A synergy of activity,stability, and inhibitor‐interaction of HIV‐1 protease mutants evolved under drug‐pressure

A clinically‐relevant, drug‐resistant mutant of HIV‐1 protease (PR), termed Flap+_(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug‐PR interactions, compared to wild‐type PR. A similar mutant, Flap+_(I54A), which evolves from Flap+_(I54V) and contains the single change at residue 54 relative to Flap+_(I54V), does not. Yet, how Flap+_(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+_(I54V), Flap+_(I54A), and Flap+_(I54), a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Therapy of Venezuelan patients with severe mucocutaneous or early lesions of diffuse cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus Calmette-Guerin: preliminary report

Severe mucocutaneous (MCL) and diffuse (DCL) forms of American cutaneous leishmaniasis (ACL) are infrequent in Venezuela. Chemotherapy produces only transitory remission in DCL, and occasional treatment failures are observed in MCL. We have evaluated therapy with an experimental vaccine in patients with severe leishmaniasis. Four patients with MCL and 3 with early DCL were treated with monthly intradermal injections of a vaccine containing promastigotes of Leishmania (Viannia) braziliensis killed by pasteurization and viable Bacillus Calmette- Guerin. Clinical and immunological responses were evaluated. Integrity of protein constituents in extracts of pasteurized promastigotes was evaluated by gel electrophoresis. Complete remission of lesions occurred after 5-9 injections in patients with MCL or 7-10 injections in patients with early DCL. DCL patients developed positive skin reactions, average size 18.7 mm. All have been free of active lesions for at least 10 months. Adverse effects of the vaccine were limited to local reactivity to BCG at the injection sites and fever in 2 patients. Extracts of pasteurized and fresh promastigotes did not reveal differences in the integrity of protein components detectable by gel electrophoresis. Immunotherapy with this modified vaccine offers an effective, safe option for the treatment of patients who do not respond to immunotherapy with vaccine containing autoclaved parasites or to chemotherapy.     

Variants in the interleukin-1 alpha and beta genes,and the risk for periodontal disease in dogs

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A >C and IL1A/1_g.521T >A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), P= 0.022; 5.76 (1.03–32.1), P= 0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G >T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.     

Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion

Cancer invasion into an extracellular matrix (ECM) results from a biophysical reciprocal interplay between the expanding cancer lesion and tissue barriers imposed by the adjacent microenvironment. In vivo, connective tissue provides both densely packed ECM barriers adjacent to channel/track-like spaces and loosely organized zones, both of which may impact cancer invasion mode and efficiency; however little is known about how three-dimensional (3D) spaces and aligned tracks present in interstitial tissue guide cell invasion. We here describe a two-photon laser ablation procedure to generate 3D microtracks in dense 3D collagen matrices that support and guide collective cancer cell invasion. Whereas collective invasion of mammary tumor (MMT) breast cancer cells into randomly organized collagen networks required matrix metalloproteinase (MMP) activity for cell-derived collagen breakdown, re-alignment and track generation, preformed tracks supported MMP-independent collective invasion down to a track caliber of 3 μm. Besides contact guidance along the track of least resistance and initial cell deformation (squeezing), MMP-independent collective cell strands led to secondary track expansion by a pushing mechanism. Thus, two-photon laser ablation is useful to generate barrier-free microtracks in a 3D ECM which guide collective invasion independently of pericellular proteolysis.     

Testing the quick meal hypothesis: The effect of pulp on hoarding and seed predation of Hymenaea courbaril by red‐rumped agoutis (Dasyprocta leporina)

Abstract: Red‐rumped agoutis (Dasyprocta leporina) are important seed dispersers/predators of Neotropical large‐seeded plants. Several species of seeds cached by agoutis have an edible reward, in contrast to temperate rodent‐dispersed diaspores. The quick meal hypothesis states that the presence of a reward such as edible pulp will enhance the efficiency of rodents as seed disperses by satiating the animal and, consequently, reducing seed predation and enhancing hoarding. In this study, this hypothesis was tested using as the reference system the pulp and seeds of Hymenaea courbaril. Seeds with and without pulp were offered to agoutis and the behaviour of each individual was recorded. Since the probability of predation and hoarding were complementary, we used the probability of predation. The proportion of agoutis that preyed on at least one seed was similar for seeds with (42.8% of individuals) and without (40.0% of individuals) pulp. In agoutis that preyed upon at least one seed, the probability that they killed a seed did not differ between seeds with (0.17 ± 0.03) and without (0.20 ± 0.08) pulp. Hence, these results do not support the ‘quick meal hypothesis’.     

Extrafloral nectaries as a deterrent mechanism against seed predators in the chemically protected weed Crotalaria pallida (Leguminosae)

Abstract In several plants, extrafloral nectaries (EFN) are located close to the reproductive structures, suggesting that ants may act as a defence against specialized seed predators that overcome chemical defences. Alternatively, ants may also deter herbivores in a generalized manner, thereby protecting the whole plant. In this work, we examined the relationship between the chemically protected weed Crotalaria pallida Ait. (Leguminosae) that bears EFN, its specialized seed predator, the larvae of the arctiid moth Utetheisa ornatrix L. (Arctiidae) and ants. We tested two hypotheses related to the type of deterrence caused by ants. The Seed Predator Deterrence Hypothesis predicts that ant deterrence is directed primarily towards herbivores that destroy seeds and other reproductive structures, without attacking herbivores on vegetative structures. The General Deterrence Hypothesis states that ants are general in their effects, equally deterring herbivores in vegetative and reproductive structures. Our results supported the predictions of the Seed Predator Deterrence Hypothesis, namely, that (i) ant activity on EFN was related to the vulnerability of reproductive structures to attack by U. ornatrix; (ii) ant patrolling was restricted almost entirely to racemes; (iii) ants removed termites used as baits more frequently on racemes than on leaves; and (iv) U. ornatrix larvae were often expulsed from the racemes. These results indicate that EFN can act as another deterrent mechanism in chemically protected plants by promoting the expulsion of specialist seed predators.     

Prediction of indirect interactions in proteins

Background  

Both direct and indirect interactions determine molecular recognition of ligands by proteins. Indirect interactions can be defined as effects on recognition controlled from distant sites in the proteins, e.g. by changes in protein conformation and mobility, whereas direct interactions occur in close proximity of the protein's amino acids and the ligand. Molecular recognition is traditionally studied using three-dimensional methods, but with such techniques it is difficult to predict the effects caused by mutational changes of amino acids located far away from the ligand-binding site. We recently developed an approach, proteochemometrics, to the study of molecular recognition that models the chemical effects involved in the recognition of ligands by proteins using statistical sampling and mathematical modelling.