Analysis of protein-carbohydrate interaction at the lower size limit of the protein part (15-mer peptide) by NMR spectroscopy,electrospray ionization mass spectrometry,and molecular modeling

Structural analysis of minimally sized lectins will offer insights into fundamentals of intermolecular recognition and potential for biomedical applications. We thus moved significantly beyond the natural limit of lectin size to determine the structure of synthetic mini-lectins in solution, their carbohydrate selectivity and the impact of ligand binding on their conformational behavior. Using three disaccharide (Thomsen-Friedenreich antigen; Gal beta 1,3GalNAc alpha 1,R)-binding pentadecapeptides without internal disulfide bridges as role models, we successfully tested a combined strategy with different techniques of NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling. In solution, the peptides invariably displayed flexibility with rather limited restrictions, shown by NMR experiments including nearly complete resonance assignments and molecular dynamics simulations. The occurrence of aromatic/nonpolar amino acids in the sequence did not lead to formation of a hydrophobic core known from microbial chitinase modules. Selectivity of disaccharide binding was independently observed by mass spectrometry and NMR analysis. Specific ligand interaction yielded characteristic NMR signal alterations but failed to reduce conformational flexibility significantly. We have thereby proven effectiveness of our approach to analyze even low-affinity interactions (not restricted to carbohydrates as ligands). It will be useful to evaluate the impact of rational manipulation of lead peptide sequences.     

Synthesis and NMR characterization of hydroxyurea and mesylglycol glycoconjugates as drug candidates for targeted cancer chemotherapy

Tumor targeting of glycoconjugated antineoplastic agents is a strategy currently under investigation for cancer chemotherapy. We have synthesized the glucosides and galactosides of the clinically established drug hydroxyurea and of mesylglycol, the reactive moiety of the anticancer drug busulfan. Glycosides of hydroxyurea were obtained by carbamoylation of hydroxylamine glycosides. The glycosides of mesylglycol were synthesized by mesylation of protected glycol glycosides. All compounds were characterized by detailed 1H and 13C NMR analysis.     

Neural Induction of the Blood–Brain Barrier: Still an Enigma

1. The study of the blood–brain barrier and its various realms offers a myriad of opportunities for scientific exploration. This review focuses on two of these areas in particular: the induction of the blood–brain barrier and the molecular mechanisms underlying this developmental process.2. The creation of the blood–brain barrier is considered a specific step in the differentiation of cerebral capillary endothelial cells, resulting in a number of biochemical and functional alterations. Although the specific endothelial properties which maintain the homeostasis in the central nervous system necessary for neuronal function have been well described, the inductive mechanisms which trigger blood–brain barrier establishment in capillary endothelial cells are unknown.3. The timetable of blood–brain barrier formation is still a matter of debate, caused largely by the use of varying experimental systems and by the general difficulty of quantitatively measuring the degree of blood–brain barrier tightness. However, there is a general consensus that a gradual formation of the blood–brain barrier starts shortly after intraneural neovascularization and that the neural microenvironment (neurons and/or astrocytes) plays a key role in inducing blood–brain barrier function in capillary endothelial cells. This view stems from numerous in vitro experiments using mostly cocultures of capillary endothelial cells and astrocytes and assays for easily measurable blood–brain barrier markers. In vivo, there are great difficulties in proving the inductive influence of the neuronal environment. Also dealt with in this article are brain tumors, the least understood in vivo systems, and the induction or noninduction of barrier function in the newly established tumor vascularization.4. Finally, this review tries to elucidate the question concerning the nature of the inductive signal eliciting blood–brain barrier formation in the cerebral microvasculature.     

Inhibition of leukotriene receptors boosts neural progenitor proliferation

Neural stem and progenitor cells serve as a reservoir for new neurons in the adult brain throughout lifetime. One of the critical steps determining the net production of new neurons is neural progenitor proliferation, which needs to be tightly controlled. Since inflammation has detrimental effects on neurogenesis and the 5-lipoxygenase/leukotriene pathway is involved in inflammatory processes, we investigated the effects of leukotrienes and montelukast, a small molecule inhibitor of the leukotriene receptors CysLT(1)R and GPR17, on neural stem and progenitor cell proliferation. We demonstrate expression of the leukotriene receptor GPR17 by neural progenitors and by neural stem cells. Stimulation with excess amounts of leukotrienes did not affect progenitor proliferation, whereas blockade of GPR17 with montelukast strongly elevated neural stem and progenitor proliferation, while maintaining their differentiation fate and potential. This effect was associated with increased ERK1/2 phosphorylation suggesting an involvement of the EGF signaling cascade. Based on our results, montelukast and the inhibition of the 5-LOX pathway might be potent candidates for future therapies employing neurogenesis to promote structural and functional improvement in neurodegeneration, neuropsychiatric disease and ageing.     

Troponin elevation in acute ischemic stroke (TRELAS) - protocol of a prospective observational trial

Background  

Levels of the cardiac muscle regulatory protein troponin T (cTnT) are frequently elevated in patients with acute ischemic stroke and elevated cTnT predicts poor outcome and mortality. The pathomechanism of troponin release may relate to co-morbid coronary artery disease and myocardial ischemia or, alternatively, to neurogenic cardiac damage due to autonomic activation after acute ischemic stroke. Therefore, there is uncertainty about how acute ischemic stroke patients with increased cTnT levels should be managed regarding diagnostic and therapeutic workup.     

Regulatory T cells and IL-10 independently counterregulate cytotoxic T lymphocyte responses induced by transcutaneous immunization

Background

The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T_reg) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.

Methodology/Principal Findings

TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3⁺ regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10^-/-) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T_reg in IL-10^-/- mice and the use of B cell deficient JHT^-/- mice, we can exclude T_reg and B cells as source of IL-10 in the setting of TCI.

Conclusion/Significance

We identify T_reg and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T_reg function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors.     

Transmigration of melanoma cells through the blood-brain barrier: role of endothelial tight junctions and melanoma-released serine proteases

Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasize to the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the only possibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the great clinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletely understood. In order to investigate this question we have used an in vitro experimental setup based on the culture of cerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able to adhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from the luminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was accelerated when they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through the endothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanoma cells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at least partially) the paracellular transmigration pathway. During this process melanoma cells produced and released large amounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitor Pefabloc® was able to decrease to 44–55% the number of melanoma cells migrating through CECs. Our results suggest that release of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main steps in the formation of brain metastases in malignant melanoma.     

Efficient synthesis of the cyclometalated complex fac-[Rh(ppy)3] (ppy = 2-phenylpyridinato)

A new convenient high-yield synthesis of the tris-cyclometalated complexes fac-[Rh(ppy)₃] (4; ppy = 2-phenylpyridinato) was developed. Complex 4 was prepared in a kind of one-pot synthesis starting from in situ prepared [Rh(acac)(coe)₂] (2) which was heated in refluxing 2-phenylpyridine for a short time. After purification by filtration over alumina, compound 4 was obtained in yields of 65%. Also [Rh(acac)(ppy)₂] (3) was prepared in a similar manner by oxidative addition of Hppy in refluxing toluene in high yields. In contrast to previous findings with the analogous iridium compounds, there was not any hint at the formation of the isomer mer-[Rh(ppy)₃] using similar reaction conditions as applied for iridium. Furthermore the compound [{Rh(μ-Cl)(ppy)₂}₂] (5) was prepared from [{Rh(μ-Cl)(coe)₂}₂] (1) and Hppy in refluxing toluene in nearly quantitative yield.