4-Methylpseudoproline analogues of cyclolinopeptide A: Synthesis, structural analysis and evaluation of their suppressive effects in selected immunological assays.

The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro3-Pro4 fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed. The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu1-Val2-(R)-(αMe)Ser(ΨPro)3-Pro4-Phe5-Phe6-Leu7-Ile8-Ile9) and c(Leu1-Val2-Pro3-(R)-(αMe)Ser(ΨPro)4-Phe5-Phe6-Leu7-Ile8-Ile9), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro3 by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro4 residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders.

Management of Concomitant Moyamoya Disease, Arterial Venous Malformation, and Intracranial Aneurysm: Case Illustration, Literature Review, and Management Algorithm.

BACKGROUND: Moyamoya disease (MMD), arterial venous malformations (AVMs), and intracranial aneurysms are distinct cerebrovascular disease processes that most commonly occur in isolation. The literature on the treatment algorithm of each individual condition is well established. An association between MMD and intracranial aneurysms is also known, but MMD in association with AVM is rare. CASE DESCRIPTIONS: The authors discuss various dilemmas in the multimodality management of these conditions when they coexist with an illustrative case of a 46-year-old woman who presented following a stroke, with 1) left-sided MMD, 2) left-sided frontal AVM, and 3) an aneurysm arising from the A1 segment of the right anterior cerebral artery. These were managed respectively by 1) the left external carotid artery to M2 segment of middle cerebral artery bypass using an autologous radial artery graft and left indirect superior temporal artery-encephaloduroarteriosynangiosis, 2) stereotactic radiosurgery, and 3) endovascular coiling of the aneurysm. Three years following intervention, cerebral angiography showed a patent bypass, complete obliteration of the AVM, and no residual filling of the coiled aneurysm. CONCLUSIONS: Our strategy of surgical revascularization for MMD, radiosurgery for AVM, and endovascular coiling for aneurysm resulted in a positive long-term clinical outcome. In view of the rarity of the condition, the authors propose a management algorithm for such patients.

Synthesis and biological activity of cyclolinopeptide A analogues modified with γ4-bis(homo-phenylalanine).

Cyclolinopeptide A (CLA), an immunosuppressive nonapeptide derived from linen seeds, was modified with S or R-γ4-bis(homo-phenylalanine) in positions 3 or 4, or both 3 and 4. These modifications changed the flexibility of new analogues and distribution of intramolecular hydrogen bonds. Analogues 11 c(Pro1-Pro2-Phe3-S-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9), 13 c(Pro1-Pro2-S-γ4-hhPhe3-R-γ4-hhPhe4-Leu5-Ile6-Ile7-Leu8-Val9) and 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) existed as a mixture of stable cis/trans isomers of Pro-Pro peptide bond. The comparison of the relative spatial orientations in crystal state of the two carbonyl groups, neighboring γ-amino acids, revealed conformational similarities to α-peptides. The addition of two -CH2- groups in γ-amino acids led to a more rigid conformation, although a more flexible one was expected. A significant difference in the relative orientation of the carbonyl groups was found for cyclic γ-peptides with a dominance of an antiparallel arrangement. As carbonyl groups may be engaged in the interactions with plausible receptors through hydrogen bonds, a similar biological activity of the modified peptides was expected. Our biological studies showed that certain cyclic, but not the corresponding linear peptides, lowered the viability of peripheral blood mononuclear cells (PBMC) at 100µg/mL concentration. The proliferation of PBMC induced by phytohemagglutinin A (PHA) was strongly inhibited by cyclic peptides only, in a dose-dependant manner. On the other hand, lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production in whole blood cell cultures was inhibited by both linear and cyclic peptides. Peptide 15 c(Pro1-Pro2-R-γ4-hhPhe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) blocked the expression of caspase-3, inhibited the expression of caspases-8 and -9 in 24h culture of Jurkat cells, and caused DNA fragmentation in these cells, as an indicator of apoptosis. Thus, we revealed a new mechanism of immunosuppressive action of a nonapeptide.

Assessing the correlation between the degree of disc degeneration on the Pfirrmann scale and the metabolites identified in HR-MAS NMR spectroscopy.

OBJECTIVE: The objective of this study is to assess the correlation between the degree of degeneration of lumbar discs according to the Pfirrmann classification system and the concentrations of metabolites determined by means of 1H high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR) spectroscopy. MATERIALS AND METHODS: Twenty-six human intervertebral lumbar discs that were operated on due to degenerative disease were analyzed. Routine preoperative 1.5T, T2-weighed magnetic resonance (MR) images were used to classify the cases according to the Pfirrmann classification system. In all the cases, during microdiscectomy, the fragments of the annulus fibrosus and nucleus pulposus were harvested and their metabolic profile was examined by means of 1H HR MAS. The grades of disc degeneration on the Pfirrmann scale were correlated with the metabolite concentrations. RESULTS: Spectral analyses of the intervertebral discs with Pfirrmann grades IV and V demonstrated significantly higher concentrations of creatine, glycine, hydroxyproline, alanine, leucine, valine, acetate, isoleucine, α,ß-glucose, and myo-inositol, and a lower intensity of the N-acetyl peak of chondroitin sulfate, compared to the spectra with Pfirrmann grade III. CONCLUSION: Our results demonstrate correlations between metabolite concentrations and the degree of lumbar disc degeneration assessed using the Pfirrmann grading system and provide another step toward the potential use of in vivo MR spectroscopy for investigation of biomarkers in lumbar disc degeneration.

Characterization of metabolites determined by means of 1H HR MAS NMR in intervertebral disc degeneration.

OBJECT: The objective of this study is the identification of metabolites by means of (1)H high resolution magic angle spinning nuclear magnetic resonance ((1)H HR MAS NMR) spectroscopy and the evaluation of their applicability in distinguishing between healthy and degenerated disc tissues. MATERIALS AND METHODS: Differences between the metabolic profiles of healthy and degenerated disc tissues were studied by means of (1)H HR MAS NMR. Analysis was performed for 81 disc tissue samples (control samples n = 21, degenerated disc tissue samples n = 60). Twenty six metabolites (amino acids, carbohydrates, and alcohols) were identified and quantified. RESULTS: The results indicate that the metabolic profile of degenerated discs is characterized by the presence of 2-propanol and the absence of scyllo-inositol and taurine. The concentrations of 2-propanol and lactate increase with age. CONCLUSION: PCA analysis of ex vivo (1)H HR MAS NMR data revealed the occurrence of two groups: healthy and degenerative disc tissues. The effects of insufficient nutrient supply of discs, leading to their degeneration and back pain, are discussed.

Prediction of survival for patients with advanced colorectal cancer using (1) H High-resolution magic angle spinning nuclear MR spectroscopy.

PURPOSE: To evaluate whether the metabolic profiles of colorectal cancer specimens can be used for prediction of survival. MATERIALS AND METHODS: The metabolic profiles of colorectal cancer tissues were determined using the high-resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) technique (16.4 T). HR MAS analysis was performed for 52 tissues taken from patients classified as survivors and nonsurvivors (30). Quantitative analysis was performed for each spectrum. Receiver operating characteristic (ROC) curves were used to evaluate the potential to predict patient survival over 5.5 years. RESULTS: Analysis of (1) H NMR spectra led to the identification and quantitative analysis of 30 metabolites. A significant increase in the Tau/Gly and Tau/MI ratios were associated with long-term survival (P = 0.004 and P = 0.003, respectively). ROC analysis indicated that the Tau/MI ratio had the best predictive value for survival (sensitivity 64.7% and specificity 100%). Good predictive value of survival was found for Tau/Gly ratio (sensitivity 63.6% and specificity 96.3%). Moreover, the Glu/Gln metabolic ratio with a cutoff level of 1.74 was predictive of survival with a sensitivity of 83.3% and a specificity of 85.7%. CONCLUSION: Our results indicate that HR MAS spectroscopy is potentially useful for survival prediction in advanced colorectal cancer.

Crystal structure of (3R)-3-benzyl-4-[(tert-but-oxy-carbon-yl)amino]-butanoic acid.

The characteristic feature of the title mol-ecule, C16H23NO4, is the syn configuration of the partially double amide C-N bond [C-N-C-O torsion angle = -14.8 (2)°]. The crystal packing is determined by inter-molecular O-H⋯O and N-H⋯O hydrogen bonds, which link the mol-ecules into a double-chain structure extending along [010].

Synthesis and biological activity of cyclolinopeptide A analogues modified with γ(3)-bis(homophenylalanine).

Cyclolinopeptide A, naturally occurring immunomodulatory nonapeptide, was modified with S or R-γ(3)-bis(homophenylalanine) in positions 3 or 4, or both 3 and 4. The replacement of one or both Phe residues by γ(3)-hhPhe led to decrease of their conformational flexibility in the analogues in comparison to CLA. All cyclic peptides, except 11, exist as isomers with the cis Pro-Pro peptide bond. Cyclic peptide 11 with single modification S-γ(3)-hhPhe(4) exists as a mixture of two isomers and the major isomer (89%) contains all peptide bonds of the trans geometry. The peptides were subjected to several immunological tests in vitro and in vivo. Linear peptides 1-8, precursors of CLA analogues 9-16, were not toxic against human peripheral blood mononuclear cells (PBMC) but cyclic analogues showed dose-dependent toxicity with exception of peptide 11. Linear peptides did not inhibit mitogen-induced PBMC proliferation whereas cyclic ones inhibited the proliferation in a dose-dependent manner. The actions of linear and cyclic peptides with regard to lipopolysaccharide (LPS) -induced tumour necrosis factor alpha (TNF α) production in whole human blood cultures were differential but particularly suppressive in the case of linear compound 6. Therefore, for in vivo tests compounds 6 and 11 were selected. The compounds showed comparable, suppressive actions in induction and effector phases of delayed type hypersensitivity as well as in the carrageenan-induced foot pad edema in mouse models. In summary, linear peptide 6 and cyclic peptide 11 are attractive as potential immune suppressor drugs.

The structure of cyclolinopeptide A in chloroform refined by RDC measurements.

Three-dimensional structures of molecules traditionally assigned from nuclear Overhauser effects and vicinal coupling constants are recently complemented by measurements of residual dipolar couplings. Residual dipolar couplings measured in a stretched poly(dimethylsiloxane) gel were used to determine the structure of cyclolinopeptide A in chloroform solution at -50 °C. After structure refinement, conformational details of main cluster were discussed in relation to crystal and nuclear Overhauser effect derived structures.

Simulation-based training improves ITU staff knowledge in the management of head injuries.

Traumatic brain injury (TBI) in the polytrauma situation is a phenomenon often seen at UK hospitals. Without immediate access to dedicated neurocritical care facilities, the potential for under-treatment of the underlying brain injury and serious neurological sequalae is high, especially if staff on the general intensive care units on which these patients are treated lack confidence in this area of practice, a reality confirmed by our baseline study. We found that by engaging staff by implementing a regular simulation-based team training programme, we were able to boost the skills, knowledge, and ultimately confidence levels in treating TBI amongst these groups of staff. "Buy-in" by those concerned was high, and we found that self-reported scores for the attributes described above were improved considerably and consistently by our intervention. This quality improvement project has been rolled out through several iterations to become sustainable, has significant cost-saving potential, and will hopefully lead to proven improved clinical outcomes for this group of patients.

4-Methylpseudoproline derived from α-methylserine - synthesis and conformational studies.

This paper presents the synthesis and solution conformational studies of the tripeptides Fmoc-Ala-(R)-(αMe)Ser(Ψ(H,H)Pro)-Ala-OBu(t) (6a) and Fmoc-Ala-(S)-(αMe)Ser(Ψ(H,H)Pro)-Ala-OBu(t) (6b). Additionally, the X-ray structure of 6a is given. NMR analysis corroborated by theoretical calculations (XPLOR) shows that in both peptides the amide bond between pseudoproline and the preceding amino acid is in the trans conformation. The same amide bond geometry was observed in the crystal state of 6a. The latter is additionally influenced by the presence of two symmetrically independent molecules in an asymmetric unit. Both molecules adopt a conformation which resembles ß-turn type II, stabilized by hydrogen bonding. The conformational preferences and prolyl cis-trans isomerization of Ac-(αMe)Ser(Ψ(H,H)Pro)-NHMe (7) were explored at the IEFPCM/B3LYP/6-31+G(d) level of theory in vacuum, water and chloroform. It has been shown that the trans isomer predominates in water solutions and the cis isomer is preferred in chloroform. The conformation of 7 is down-puckered independently of the geometry of the amide bonds, with lower puckering in the transition state of the cis-trans isomerization.

Problems with molecular mechanics implementations on the example of 4-benzoyl-1-(4-methyl-imidazol-5-yl)-carbonylthiosemicarbazide.

Results from force fields implemented in HyperChem, a program frequently used in studies of bioactive compounds, have been compared using the example of the conformational analysis of a 1-carbonylthiosemicarbazide that exhibits strong antibacterial activity. By comparing these results with the original force fields and the experimental NMR ROESY spectrum, it was shown that these implementations can lead to erroneous results.

Analysis of cancer tissues by means of spectroscopic methods.

The personalized approach in cancer treatment stimulates the search for new analytical techniques, including spectroscopic methods such as Raman spectroscopy, mass spectrometry MALDI (matrix-assisted laser desorption/ionization) imaging and high-resolution magic angle spinning nuclear magnetic resonance (HR MAS NMR). The purpose of these studies is determination of metabolic profiles of cancer tissues, and their application in diagnostics and therapy of cancers. The review is mainly focused on application of HR MAS NMR technique. Qualitative and quantitative analysis of metabolites by means of this method is described for breast cancer tissues. In the near future HR MAS NMR in vitro studies of metabolic profiles combined with in vivo studies using MRI scanners may be applied as a new diagnostic tool.

Measurements of heavy-atom isotope effects using 1H NMR spectroscopy.

A novel method for measuring heavy-atom KIEs for magnetically active isotopes using (1)H NMR is presented. It takes advantage of the resonance split of the protons coupled with the heavy atom in the (1)H spectrum. The method is validated by the example of the (13)C-KIE on the hydroamination of styrene with aniline, catalyzed by phosphine-ligated palladium triflates.

Cyclolinopeptide derivatives modify methotrexate-induced suppression of the humoral immune response in mice.

High doses of chemotherapeutics in clinical treatment, leading to cell toxicity, can be lowered by co-administration of other immunoregulatory drugs. The aim of this study was to evaluate effects of several derivatives of cyclolinopeptide A (CLA), derived from linen seeds, on the suppressive action of metothrexate (MTX) in a mouse model of humoral immune response in vitro. New CLA analogues 1 and 2, and their linear precursors 3 and 4, containing conformationally restricted dipeptide fragment Phe-Phe or D-Phe-D-Phe with ethylene bridge (-CH(2)-CH(2)-) between phenylalanine nitrogens were synthesized. NMR studies and theoretical calculations showed that introduction of locally constraining fragment into CLA molecule increased its overall conformational flexibility. The bioactivity of new CLA analogues was examined in the mouse model of the in vitro secondary humoral immune response, suppressed by methotrexate (MTX). The results revealed differential actions of the peptides such as 1/augmentation of the suppressive activity of MTX or 2/antagonistic effects of the peptides on MTX-induced suppression. Potential advantages for the application of CLA-derived peptides in therapy and structure-activity relationships were discussed.

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides.

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.

Biological and docking studies of topoisomerase IV inhibition by thiosemicarbazides.

4-Benzoyl-1-(4-methyl-imidazol-5-yl)-carbonylthiosemicarbazide (1) was synthesized, and its antibacterial and type IIA topoisomerase (DNA gyrase and topoisomerase IV) activity evaluated. (1) was found to have high therapeutic potential against opportunistic Gram-positive bacteria, and inhibitory activity against topoisomerase IV (IC(50)=90 µM) but not against DNA gyrase. An increase in activity against topoisomerase IV (IC(50)=14 µM) was observed when the imidazole moiety of (1) was replaced with the indole group in 4-benzoyl-1-(indol-2-yl)-carbonylthiosemicarbazide (2). However, (2) showed only weak antibacterial activity. Although the results of the bacterial type IIA topoisomerases inhibition study did not parallel antibacterial activities, our observations strongly imply that a 4-benzoylthiosemicarbazide scaffold can be developed into an efficient Gram-positive antibacterial targeting topoisomerase IV. The difference in activity against type IIA topoisomerases between (1) and (2) was further investigated by docking studies, which suggested that these compounds target the ATP binding pocket.

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73.

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer.  Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation.  Two p53 homologues, p63 and p73, have been described in humans.  Unlike p53, these proteins regulate developmental processes rather than genome stability.  Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx.  Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined.  Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.

Synthesis and immunosuppressive activity of cyclolinopeptide A analogues containing homophenylalanine.

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. Cyclolinopeptide A, naturally existing immunomodulatory peptide, was modified with homophenylalanine in positions 3 (4), 4 (5) or both 3 and 4 (6). The conformational influence of the replacement of Phe by Hphe was analyzed by NMR spectroscopy. Peptides 4-6 exist as single isomers with all trans peptide bonds except cis Pro-Pro peptide bond. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug--cyclosporine A. The substitution of Phe with Hphe in positions 3 and 4 of CLA led to three different activities in the studied immunological assays. Very potent inhibition of AFC number of peptide 4 was not associated with cell toxicity. This compound caused a complete block of T- and B-cell proliferation. Peptides 5 and 6, containing Hphe in position 3 or 3 and 4, respectively, gave similar effects on the proliferative response of splenocytes to mitogens. Peptide 6 was a moderate suppressor of the humoral immune response, peptide 5 was exceptionally inhibitory. The presence of Hphe in position 4 of CLA backbone markedly reduced the viability of the tested cell line, however addition of the second Hphe in position 3 improved cell survival in comparison with the solvent.

Synthesis, conformational analysis and immunological activity of beta3Phe-substituted Cyclolinopeptide A analogues.

CLA, a natural, highly hydrophobic cyclic nonapeptide with sequence c(Pro(1)-Pro(2)-Phe(3)-Phe(4)-Leu(5)-Ile(6)-Ile(7)-Leu(8)-Val(9)-), isolated from linseed oil, was found to possess a strong immunosuppressive activity comparable, in low doses, with that of CsA, with a mechanism that depends on the inhibition of the interleukin-1 and interleukin-2 action. Structural analysis of CLA and its related compounds has underlined that the presence of the tetrapeptide Pro-Pro-Phe-Phe sequence, the Pro-Pro cis amide bond, and the 'edge-to-face' interaction are possible important features for the immunosuppressive activity of CLA. To evaluate the role and significance of 'edge-to-face' interaction in the process of molecular recognition by receptors, we have synthesised three linear precursors and three cyclic analogues of CLA, in which one or both Phe residues have been replaced by beta(3)Phe residues. A conformational analysis by NMR in CD(3)CN/H(2)O mixture has been carried out on the CLA analogue, in which Phe(3) has been replaced by a betaPhe, to study the influence of the mutation on the three-dimensional structure. All linear and cyclic CLA analogues containing betaPhe have been tested in the humoral and cellular immune response in vivo assays in mice. The peptide activities have been compared with CsA, as a reference drug.