hormone aggregation inhibitors is ongoing.

Biological Activity of Hydrophilic Extract of Chlorella vulgaris Grown on Post-Fermentation Leachate from a Biogas Plant Supplied with Stillage and Maize Silage.

Algae are employed commonly in cosmetics, food and pharmaceuticals, as well as in feed production and biorefinery processes. In this study, post-fermentation leachate from a biogas plant which exploits stillage and maize silage was utilized as a culture medium for Chlorella vulgaris. The content of polyphenols in hydrophilic extracts of the Chlorella vulgaris biomass was determined, and the extracts were evaluated for their antioxidant activity (DPPH assay), antibacterial activity (against Escherichia coli, Lactobacillus plantarum, Staphylococcus aureus, Staphylococcus epidermidis) and antifungal activity (against Aspergillus niger, Candida albicans, Saccharomyces cerevisiae). The use of the post-fermentation leachate was not found to affect the biological activity of the microalgae. The aqueous extract of Chlorella vulgaris biomass was also observed to exhibit activity against nematodes. The results of this study suggest that Chlorella vulgaris biomass cultured on post-fermentation leachate from a biogas plant can be successfully employed as a source of natural antioxidants, food supplements, feed, natural antibacterial and antifungal compounds, as well as in natural methods of plant protection.

The cellular effects of novel triazine nitrogen mustards in glioblastoma LBC3, LN-18 and LN-229 cell lines.

1,3,5-triazine is an important heterocyclic skeleton for mono, two or three 2-chloroethylamine groups. The study presented here provides novel information on cellular effects of 1,3,5-triazine with mono, two or three 2-chloroethylamine groups in glioblastoma LBC3, LN-18 and LN-229 cell lines. In our study, the most cytotoxic effect was observed in 1,3,5-triazine with three 2-chloroethylamine groups (12f compound). It has been demonstrated that 12f induce time- and dose-dependent cytotoxicity in all investigated glioma cell lines. Apart from that in glioblastoma cells, treated with 12f compound, we noticed strong induction of apoptosis. In conclusion, this research provides novel information concerning cellular effects of apoptosis in LBC3, LN-18 and LN-229 cell lines. Moreover, we suggest that 12f compound may be a candidate for further evaluation as an effective chemotherapeutic agent for human glioblastoma cells.

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues.

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C-terminal, N-terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H-PheCysTrp-OH, H-PheCysTyr-OH, H-PheTyrMet-OH, and H-TrpTyr-OH.

Study on the Materials Formed by Self-Assembling Hydrophobic, Aromatic Peptides Dedicated to Be Used for Regenerative Medicine.

The aims of this study were to identify the short aromatic peptides which are able to form highly ordered amyloid-like structures in self-assembling processes, to test the influence of length of hydrophobic peptides on tendency to aggregation, and to check if aggregated peptides fulfill requirements expected for materials useful for scaffolding. All tested hydrophobic peptides were prepared on solid phase by using DMT/NMM/TsO- as a coupling reagent. The progress of aggregation was studied by set of independent tests. All aggregated peptides were found stable under in vitro conditions. All fibrous material formed by self-assembling of peptides does not show any cytotoxic effects on L929 fibroblast cells. Peptides containing tyrosine and tryptophan residues even effectively accelerated the proliferation and stimulated the activity of L929 fibroblasts.

Insulin Hot-Spot Analogs Formed with N-Methylated Amino Acid Residues Inhibit Aggregation of Native Hormone.

In this study, N-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin "amyloidogenic" analogs containing N-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3-7). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation.

Search for New Aggregable Fragments of Human Insulin.

In this study, three independent methods were used to identify short fragment of both chains of human insulin which are prone for aggregation. In addition, circular dichroism (CD) research was conducted to understand the progress of aggregation over time. The insulin fragments (deca- and pepta-peptides) were obtained by solid-phase synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) as a coupling reagent. Systematic studies allowed identification of the new fragments, expected to be engaged in triggering aggregation of the entire structure of human insulin under physiological conditions. It was found that the aggregation process occurs through various structural conformers and may favor the formation of a fibrous structure of aggregate.

New methodology for automated SPOT synthesis of peptides on cellulose using 1,3,5-triazine derivatives as linkers and as coupling reagents.

Two new rigid bi-aromatic linkers for synthesis of peptide arrays by SPOT methodology were obtained from cellulose treated with 2,4-dichloro-6-methoxy-1,3,5-triazine. Reaction with m-phenylenediamine gave non-cleavable TYPE I linker which enabled attachment of the peptides via resistant to harsh reaction conditions amide, ether, and amine bonds. Reaction with 3-Fmoc-aminobenzoic acid followed by thermal isomerization of the intermediate "superactive" ester producing an amide-like bond gave TYPE II linker that was very stable during peptide synthesis. However, the peptide was cleavable, with fragment of the linker, in the presence of 1 M LiOH solution. The uniform loading of the cellulose and efficient synthesis of the peptide array was achieved by using N-(4,6-dimethoxy-1,3,5-triazin-1-yl)-N-methylmorpholinium 4-toluenesulfonate as the coupling reagent.

Novel tool in rheumatoid arthritis diagnosis-The usage of urease flap region peptidomimetics.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and non-RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms. Thus, to better understand the role of bacteria in RA, we synthetized 6 peptidomimetics of bacterial ureases' flap region. These peptides were then used to distinguish RA patients from healthy people sera by immunoblotting. Most patients' sera were bound to peptidomimetic characteristic for Enterobacter sp. and Klebsiella sp. flap urease. We also found similarities between peptidomimetic sequence and human proteins connected with RA. This pilot study suggests that bacteria may trigger RA via mechanism of molecular mimicry of urease to host proteins and ureases flap peptidomimetics may be potential candidate as a new additional diagnostic test.

Cross-Reactivity of Polyclonal Antibodies against Canavalia ensiformis (Jack Bean) Urease and Helicobacter pylori Urease Subunit A Fragments.

Overlapping decapeptide fragments of H. pylori urease subunit A (UreA) were synthesized and tested with polyclonal antibodies against Canavalia ensiformis (Jack bean) urease. The linear epitopes of UreA identified using the dot blot method were then examined using epitope mapping. For this purpose, series of overlapping fragments of UreA, frameshifted ± four amino acid residues were synthesized. Most of the UreA epitopes which reacted with the Jack bean urease polyclonal antibodies had been recognized in previous studies by monoclonal antibodies against H. pylori urease. Fragments 11 - 24, 21 - 33, and 31 - 42 were able to interact with the Jack bean urease antibodies, giving stable immunological complexes. However, the lack of recognition by these antibodies of all the components in the peptide map strongly suggests that a non-continuous (nonlinear) epitope is located on the N-terminal domain of UreA.

Towards Intelligent Drug Design System: Application of Artificial Dipeptide Receptor Library in QSAR-Oriented Studies.

The pharmacophore properties of a new series of potential purinoreceptor (P2X) inhibitors determined using a coupled neural network and the partial least squares method with iterative variable elimination (IVE-PLS) are presented in a ligand-based comparative study of the molecular surface by comparative molecular surface analysis (CoMSA). Moreover, we focused on the interpretation of noticeable variations in the potential selectiveness of interactions of individual inhibitor-receptors due to their physicochemical properties; therefore, the library of artificial dipeptide receptors (ADP) was designed and examined. The resulting library response to individual inhibitors was arranged in the array, preprocessed and transformed by the principal component analysis (PCA) and PLS procedures. A dominant absolute contribution to PC1 of the Glu attached to heptanoic gating acid and Phe bonded to the linker m-phenylenediamine/triazine scaffold was revealed by the PCA. The IVE-PLS procedure indicated the receptor systems with predominant Pro bonded to the linker and Glu, Gln, Cys and Val directly attached to the gating acid. The proposed comprehensive ligand-based and simplified structure-based methodology allows the in-depth study of the performance of peptide receptors against the tested set of compounds.

Search for Fibrous Aggregates Potentially Useful in Regenerative Medicine Formed under Physiological Conditions by Self-Assembling Short Peptides Containing Two Identical Aromatic Amino Acid Residues.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H-dPhedPhe-OH, H-dCysdPhedPhe-OH, H-CysTyrTyr-OH, H-dPhedPhedCys-OH, H-TyrTyrMet-OH, and H-TyrMetTyr-OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

Visible-Light Microscopic Discovery of Up to 150 µm Long Helical Amyloid Fibrils Built of the Dodecapeptide H-(Val-Ala-Leu)4 -OH and of Decapeptides Derived from Insulin.

In the formation of amyloid fibrils from small peptides, the appearance of superhelices of (P)- or (M)-helicity has been observed for the first time; high concentrations of the peptides and extended periods of incubation at physiological pH appear to be important for this phenomenon. In view of the general importance of peptide and protein aggregation, we give a brief overview with selected examples for demonstration.

Synthesis of linear and cyclic opioid-based peptide analogs containing multiple N-methylated amino acid residues.

A series of six novel opioid peptide analogs containing one to three N-methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid-phase method. Introduction of two consecutive N-methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine-based coupling reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by-products than observed with the standard coupling reagents such as TBTU or HATU.

Preliminary studies on application of library of artificial receptors for differentiation of metabolites in urine of healthy and cancer bearing mice.

A library of artificial receptors formed by the self-organization of N-lipidated peptides attached to the cellulose via m-aminophenylamino-1,3,5-triazine was used for differentiation of metabolites in urine of healthy and cancer bearing mice. The interactions of urine metabolites with the receptors were visualized by using competitive adsorption-desorption of an appropriate reporter dye. Analysis of the binding pattern (fingerprint) of urine metabolites from healthy and from cancer suffering mice showed that there were several structures among 120-elements molecular receptors which were able to differentiate bonded ligands depending on the healthy state. For all three tested types of cancers two structures: Lipid-Pro-Ala-NH-C6H4-NH-DMT-cellulose and Lipid-Arg-Pro-NH-C6H4-NH-DMT-cellulose were selected as diagnostic.

Synthesis of chiral triazine coupling reagents based on esters of N-alkylproline and their application in the enantioselective incorporation of D or L amino acid residue directly from racemic substrate.

Esters of N-methylproline and N-allylproline were prepared and used as component for synthesis of chiral triazine based coupling reagents. N-Triazinylammonium tetrafluoroborate obtained from methylester of L-N-methylproline, 2-chloro-4,6-dimethozxy-1,3,5-triazine and tetrafluoroboric acid in the coupling of rac-Z- A1a-OH with glycine methylester preferred formation of D-Z-AlaGly-OMe with L/D ratio 21/79. Coupling reagent prepared from D enantiomer of N-methylproline gave L-Z-AlaGly-OMe with L/D ratio 75/25.

Assessment of individual dose equivalents Hp(0.07) of medical staff occupationally exposed to ionizing radiation in 2012.

BACKGROUND: The paper presents the Nofer Institutes of Occupational Medicine in Lódz's results of the assessment of individual dose equivalents Hp(0.07) of medical staff exposed to X-rays in Poland in 2012. In addition, the collected data was analysed in terms of types of medical units performing medical procedures and the categorization of personnel. MATERIAL AND METHODS: Dosimetric service was provided for medical staff of interventional radiology departments occupationally exposed to ionizing radiation in terms of individual dose equivalents Hp(0.07). In 2012, personal dosimetry Hp(0.07) determinations were performed by the Nofer Institute of Occupational Medicine in Lódz and covered 2044 employees from 174 health facilities. The determinations were performed using thermoluminescence dosimetry according to the procedure accredited by the Polish Centre for Accreditation (document number AB 327). The measurements were performed using ring-dosimeters in the periods of 1 or 2 months. RESULTS: Mean annual individual dose equivalent Hp(0.07) in 2012 was equal to 3.3 mSv (annual limit for Hp(0.07) is 500 mSv). The average value of annual individual dose equivalent Hp(0.07) decreased comparing to the previous year. In 2012, no single case of exceeding the annual limit for Hp(0.07) was reported. Data stored in the file indicates that more than 96% of all of the annual doses did not exceed the level of 10 mSv. CONCLUSIONS: The analysis of data on occupational exposure to ionizing radiation confirms a stable level of exposure and satisfactory radiological protection in interventional radiology facilities monitored by the Nofer Institute of Occupational Medicine in Lódz in Poland in 2012.