Population shift vs induced fit: the case of bovine seminal ribonuclease swapping dimer

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. This enzyme exists as two conformational isomers with distinctive biological properties. The structure of the major isomer is characterized by the swapping of the N-terminal segment (MxM BS-RNase). In this article, the crystal structures of the ligand-free MxM BS-RNase and its complex with 2'-deoxycitidylyl(3',5')-2'-deoxyadenosine derived from isomorphous crystals have been refined. Interestingly, the comparison between this novel ligand-free form and the previously published sulfate-bound structure reveals significant differences. In particular, the ligand-free MxM BS-RNase is closer to the structure of MxM BS-RNase productive complexes than to the sulfate-bound form. These results reveal that MxM BS-RNase presents a remarkable flexibility, despite the structural constraints of the interchain disulfide bridges and the swapping of the N-terminal helices. These findings have important implications to the ligand binding mechanism of MxM BS-RNase. Indeed, a population shift rather than a substrate-induced conformational transition may occur in the MxM BS-RNase ligand binding process.     

Influence of conformational flexibility on biological activity in cyclic astin analogues

The astins, a family of natural antitumor cyclopeptides, from the roots of Aster tataricus, consist of a 16-membered ring system containing uncoded amino acid residues. The backbone conformation, with a cis-3,4-dichlorinated proline residue, plays an important role in antineoplastic activity. The acyclic astins, on the other hand, do not show antitumor activity, suggesting that the cyclic nature of astins may be a key role in their biological properties. Although the antineoplastic activity of natural astins has been screened in vitro and in vivo, the mechanism of action has never been investigated. With the aim at elucidating the influence of conformational flexibility on biological activity, we have designed and synthesized several astin analogues containing either Aib and the nonproteinogenic Abu and (S)beta3-hPhe residues, able to modify the peptide backbone structure, or the peptide bond surrogate -SO2-NH-. Tested for their antitumor effect, our astin-related cyclopeptides are able to inhibit the growth of tumor cell lines, while the acyclic astins are inefficacious. The present work reports on the structure-activity study of a selected synthetic cyclotetrapeptide corresponding to the sequence c[Thr-Aib-(S)beta3-hPhePsi(CH2-SO2-NH)-Abu], synthesized by classical methods and characterized conformationally by two-dimensional NMR and molecular dynamics analyses.     

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.     

A new pentastomid from the black vulture

This article describes a new pentastomid species from the abdominal air sacs of a black vulture (Aegypius monachus Linnaeus, 1766) from central Spain. The parasite's morphological characteristics (as shown by light and scanning electron microscopy) suggest that it should be classified in the new genus. It is the third pentastomid species described in birds and the first for the Accipitridae. The mouth is almost terminal, there are 2 pairs of hooks behind the mouth, and the genital pore is immediately posterior to these structures, placing the new species within the Cephalobaenida. The anterior and posterior hooks are similar in size and are flanked by parapodial lob. Cuticular tubercles are absent, false annulations can be seen, and the parasite's eggs have 2 layers.     

Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism

Ca(V)1.2, the cardiac L-type calcium channel, is important for excitation and contraction of the heart. Its role in other tissues is unclear. Here we present Timothy syndrome, a novel disorder characterized by multiorgan dysfunction including lethal arrhythmias, webbing of fingers and toes, congenital heart disease, immune deficiency, intermittent hypoglycemia, cognitive abnormalities, and autism. In every case, Timothy syndrome results from the identical, de novo Ca(V)1.2 missense mutation G406R. Ca(V)1.2 is expressed in all affected tissues. Functional expression reveals that G406R produces maintained inward Ca(2+) currents by causing nearly complete loss of voltage-dependent channel inactivation. This likely induces intracellular Ca(2+) overload in multiple cell types. In the heart, prolonged Ca(2+) current delays cardiomyocyte repolarization and increases risk of arrhythmia, the ultimate cause of death in this disorder. These discoveries establish the importance of Ca(V)1.2 in human physiology and development and implicate Ca(2+) signaling in autism.     

Comparison of the predicted in vivo behaviour of the Sn(II)-APDDMP complex and the results as studied in a rodent model

In a quest for more effective radiopharmaceuticals for pain palliation of metastatic bone cancer, this paper relates results obtained with ((117m)Sn labelled) Sn(II) complexed to the bone seeking bisphosphonate, N,N-dimethylenephosphonate-1-hydroxy-3-aminopropylidenediphosphonate (APDDMP). APDDMP is synthesised from the known bone cancer pain palliation agent 1-hydroxy-3-aminopropylidenediphosphonate (APD, Pamindronate). This work is performed to utilise the idea that the low bone marrow radio toxicity of (117m)Sn could afford a highly effective radiopharmaceutical in pain palliation but also in the curative treatment of bone metastasis. Complex-formation constants of APDDMP with the important blood plasma metal-ions, Ca(2+), Mg(2+), Zn(2+) as well as the added metal ion, Sn(2+) were measured by glass electrode potentiometry at 25 degrees C and I = 150 mM. Blood plasma models were constructed using the computer code ECCLES and the results compared with those gathered from tests on a rodent model. The ((117m)Sn-labelled) Sn(II)-APDDMP complex was found to have only some liver and bone uptake although a high trabecular to normal bone ratio was recorded. From the blood plasma model this was shown to be primarily due to the high affinity of APDDMP for Ca(II) causing some of the Sn(II)-APDDMP complex to dissociate. High kidney uptake and excretion as well as high bladder uptake was recorded which was shown to be due to the dissociation of the Sn(II)-APDDMP complex in blood plasma. Animal model observations could be explained by the blood plasma modelling.     

Comparative effect of ion calcium and magnesium in the activation and infection of the murine macrophage by Leishmania major

Amastigotes of Leishmania major have a great ability to evade destruction in host cells. This study investigated the activation in resident, inflammatory macrophages and J774 cells in vitro treated with lipopolysaccharide (LPS), soluble Leishmania antigen (SLA), calcium ionophore (CaI) and magnesium (Mg2+) alone or combined. An increase in nitric oxide (NO) production was observed in J774 or inflammatory macrophages treated with LPS alone or in combination with SLA and CaI. The same treatments did not affect the NO release by resident macrophages. There was no interference in uptake of L. major but CaI decreased intracellular proliferation of the parasite. This study demonstrated the importance of CaI in decreasing L. major proliferation inside murine macrophages while Mg2+ seemed to increase parasite proliferation. These finding may help to understand the events involved in host cells' clearance of this pathogen.     

Isoenzyme patterns: a valuable molecular tool for the differentiation of Zygosaccharomyces species and detection of misidentified isolates

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.     

Amperometric measurements of nitric oxide in erythrocytes

In the recent years, there has been an increase in the development of new biosensors that could be helpful in the study of various physiological processes. In this study, we report the development of a new in vitro experimental design for real-time nitric oxide (NO) amperometric measurements in erythrocyte suspensions. To achieve this, we employed human erythrocyte suspensions in sodium chloride 0.9%, pH 7 (haematocrit 0.05%). The production of NO by erythrocytes was measured with a commercial NO sensor during stimulation by L-arginine, acetylcholine, choline, atropine and velnacrine maleate (10 microM of final concentrations). We also measured the nitrite and nitrate concentrations produced by erythrocyte suspensions stimulated with the above effectors by means of the Griess reaction method. We observed that there was a direct relation between the electric current produced by the NO sensor, and the NO standard concentrations, thereby leading to a good calibration curve. The in vitro erythrocytes produced significant amperometric NO values in response to a wide range of effectors and these results have the same variation profile of the nitrites and nitrates results achieved with the Griess method. In conclusion, the amperometric NO sensor constitutes a reliable method for direct, and real-time measurement in vitro of the NO production of erythrocyte suspensions, As such, it offers a potential diagnostic technique for the evaluation of diseases, and the therapeutic progression of diseases, related to intracellular NO metabolism.     

Role of pre-existing redox profile of human macrophages on lipid synthesis and cholesteryl ester cycle in presence of native, acetylated and oxidised low density lipoprotein

The importance of the interactions of modified lipids and macrophages in foam cell generation is clear; however, little attention has been paid to the role of intra-macrophagic redox potential as a modulator of their lipid synthesis and metabolism. In this study, the effects of previously induced non-toxic manipulations of intracellular redox balance on lipid synthesis in human monocyte-derived macrophages (HMDM) was evaluated. Cells, pre-treated with 2.5 microM of the pro-oxidising agent CuSO(4) or with 5 mM of the antioxidant and thiol supplier N-acetylcysteine (NAC), were exposed to radiolabelled oleic acid alone or in combination with native low density lipoprotein (LDL) or modified LDL to evaluate the incorporation of radioactivity into cholesteryl ester, triacylglycerols and phospholipids. CuSO(4)-treated macrophages synthesised more lipids than NAC-treated cells in absence of exogenous lipid, and, generally, in the presence of native or acetylated, but oxidised LDL. In addition, the activities of the enzymes involved in cholesteryl ester storage were also influenced by the pro-oxidant condition. The ratio values between acyl-coenzyme A:cholesterol acyl transferase and cholesteryl ester hydrolase activity suggest that in CuSO(4)-treated macrophages the hydrolysis of cholesteryl ester is favoured with respect to esterification. The interaction of HMDM with oxidised LDL showed a significant different pattern in term of lipid synthesis with respect to those induced by native or acetylated LDL, disrespectful of the initial redox profile of the cells. On the whole, these results suggest that the pre-existing internal redox condition is a further parameter able to modulate the effects of native or acetylated LDL-cell interaction, influencing both HMDM lipid synthesis profile and cholesterol storage. Moreover, oxidised LDL represent a carrier of additional factor(s) able per se to introduce perturbation in the synthetic pathway of lipids, which is not influenced by the redox potential of the macrophage.