Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.     

Domesticity of Lutzomyia whitmani (Diptera: psychodidae) populations: field experiments indicate behavioural differences

The sandfly Lutzomyia whitmani (Antunes & Coutinho) is an important vector for cutaneous leishmaniasis throughout much of Brazil, and has recently been shown to consist of more than one mitochondrial lineage. It has frequently been asserted that the degree of adaptation of L. whitmani to human environments varies across its range. As a standardized test of indoor feeding for three geographically distant populations of L. whitmani, catches inside experimental chicken sheds of varying degrees of wall closure (0%, 33%, 67% and 98%) were compared. Each increment in shed closure reduced catches of females (relative to the most open shed) by a similar degree for each population: geometric mean catches dropped by 11-40% with 33% closure, by 41-62% with 67% closure, and by 69-100% with 98% closure. Geometric mean catches of males from the two more northerly populations also decreased with increasing shed closure, by 18% and 22% for 33% closure, 58% and 69% for 67% closure, 91% and 93% for 98% closure. Males from the most southerly population showed significantly different behaviour, with 33% closure causing a 54% increase in geometric mean catch, 67% closure causing a 6% increase, and 98% closure causing a 32% reduction. For this southerly population, sex ratios became more male biased with increasing density in more closed sheds, suggesting aggregation driven by intra-specific communication. Lutzomyia intermedia (Lutz & Neiva) was relatively more likely than L. whitmani to approach baits in the three more closed sheds, rather than the most open shed, offering a behavioural explanation for observed differences in indoor biting rates between the species.     

Inhibition of Human Dyskerin as a New Approach to Target Ribosome Biogenesis

The product of the DKC1 gene, dyskerin, is required for both ribosome biogenesis and telomerase complex stabilization. Targeting these cellular processes has been explored for the development of drugs to selectively or preferentially kill cancer cells. Presently, intense research is conducted involving the identification of new biological targets whose modulation may simultaneously interfere with multiple cellular functions that are known to be hyper-activated by neoplastic transformations. Here, we report, for the first time, the computational identification of small molecules able to inhibit dyskerin catalytic activity. Different in silico techniques were applied to select compounds and analyze the binding modes and the interaction patterns of ligands in the human dyskerin catalytic site. We also describe a newly developed and optimized fast real-time PCR assay that was used to detect dyskerin pseudouridylation activity in vitro. The identification of new dyskerin inhibitors constitutes the first proof of principle that the pseudouridylation activity can be modulated by means of small molecule agents. Therefore, the presented results, obtained through the usage of computational tools and experimental validation, indicate an alternative therapeutic strategy to target ribosome biogenesis pathway.     

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders

Cannabidiol (CBD) is a major phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ⁹-tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1A-mediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamide-mediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed with CBD. Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARγ receptors agonism, intracellular (Ca²⁺) increase, etc.), on CBD behavioural effects.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Reserve selection and persistence: complementing the existing Atlantic Forest reserve system

This study is an exercise to check the efficiency of the existing reserve system, and to show how systematic conservation planning—using information available and the complementarity concept—can improve the basis for decisions and minimize costs. We verified the performance, in number of cells and primate species representation, of the existing Atlantic Forest (Brazil) reserve network with a quarter-degree resolution grid, with 1,884 cells. We used occurrence data of 20 endemic primate species, and the maps of 237 existing reserves. Reserve networks were selected to represent primate species first considering no pre-existing reserves in Atlantic Forest, and then, considering the existing reserve system, taking into account the minimum area for viable population of the larger species (Northern muriqui Brachyteles hypoxanthus). Reserve selection was carried out using the complementarity concept implemented by a simulated annealing algorithm. Primate species representation (at least one occurrence in the network) could be achieved with 8% of the existing reserve system (nine cells in relation to the 120 in the existing reserve system). We found that today’s reserve system represents 89% of endemic primate species, excluding the species Coimbra Filho’s titi monkey (Callicebus coimbrai) and Marcgraf’s capuchin (Cebus flavius). The networks selected without considering existing reserves contained nine cells. The networks selected considering existing reserves (120 cells), had two new cells necessary to represent all the primates. This does not mean that a viable alternative is to start from zero (i.e., nonexistent reserves). Identifying critical supplementary areas using biodiversity information to fill the gaps and then starting “conservation in practice” in these areas should be priorities.     

Adenosine Inhibition of 45Ca Uptake by Synaptosomes as Function of Time of Electrical Stimulation

Abstract

The effect of adenosine on ⁴⁵Ca uptake by rat brain synaptosomes electrically stimulated was studied as function of time of stimulation (10, 30, 120 s). Inhibition of ⁴⁵Ca uptake was more evident for 120 s.     

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal differences in the kinetics of anti-NBL and ML Ab responses. While anti-NBL Abs declined slowly from day 19 until the end of the experiment, Abs to ML antigen remained high in the same period. It is remarkable the optimal Ab response to NBL antigens with 2000 ML infective dose and the reduced number of NBL antigens identified throughout the experimental T. spiralis infection, standing out the immunodominant 49 kDa antigen. Interestingly, this antigen, which was prominently expressed in NBL somatic proteins, was also detected in NBL-ESP.