3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.     

Remission of established disease in diabetic NOD mice induced by anti-CD3 monoclonal antibody]

Anti-CD3 monoclonal antibodies are potent immunosuppressants widely used in clinical transplantation to prevent or treat acute allograft rejection. We have used a hamster monoclonal antibody (145 2C11) specific for the epsilon chain of the murine CD3 complex to treat autoimmune insulin-dependent diabetes appearing spontaneously by 15 to 30 weeks of age in female Non Obese Diabetic (NOD) mice. Mice showing overt disease (glycosuria and glycemia > or = 4 g/l) were randomized in two groups receiving either anti-CD3 (5 micrograms/day i.v. for 5 consecutive days) or an identical dose of hamster polyclonal immunoglobulins. Progressive remission of disease was observed, 3 to 6 weeks after the end of treatment, in 80% of anti-CD3 treated mice as compared to 6% of mice in the control group. This remission was maintained long term namely, during the 4 to 5 months (after the end of treatment) observation period. These results open interesting perspectives on the possibility to treat recently diagnosed diabetic patients with therapy showing long term efficacy and no chronic toxicity.     

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroligin 1 Induces Blood Vessel Maturation by Cooperating with the α6 Integrin

The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.     

Jensen’s inequality as a tool for explaining the effect of oscillations on the average cytosolic calcium concentration

It has often been asked which physiological advantages calcium (Ca²⁺) oscillations in non-excitable cells may have as compared to an adjustable stationary Ca²⁺ signal. One of the proposed answers is that an oscillatory regime allows a lowering of the average Ca²⁺ concentration, which is likely to be advantageous because Ca²⁺ is harmful to the cell in high concentrations. To check this hypothesis, we apply Jensen’s inequality to study the relation between the average Ca²⁺ concentration during oscillations and the Ca²⁺ concentration at the (unstable) steady state. Jensen’s inequality states that for a (strictly) convex function, the function value of the average of a set of argument values is lower than the average of the function values of the arguments from that set. We show that the kinetics of the Ca²⁺ efflux out of the cell is crucial in this context. By analytical calculations we derive that, if the Ca²⁺ efflux is a convex function of the cytosolic Ca²⁺ concentration, then oscillations lower the average Ca²⁺ concentration in comparison to the unstable steady state. If it is a concave function, the average Ca²⁺ concentration is increased, while it remains the same if that function is linear. We also analyse the case where the efflux obeys a Hill kinetics, which involves both a convex and a concave part. The results are illustrated by numerical simulations and simple example models. The theoretical predictions are tested with three experimental data sets from the literature. In two of them, the average appears to be higher than the steady-state value, while the third points to approximate equality. Thus oscillations may be used in real cells to tune the average Ca²⁺ concentration in both directions.     

Relationships between xylem anatomy,root hydraulic conductivity,leaf/root ratio and transpiration in citrus trees on different rootstocks

The aim of the study was to determine the extent in which leaf and whole plant transpiration (Tp) were influenced by root hydraulic conductance (K_r), leaf to root ratio and leaf mass. Also, the relationships between the anatomic characteristics of roots and K_r were investigated. To this end, 9‐month‐old seedlings of the citrus rootstocks Cleopatra mandarin (CM), Poncirus trifoliata (PT), and their hybrids Forner‐Alcaide no 5 (FA‐5) and Forner‐Alcaide no 13 (FA‐13) and 15‐month‐old trees of Valencia orange budded on these four rootstocks were tested. The hybrid FA‐13 and PT had higher values of K_r and leaf transpiration rates (E) than FA‐5 and CM. There was a positive curvilinear correlation between E and K_r. Furthermore, E levels in the different types of plants decreased with increased leaf/root (L/R) ratios. Pruning of the roots and defoliation confirmed that transpiration rates were strongly influenced by the L/R ratio. However, variations in E because of differences in L/R ratios were less pronounced in trees budded on FA‐13 and PT than on the other two rootstocks. In addition, there was a positive correlation between Tp and leaf biomass, although differences between rootstocks may be attributed to differences in K_r. The average lumen diameter of xylem vessels was greater in rootstocks with high K_r. Size of epidermal and hypodermal cells of fibrous roots may also restrict K_r.     

Phylogenetic relationships and biogeographical patterns in Circum-Mediterranean subfamily Leuciscinae (Teleostei,Cyprinidae) inferred from both mitochondrial and nuclear data

Background  

Leuciscinae is a subfamily belonging to the Cyprinidae fish family that is widely distributed in Circum-Mediterranean region. Many efforts have been carried out to deciphering the evolutionary history of this group. Thus, different biogeographical scenarios have tried to explain the colonization of Europe and Mediterranean area by cyprinids, such as the "north dispersal" or the "Lago Mare dispersal" models. Most recently, Pleistocene glaciations influenced the distribution of leuciscins, especially in North and Central Europe. Weighing up these biogeographical scenarios, this paper constitutes not only the first attempt at deciphering the mitochondrial and nuclear relationships of Mediterranean leuciscins but also a test of biogeographical hypotheses that could have determined the current distribution of Circum-Mediterranean leuciscins.     

Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides

Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.     

Taeniosis-cysticercosis complex in individuals of a peasants' settlement (Teodoro Sampaio, Pontal of Paranapanema, SP, Brazil)

In order to evaluate the taeniosis-cysticercosis complex in a population of a peasants' settlement, located at Teodoro Sampaio, state of S?o Paulo, Brazil (longitude 52 degrees 36'12 ", latitude 22 degrees 17'12 ") a series of laboratory markers were determined. After signing an informed consent, participants answered a standardized questionnaire. To determine anti-Taenia solium cysticercus antibodies, the samples were tested by enzyme linked immunoabsorbent assay using 18-and 14-kDa antigen proteins from vesicular fluid of Taenia crassiceps (VF-Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Total IgE levels were determined by chemmiluminescence's assay and hemogram by flow cytometer flux counter. A total of 84 individuals, 5.9% presented anti-T. solium cysticercus antibodies in ELISA and 3.6% were strongly reactive in the 18/14 kDa immunoblotting confirmatory test. All of the individuals with positive antibodies showed elevated Total IgE levels. We conclude that the frequency of anti-T. solium cysticercus antibodies in this population is higher than other regions considered endemic in S?o Paulo. Thus, it is important to carry out surveys in Peasants' settlement areas with the objective of establishing public health measures for prevention and control of infectious diseases such as taeniosis-cysticercosis.     

Essential Roles of Hydrophobic Residues in Both MD-2 and Toll-like Receptor 4 in Activation by Endotoxin

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4·MD-2 complex. Direct binding to MD-2 of LPS and LPS analogues that act as TLR4 agonists or antagonists is well established, but the role of MD-2 and TLR4 in receptor activation is much less clear. We have identified residues within the hairpin of MD-2 between strands five and six that, although not contacting acyl chains of tetraacylated lipid IVa (a TLR4 antagonist), influence activation of TLR4 by hexaacylated lipid A. We show that hydrophobic residues at positions 82, 85, and 87 of MD-2 are essential both for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation. We also identified a pair of conserved hydrophobic residues (Phe-440 and Phe-463) in leucine-rich repeats 16 and 17 of the TLR4 ectodomain, which are essential for activation of TLR4 by LPS. F440A or F463A mutants of TLR4 were inactive, whereas the F440W mutant retained full activity. Charge reversal of neighboring cationic groups in the TLR4 ectodomain (Lys-388 and Lys-435), in contrast, did not affect cell activation. Our mutagenesis studies are consistent with a molecular model in which Val-82, Met-85, and Leu-87 in MD-2 and distal portions of a secondary acyl chain of hexaacylated lipid A that do not fit into the hydrophobic binding pocket of MD-2 form a hydrophobic surface that interacts with Phe-440 and Phe-463 on a neighboring TLR4·MD-2·LPS complex, driving TLR4 activation.Bacterial lipopolysaccharide (LPS)³ is recognized by the innate immune system of vertebrates via an elaborate mechanism involving the membrane receptor TLR4 (1, 2). The extracellular (or cell surface) proteins LPS-binding protein and CD14 promote extraction and transfer of individual molecules of LPS from the Gram-negative bacterial outer membrane to MD-2, either secreted monomeric soluble (s)MD-2 or MD-2 bound with high affinity to the ectodomain of TLR4 (3–7). In contrast to other Toll-like receptors, TLR4 requires an additional molecule, MD-2, for ligand recognition (8). In contrast to MD-2, there has been no evidence of direct binding of LPS to TLR4 (9, 10). Although LPS, and particularly the lipid A portion of LPS, is generally conserved among Gram-negative bacteria, there are many variables in LPS structure that affect TLR4 activation. Most important is the acylation pattern of the lipid A moiety, which represents the minimal segment of LPS that can trigger activation of TLR4 (11). Comparison of crystal structures of MD-2 with and without bound tetraacylated lipid IVa indicates no significant alteration of the protein fold in the absence or presence of bound ligand (12). It has been proposed that both LPS and MD-2 are key to the different effects of tetra- versus hexaacylated LPS on TLR4 (8, 13, 14). Lipid IVa complexed to murine MD-2 has weak agonist effects on murine TLR4 but acts as a receptor antagonist in the same complex containing human MD-2. Hexaacylated endotoxins complexed to human or murine MD-2 act as potent TLR4 agonists. The crystal structure of the TLR4·MD-2·eritoran complex revealed that MD-2 binds to the N-terminal region of TLR4 (15). It seems likely that for TLR4 activation, there needs to be an additional interaction between two ternary TLR4·MD-2·LPS complexes, which is agonist-dependent (15–17). Because tetraacylated and hexaacylated endotoxins that act, respectively, as TLR4 antagonists and agonists differ only in their acylation pattern, we speculated that hydrophobic protein-lipid A interactions are essential in the agonist properties of hexaacylated lipid A. To pursue this hypothesis, we used molecular modeling to select and test the involvement of solvent-exposed hydrophobic residues of MD-2 and TLR4, which we reasoned could be needed for TLR4 activation. We show by mutagenesis studies that residues on the solvent-exposed hairpin of MD-2 support transfer of endotoxin from CD14 to MD-2 and TLR4 activation only when these sites contain hydrophobic residues. In the ectodomain of TLR4, we have identified two neighboring phenylalanine residues located on the convex face of consecutive leucine rich repeats that are required for LPS-triggered TLR4 activation. From those results and molecular docking, we propose that amino acid side chains of both MD-2 and TLR4 ectodomain form an acyl chain binding site, which envelops part of an acyl chain of lipid A that cannot fit into the binding pocket of MD-2 in a TLR4·MD-2 complex and represents a key to LPS-induced TLR4 activation.