PDZ/PDZ interaction between PSD‐95 and nNOS neuronal proteins

N‐Methyl‐D‐aspartate (NMDA) receptors are key components in synaptic communication and are highly relevant in central nervous disorders, where they trigger excessive calcium entry into the neuronal cells causing harmful overproduction of nitric oxide by the neuronal nitric oxide synthase (nNOS) protein. Remarkably, NMDA receptor activation is aided by a second protein, postsynaptic density of 95 kDa (PSD95), forming the ternary protein complex NMDA/PSD95/nNOS. To minimize the potential side effects derived from blocking this ternary complex or either of its protein components, a promising approach points to the disruption of the PSD‐95/nNOS interaction which is mediated by a PDZ/PDZ domain complex. Since the rational development of molecules targeting such protein‐protein interaction relies on energetic and structural information herein, we include a thermodynamic and structural analysis of the PSD95‐PDZ2/nNOS‐PDZ. Two energetically relevant events are structurally linked to a “two‐faced” or two areas of recognition between both domains. First, the assembly of a four‐stranded antiparallel β‐sheet between the β hairpins of nNOS and of PSD95‐PDZ2, mainly enthalpic in nature, contributes 80% to the affinity. Second, binding is entropically reinforced by the hydrophobic interaction between side chains of the same nNOS β‐hairpin with the side chains of α2‐helix at the binding site of PSD95‐PDZ2, contributing the remaining 20% of the total affinity. These results suggest strategies for the future rational design of molecules able to disrupt this complex and constitute the first exhaustive thermodynamic analysis of a PDZ/PDZ interaction.     

Exploring the carbohydrate‐binding ability of Canavalia bonariensis lectin in inflammation models

Lectins are a group of proteins of non‐immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA‐like mannose/glucose‐specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α‐methyl‐D‐mannoside (MMA) and mannose‐1,3‐α‐D‐mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose‐type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high‐mannose N‐glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

Recognition motifs rather than phylogenetic origin influence the ability of targeting peptides to import nuclear-encoded recombinant proteins into rice mitochondria

Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.

     

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Role of B lymphocytes in the infarcted mass in patients with acute myocardial infarction

Despite early reperfusion, patients with ST segment elevation myocardial infarction (STEMI) may present large myocardial necrosis and significant impairment of ventricular function. The present study aimed to evaluate the role of subtypes of B lymphocytes and related cytokines in the infarcted mass and left ventricular ejection fraction obtained by cardiac magnetic resonance imaging performed after 30 days of STEMI. This prospective study included 120 subjects with STEMI submitted to pharmacoinvasive strategy. Blood samples were collected in subjects in the first (D1) and 30th (D30) days post STEMI. The amount of CD11b+ B1 lymphocytes (cells/ml) at D1 were related to the infarcted mass (rho = 0.43; P=0.033), measured by cardiac MRI at D30. These B1 cells were associated with CD4+ T lymphocytes at D1 and D30, while B2 classic lymphocytes at day 30 were related to left ventricular ejection fraction (LVEF). Higher titers of circulating IL-4 and IL-10 were observed at D30 versus D1 (P=0.013 and P<0.001, respectively). Titers of IL-6 at D1 were associated with infarcted mass (rho = 0.41, P<0.001) and inversely related to LVEF (rho = −0.38, P<0.001). After multiple linear regression analysis, high-sensitivity troponin T and IL-6 collected at day 1 were independent predictors of infarcted mass and, at day 30, only HDL-C. Regarding LVEF, high-sensitivity troponin T and high-sensitivity C-reactive protein were independent predictors at day 1, and B2 classic lymphocytes, at day 30. In subjects with STEMI, despite early reperfusion, the amount of infarcted mass and ventricular performance were related to inflammatory responses triggered by circulating B lymphocytes.     

S-adenosylmethionine induces mitochondrial dysfunction,permeability transition pore opening and redox imbalance in subcellular preparations of rat liver

Journal of Bioenergetics and Biomembranes - S-adenosylmethionine (AdoMet) predominantly accumulates in tissues and biological fluids of patients affected by liver dysmethylating diseases,...     

Validation of a Dehydroepiandrosterone-Sulfate Assay in Three Platyrrhine Primates (Alouatta caraya,Aotus azarae infulatus,and Sapajus apella)

International Journal of Primatology - The hormone dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) are the most abundant circulating steroids in human and some nonhuman primates, and...     

Characterization of substrate specificity and inhibitory mechanism of bile salt hydrolase from Lactobacillus reuteri CRL 1098 using molecular docking analysis

Objectives

To elucidate the molecular mechanisms involved in the substrate interaction of the bile salt hydrolase of Lactobacillus reuteri CRL 1098 (LrBSH) with bile acids (BAs) and to evaluate potential enzyme inhibitors based on computer and in vitro modeling assays.

Results

Asp19, Asn79, and Asn171 participated in the LrBSH interaction with all BAs tested while Leu56 and Glu 222 played an important role in the interaction with glyco- and tauro-conjugated BAs, respectively. A great percentage of hydrophobic and polar interactions were responsible for the binding of LrBSH with glyco- and tauro-conjugated BAs, respectively. Remarkably, the four binding pocket loops participated in the substrate binding site of LrBSH unlike most of the reported BSHs. Inhibition assays showed that ascorbic acid, citric acid, penicillin G, and ciprofloxacin decreased LrBSH activity by 47.1%, 40.14%, 28.8%, and 9%, respectively. Docking analysis revealed that tetracycline and caffeic acid phenethyl ester had the low binding energy (?7.32 and ?7.19 kcal/mol, respectively) and resembled the interaction pattern of GDCA (?6.88 kcal/mol) while penicillin (?6.25 kcal/mol) and ascorbic acid (?5.98 kcal/mol) interacted at a longer distance.

Conclusion

This study helps to delve into the molecular mechanisms involved in the recognition of substrates and potential inhibitors of LrBSH.

     

Length-weight relationships of ornamental fish species from Amanã Lake,Amanã Reserve,Amazonas, Brazil

Length-weight relationships (LWRs) were estimated for 16 ornamental fish species from Amanã Lake and nine tributary streams, in Central Brazilian Amazonia. Sampling was carried out bimonthly for a year (2007–2008), using two sets of four gillnets (20 m × 2 m, 2,5; 3,5; 04; 4,5 cm stretch mesh size); trawls were performed with seine nets (35 m × 6 m, 3 mm stretch mesh size); native fish-traps (fyke-net like fish-traps woven from local lianas), and dip nets (0.5 m × 0.5 m stretch mesh size). Measurements were done for standard length (SL – 0.1 cm precision) and total weight (Wt – 0.01 g precision). This study provides information on the length–weight relationships for all sampled species and, in addition, provides new maximum standard lengths for six species.