Synthesis, Biological Activity, and NMR-Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New a,a-Disubstituted Glycines

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)-a-benzyl-b-azidoalanine, a-benzyl-b- (1-pyrrolidinyl)alanine, a-benzyl-b-(1-piperidinyl)alanine, and a-benzyl-b-(4-morpholinyl)-alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [3H]DAMGO (a l ligand) and [3H]DELT (a d ligand). The affinity of analogs containing (R) or (S)-abenzyl- b-azidoalanine in position 3 to d-receptors strongly depended on the chirality of the a,a-disubstituted residue. The conformational behavior of peptides modified with (R) or (S)-a-benzyl-b-(1-piperidinyl)Ala, which displays the opposite selectivity, was analyzed by 1H and 13C NMR. The l-selective Tyr-D-Ala-(R)- a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 lacks the helical conformation observed in the d-selective Tyr- D-Ala-(S)-a-benzyl-b-(1-piperidinyl)Ala-Asp-Val-Val-Gly- NH2. Our results support the proposal that differences between d- and l-selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N-terminal message domain and the C-terminal address domain

Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers.

It is suggested that tumour stromal myofibroblasts exert an unfavourable effect on the biology of breast cancer. We are aware of only a single study which examined relationships between manifestation of myofibroblasts in the stroma of breast cancer and clinicopathological data of the patients. The present study was aimed at estimation of the effect exerted by myofibroblasts present in the tumour stroma on principal pathological parameters and on expression of Ki67, P53 and HER-2 proteins in the group of the most frequent breast cancers, the ductal cancers. In paraffin sections of 60 ductal breast cancers (20 cases in G1, 20 in G2 and 20 in G3), immunohistochemical reactions were performed to detect expression of smooth muscle actin (SMA) in order to visualize myofibroblasts, Ki67, P53 and HER-2. The studies demonstrated that the most numerous myofibroblasts were present in G3 cases and they were the least frequent in G1 cases (P = 0.02). Positive correlations were observed between the presence of myofibroblasts in tumour stroma and expression of Ki67 and HER-2 in breast cancer cells in the entire group (P < 0.001 and P = 0.001, respectively), in G2 cases (P = 0.003 and P = 0.03) and in G3 cases (P = 0.01 and P = 0.03). Considering that the higher grade, Ki67 and HER-2 are thought to represent unfavourable prognostic factors, the elevated content of myofibroblasts in tumour stroma is probably typical for cases with worse prognosis

A practical approach to the ESC 2022 cardio-oncology guidelines. Comments by a team of experts: cardiologists and oncologists

The 2022 European Society of Cardiology (ESC) guidelines [1] are a comprehensive document, prepared jointly by experts in cardiology and oncology. In the case of an oncological patient, it is necessary to individualize care in relation to the cardiological condition, the stage of the cancer and the type of potential anti-cancer therapy. Cardiac care optimisation should be undertaken before the start of oncological therapy, and continued during oncological therapy, as well as long-term after its completion [2]. The published ESC Guidelines were supplemented with a practical comments of a team of polish cardiology and oncology experts

Zn(II) Complexes of Glutathione Disulfide: Structural Basis of Elevated Stabilities

Glutathione disulfide (GSSG), a long disregarded redox partner of glutathione (GSH), is thought to participate in intracellular zinc homeostasis. We performed a concerted potentiometric and NMR spectroscopic study of protonation and Zn(II) binding properties of GSSG ((γECG)2) and a series of its nine analogs with C-terminal modifications, tripeptide disulfides: (γECS)2, (γECE)2, (γECG-NH2)2, (γECG-OEt)2, and (γEcG)2; dipeptide disulfides, (γEC)2and (γEC-OEt)2; and mixed disulfides, γECG-γEC and ECG-γEC-OEt. The acid-base and Zn(II) complexation properties in this group of compounds are strictly correlated to average C-terminal electrostatic charges. In particular, it was demonstrated that GSSG assumes a bent (head-to-tail) conformation in solution at neutral pH, which is controlled by electrostatic attraction between the protonated γ-amino groups of the Glu residue and the deprotonated C-terminal Gly carboxylates. This interaction modulates the ability of GSSG to coordinate Zn(II), both indirectly, by affecting the basicities of the amino groups, and directly, through the participation of the Gly carboxylates in the outer coordination sphere of the Zn(II) ion. A specific coiled structure of the major [Zn-GSSG]2- complex is additionally stabilized by the formation of hydrogen bonds between glycinyl carboxylates and two Zn(II)-coordinated water molecules. The elevated stability of Zn(II)-GSSG complexes was demonstrated by competition with FluoZin-3, a fluorescent sensor with high Zn(II) affinity, commonly used in in vitro and in vivo studies. The potential biological functions and reactivity of GSSG complexes of Zn(II) ions are discussed

Free energy of helix propagation in short polyalanine chains determined from peptide growth simulations of La3+-binding model peptides. Comparison with experimental data

Molecular dynamics (MD) is, at present, a unique tool making it possible to study, at the atomic level, conformational transitions in peptides and proteins. Nevertheless, because MD calculations are always based on a more or less approximate physical model, using a set of approximate parameters, their reliability must be tested by comparison with experimental data. Unfortunately, it is very difficult to find a peptide system in which conformational transitions can be studied both experimentally and using MD simulations so that a direct comparison of the results obtained in both ways could be made. Such a system, containing a rigid α-helix nucleus stabilized by La3+ coordination to a 12-residue sequence taken from an EF-hand protein has recently been used to determine experimentally the helix propagation parameters in very short polyalanine segments (Goch et al. (2003) Biochemistry 42: 6840-6847). The same parameters were calculated here for the same peptide system using the peptide growth simulation method with, alternatively, charmm 22 and cedar potential energy functions. The calculated free energies of the helix-coil transition are about two times too large for cedar and even three times too large for charmm 22, as compared with the experimental values. We suggest that these discrepancies have their origin in the incorrect representation of unfolded peptide backbone in solution by the molecular mechanics force fields

Halcyon accelerator quality control system optimization

Cel. Celem pracy jest przedstawienie metody optymalizacji kontroli jakości wiązki oraz poprawności działania kolimatora MLC akceleratora Halcyon. Punktem wyjścia do optymalizacji systemu kontroli był algorytm MPC (Machine Performance Check). Opracowanie niezależnych metod weryfikacji działania kolimatora SX2 oraz kontroli stabilności wiązki akceleratora Halcyon oparto na niezależnych od MPC algorytmach. Opracowany system kontroli jakości skonfrontowano z systemem MPC pod względem jakościowym i ilościowym. Materiały i metoda. Optymalizacja kontroli jakości stabilności wiązki oraz poprawności działania kolimatora SX2 oparta została o algorytmy aplikacji MyQA. Testy dla wiązki promieniowania objęły sprawdzenie stabilności wydajności oraz stałości energii. Druga grupa testów związanych z wiązką to testy geometryczne obejmujące analizę takich parametrów, jak: symetria, płaskość, półcień, rozmiar oraz centrum wiązki promieniowania. Testy kolimatora SX2 to testy stabilności, powtarzalności i dokładności pozycjonowania listków MLC oraz poprawność pracy kolimatora MLC dla złożonych układów, dla techniki IMRT oraz VMAT. Oprogramowanie myQA bazuje na predefiniowanych algorytmach opartych na wytycznych raportu grupy zadaniowej AAPM 142. W celu dostosowania wytycznych raportu AAPM-142 do potrzeb rozwiązań konstrukcyjnych aparatu Halcyon zbudowano bazę niezależnych testów opartych na algorytmach aplikacji myQA i porównano z testami zaproponowanymi przez firmę Varian w oprogramowaniu MPC. Wyniki. Analiza parametrów wiązki dla zbudowanego sytemu QA okazała się głębsza niż w przypadku testów dla algorytmu MPC (obraz 1a i b). Testy MPC dotyczące jakości wiązki to stałość wydajności oraz jednorodności. Wyniki obu testów przedstawione są w formie pojedynczej wartości z ustalonymi poziomami tolerancji: 4% i 2%. Zaproponowany system kontroli, oprócz testu stałości wydajności (obraz 2a) z dwoma poziomami tolerancji (warning 1,0%, error 1,5%), rozszerza kontrole wiązki o stałości energii oraz symetrii, płaskości, półcienia, rozmiaru i położenia środka (obraz 2b). Poziomy tolerancji dla testów jakościowych wiązki, w odróżnieniu od systemu MPC, to szereg wartości opartych na wytycznych raportu IAEA TRS 389. Kontrola pracy kolimatora SX2 w systemie MPC to analiza dokładności i odtwarzalności ustawienia dla pojedynczych listków, dla układów statycznych. Zaproponowany system QA pozwala testować stabilność, powtarzalność i dokładności pozycjonowania listków MLC dla układów dynamicznych. Dyskusja. Zaproponowany, niezależny system QA, w odróżnieniu od systemu MPC, pozwala na znaczące zmniejszenie poziomów tolerancji dla stałości wydajności oraz wprowadza ich zróżnicowanie dla poszczególnych parametrów geometrycznych wiązki. Przedstawiony system QA pozwala na testowanie dokładności pracy kolimatora MLC dla układów dynamicznych stosowanych w technikach IMRT oraz VMAT. Czas realizacji i dostępność czasowa wyników, jak również możliwość analizy trendów czasowych to mocna strona obu systemów.Purpose/Objective. The purpose of the work is to present a method for optimizing beam quality control and correct operation of the Halcyon MLC collimator. The starting point for optimizing the control system was the MPC (Machine Performance Check) algorithm. The development of independent methods for verifying the operation of the SX2 collimator and checking the beam stability of the Halcyon accelerator was based on algorithms independent of MPC. Material and methods. Optimization of quality control of beam stability and correctness of SX2 collimator operation was based on MyQA application algorithms. Tests for the radiation beam included checking performance stability and energy constancy. The second group of beam-related tests are geometric tests involving the analysis of parameters such as symmetry, flatness, penumbra, size and center of the radiation beam. SX2 collimator tests are tests of stability, repeatability and accuracy of MLC leaf positioning, and correctness of MLC collimator operation for complex systems, for IMRT and VMAT techniques. MyQA software is based on predefined algorithms based on the guidelines of the A APM 142 task group report. In order to adapt the guidelines of the AAPM-142 report to the needs of the Halcyon design solutions, a database of independent tests based on the myQA application algorithms has been built. Results. The analysis of radiation beam parameters for the built-in control system turned out to be more insightful than in the case of tests proposed by the MPC algorithm (image no. 1). Two MPC tests directly addressing the quality of the radiation beam are stability performance and homogeneity, presented in the form of two numerical values along with time trend analysis. The values obtained in the case of the proposed control system give a much deeper analysis, extended to control energy constancy and symmetry, flatness, penumbra, size and center of the radiation beam (image no. 2). Control of the SX2 collimator operation, in the case of the MPC system, is an analysis of the accuracy and reproducibility of the settings of each individual leaf for static MLC systems. The proposed system allows testing the stability, repeatability and accuracy of MLC leaf positioning for dynamic systems that reflect clinical working conditions. Conclusion. The time needed for the implementation of the presented range of tests is similar for both systems. Both systems offer analysis of time trends and the creation of test reports. The proposed system for controlling the basic parameters of the Halcyon accelerator, unlike the MPC system, allows for a deeper analysis of the beam quality and correctness of the MLC collimator in conditions similar to those prevailing during the implementation of the treatment plan

Dynamical insight into Caenorhabditis elegans eIF4E recognition specificity for mono-and trimethylated structures of mRNA 5′ cap

Abstract Specific recognition and binding of the ribonucleic acid 5′ termini (mRNA 5′ cap) by the eukaryotic translation initiation factor 4E (eIF4E) is a key, rate limiting step in translation initiation. Contrary to mammalian and yeast eIF4Es that discriminate in favor of 7-methylguanosine cap, three out of five eIF4E isoforms from the nematode Caenorhabditis elegans as well as eIF4Es from the parasites Schistosome mansoni and Ascaris suum, exhibit dual binding specificity for both 7-methylguanosine-and N2,N2,7-trimethylguanosine cap. To address the problem of the differences in the mechanism of the cap recognition by those highly homologic proteins, we carried out molecular dynamics simulations in water of three factors, IFE-3 and IFE-5 isoforms from C. elegans and murine eIF4E, in the apo form as well as in the complexes with 7-methyl-GDP and N2, N2,7-trimethyl-GDP. The results clearly pointed to a dynamical mechanism of discrimination between each type of the cap, viz. differences in mobility of the loops located at the entrance into the protein binding pockets during the cap association and dissociation. Additionally, our data showed that the hydrogen bond involving the N2-amino group of 7-methylguanosine and the carboxylate of glutamic acid was not stable. The dynamic mechanism proposed here differs from a typical, static one in that the differences in the protein-ligand binding specificity cannot be ascribed to formation and/or disruption of well defined stabilizing contacts

Solution structure of conformationally restricted vasopressin analogues.

In recent years, a massive effort has been directed towards designing potent and selective antagonists of neurohypophyseal hormones substituted at position 3. Modification of vasopressin at position 3 with 4,4'-biphenylalanine results in pharmacologically inactive analogues. Chemically, this substitution appears to vary only slightly from those previously made by us (1-Nal or 2-Nal), which afforded potent agonists of V2 receptors. In this situation, it seemed worthwhile to study the structure of the analogues with 4,4'-biphenylalanine (BPhe) at position 3 in aqueous solution using NMR spectroscopy and total conformational analysis. This contribution is part of extensive studies aimed at understanding spatial structures of 3-substituted [Arg8]vasopressin analogues of different pharmacological properties. NMR data were used to calculate 3D structures for all the analogues using two methods, EDMC with the ECEPP/3 force field, and molecular dynamic with the simulated annealing (SA) algorithm. The structures obtained by the first method show a better fit between the NMR spectral evidence and the calculation for all the peptides