Molecular and Supramolecular Architecture of theSalmo gairdneriProteinaceous Eggshell during Development

A detailed developmental study of eggshell architecture of the fishSalmo gairdneri(rainbow trout) was performed using transmission and scanning electron microscopy. Thioglycollic acid treatment and freeze-fracturing reveal that fibrils ca. 5–10 nm in diameter constitute each lamella of the helicoidal eggshell. Freeze-fracturing also permits a direct visualization of the helicoidal architecture. Laser-Raman studies of the eggshell indicate abundant antiparallel β-pleated-sheet conformation in the eggshell proteins ofS. gairdneriduring all developmental stages. Apparently, this conformation dictates formation of the helicoidal structure. Disulfide bonds, together with isopeptide bonds, cross-linkS. gairdnerieggshell proteins throughout development.     

Design and synthesis of novel series of pyrrole based chemotypes and their evaluation as selective aldose reductase inhibitors. A case of bioisosterism between a carboxylic acid moiety and that of a tetrazole

Pyrrolyl-propionic and butyric-acid derivatives 1 and 2 were synthesized in order to study the effect of the variation of the methylene chain in comparison to the previously reported pyrrolyl-acetic acid compound I, which was found as potent aldose reductase inhibitor, while the pyrrolyl-tetrazole derivatives 3–5 were prepared as a non-classical bioisosteres of a carboxylic acid moiety. Also, pyrrolyl-tetrazole isomers 6 and 7 without an alkyl chain between the two aromatic rings were synthesized. The in vitro aldose reductase inhibitory activity of the prepared 1–7 compounds were estimated and compared with that of the initial compound (I). Overall, the data indicate that the presented chemotypes 6 and 7 are a promising lead compounds for the development of selective aldose reductase inhibitors, aiming to the long-term complications of diabetes mellitus.     

Contribution of neural crest-derived cells in the embryonic and adult thymus

Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26(eYfp) reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.     

Carboxymethylated pyridoindole antioxidants as aldose reductase inhibitors: Synthesis, activity, partitioning, and molecular modeling

Starting from the efficient hexahydropyridoindole antioxidant stobadine, a series of carboxymethylated tetrahydro- and hexahydropyridoindole derivatives was synthesized and tested for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibiton of rat lens aldose reductase was determined by a conventional method. Kinetic analysis of (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (5b) and (2-phenethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (5c), the most potent compounds in this series with activities in micromolar range, showed uncompetitive inhibition. In addition to the importance of the acidic function, the inhibition efficacy was highly influenced by the steric conformation of the lipophilic aromatic backbone when comparing tetrahydro- and hexahydropyridoindole congeners. Selectivity with respect to the closely related aldehyde reductase was determined by measuring the corresponding inhibitory activities. Antioxidant action of the novel compounds was documented in a DPPH test and in a liposomal membrane model, oxidatively stressed by peroxyl radicals. The presence of a basicity center at the tertiary nitrogen, in addition to the acidic carboxylic function, predisposes these compounds to form double charged zwitterionic species, a characteristic which may remarkably affect their pH-lipophilicity profile. For compounds 5b and 5c, a maximal distribution ratio in a system comprised of 1-octanol/phosphate buffer was recorded near the neutral physiological pH, the region where the isoelectric point lies. Molecular docking simulations into the ALR2 active site performed for the zwitterionic species provided an explanation for the observed structure–activity relationships and the calculated parameters were in agreement with characteristic differences in the stereoelectronic profiles of the tetrahydro- versus hexahydropyridoindoles. ‘Drug-likeness’ of the novel aldose reductase inhibitors was assessed by applying the criteria of Lipinski’s ‘rule of five’.     

Design and synthesis of N-(3,5-difluoro-4-hydroxyphenyl)benzenesulfonamides as aldose reductase inhibitors

N-(3,5-Difluoro-4-hydroxyphenyl)benzenesulfonamide (4) and its derivatives 5-7 were prepared as putative bioisosteres of the previously reported aldose reductase inhibitors, which are the N-benzenesulfonylglycine derivatives I-IV. The in vitro aldose reductase inhibitory activity of the prepared compounds is higher than that of the respective glycine derivatives. Furthermore, the parent compound 4 reveals high antioxidant potential. Additionally, the intestine permeability of 4 is determined, and there is initial evidence that there is an operating influx mechanism. Overall, the data indicate that the presented chemotype could serve as a core structure for the design of putative pharmacotherapeutic agents, aiming to the long-term complications of diabetes mellitus.     

Nitric oxide and MCP-1 regulation in LPS activated rat Kupffer cells

Nitric oxide (NO) and Monocyte Chemoattractant Protein (MCP)-1 co-regulation has been found in endotoxin-activated macrophages. Kupffer cells (KC) are a main source of soluble-mediators production in liver abnormalities. We investigated in vitro similar co-regulation of NO and MCP-1 production in rat activated KC. Isolated rat KC were cultured in the presence of 1 microg/ml LPS and various concentrations of Wortmannin (0-300 nM), L-NAME (0-500 microM) or MCP-1 (0-100 ng/ml). Production of MCP-1 and NO were measured in supernatants, by ELISA and a modification of the Griess reaction, respectively. Growth arrested KC, stimulated with vehicle, produced a basal amount of NO and MCP-1. In the presence of LPS, cultured KC secreted significantly (P < 0.01) increased amounts of MCP-1 and NO. Pre-treatment of KC with various concentrations of L-NAME significantly (P < 0.05) reduced the LPS-induced secretion of NO in a concentration dependent manner, but the MCP-1 production remained unaffected. Pre-treatment with Wortmannin significantly (P < 0.05) inhibited LPS-induced secretion of MCP-1 and NO in a concentration dependent manner. Linear regression analysis revealed a positive correlation between MCP-1 and NO in the LPS (r = 0.59171, P < 0.0001) and Wortmannin (r = 0.9215, P = 0.009) treated groups, but not in the L-NAME (r = -0.08513, P = 0.873). Incubation of KC with various concentrations of MCP-1 did not increase the NO production. These results indicate that KC might be the main source of NO and MCP-1 production in liver disorders, probably through the induction of PI3-kinase(s) and without any co-regulation between these molecules, which might represent two independent immunoregulatory pathways in the role of KC in hepatic disorders.     

Biophysical properties of apolipoprotein E4 variants: implications in molecular mechanisms of correction of hypertriglyceridemia

In humans and animal models, high plasma concentrations of apolipoprotein (apo) E are associated with hypertriglyceridemia. It has been shown that overexpression of human wild-type (WT) apoE4 in apoE-deficient mice induces hypertriglyceridemia. In contrast, overexpression of an apoE4 variant, apoE4-mut1 (apoE4(L261A, W264A, F265A, L268A, V269A)), does not induce hypertriglyceridemia and corrects hypercholesterolemia. Furthermore, overexpression of another variant, apoE4-mut2 (apoE4(W276A, L279A, V280A, V283A)), induces mild hypertriglyceridemia and does not correct hypercholesterolemia. To better understand how these mutations improve the function of apoE4, we investigated the conformation and stability of apoE4-mut1 and apoE4-mut2 and their binding to dimyristoyl phosphatidylcholine (DMPC) vesicles and to triglyceride (TG)-rich emulsion particles. We found that the mutations introduced in apoE4-mut1 lead to a more stable and compactly folded conformation of apoE4. These structural changes are associated with a slower rate of solubilization of DMPC vesicles by apoE4-mut1 and reduced binding of the protein to emulsion particles compared with WT apoE4. Under conditions of apoE4 overexpression, the reduced binding of apoE4-mut1 to TG-rich lipoprotein particles may facilitate the lipolysis of these particles and may alter the conformation of the lipoprotein-bound apoE in a way that favors the efficient clearance of the lipoprotein remnants. Mutations introduced in apoE4-mut2 result in smaller structural alterations compared with those observed in apoE4-mut1. The slightly altered structural properties of apoE4-mut2 are associated with slightly reduced binding of this protein to TG-rich lipoprotein particles and milder hypertriglyceridemia as compared with WT apoE4.