Identification of amino acid residues critical for the substrate translocation in lactate permease JEN1p of saccharomyces cerevisiae

Lactic, acetic and propionic acids have been used for many years in industrial and pharmaceutical companies. In Saccharomyces cerevisiae, Jen1p is a major monocarboxylate:H+ symporter specific primarily for lactate, pyruvate and for acetate (TC # 2.A.1.12.2) (Casal et al., 1999). A phylogenetic tree of ScJen1p homologues (Casal et al., 2008) showed the existence of two main clusters: a Jen1 group (monocarboxylate transporters) and a Jen2-like (dicarboxylate transporters). Structure-function relationships in Jen1p have been approached by using a rational mutational analysis of conserved amino acid residues (Soares-Silva et al., 2007). Analysis of the conserved sequence 379NXX[S/T]HX[S/T]QDXXXT391, located in transmembrane segment seven (TMS-VII), showed that residues N379, H383 or D387 are necessary for function and specificity, while Q386 is important for the kinetics of Jen1p-mediated transport. In this work, we rationally designed and analyzed novel mutations in conserved regions located in TMS-II, TMS-V and TMS-XI of Jen1p, which we predicted to affect Jen1p specificity (distinction between mono and dicarboxylates) and function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is critical for function. Using a model based on Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI are aligned along the protein pore and substrate docking studies reveal a potential substrate translocation trajectory consisting mostly of the polar residues genetically identified as important for function. Overall, our results constitute a first step towards the genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily and a tool for the in silico prediction of the function of Jen1p homologues in other fungi (Soares-Silva et al., 2011).I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

Modeling, substrate docking, and mutational analysis identify residues essential for the function and specificity of a eukaryotic purine-cytosine NCS1 transporter

Background: The purine-cytosine FcyB transporter is a prototype member of the NCS1 family. Results: Using homology modeling, substrate docking, and rational mutational analysis, we identify residues critical for function and specificity. Conclusion: Important aspects concerning the molecular mechanism and evolution of transporter specificity are revealed. Significance: The first systematic approach on structure-function-specificity relationships in a eukaryotic NCS1 member is shown

An autopsy study of a familial oculopharyngeal muscular dystrophy (OPMD) with distal spread and neurogenic involvement

An 81-year-old man from a family with a history of oculopharyngeal muscular dystrophy (OPMD) involving 6 members over 4 generations is described. The patient first noted drooping of his eyelids at the age of 65. Dysphagia and dysarthria occurred soon thereafter. At age 78, impairment of gait developed and progressive wasting occurred in the limbs with an initial distal distribution. Electromyography of several limb muscles displayed a mixed myopathic and neurogenic pattern with giant potentials. Examination at autopsy revealed slight loss of neurons in the anterior horns of the spinal cord, with scanty ghost cells, neuronophagia, and central chromatolysis. By light microscopy the limb muscles showed moderate small-group atrophy with severe myopathy and target fibers. The viscerocranial muscles, including the ocular, vocal, and tongue muscles, demonstrated only myopathic change with the typical features of progressive muscular dystrophy. Advanced replacement by fibrous connective tissue and fat had occurred in both the viscerocranial and the lower limb muscles. The significance of neurogenic involvement in OPMD is discussed

Improving pre-service teachers\u27 preparation to integrate music and movement in early childhood classrooms

This study examines student teachers\u27 fieldwork experiences, in order to better support their efforts to integrate music and movement in early childhood classrooms

Clinical biological and genetic heterogeneity of the inborn errors of pulmonary surfactant metabolism

Pulmonary surfactant is a multimolecular complex located at the air-water interface within the alveolus to which a range of physical (surface-active properties) and immune functions has been assigned. This complex consists of a surface-active lipid layer (consisting mainly of phospholipids), and of an aqueous subphase. From discrete surfactant sub-fractions one can isolate strongly hydrophobic surf acta nt proteins B (SP-B) and C (SP-C) as well as collectins SP-A and SP-D, which were shown to have specific structural, metabolic, or immune properties. Inborn or acquired abnormalities of the surfactant, qualitative or quantitative in nature, account for a number of human diseases. Beside hyaline membrane disease of the preterm neonate, a cluster of hereditary or acquired lung diseases has been characterized by periodic acid-Schiff-positive material filling the alveoli. From this heterogeneous nosologic group, at least two discrete entities presently emerge. The first is the SP-B deficiency, in which an essentially proteinaceous material is stored within the alveoli, and which represents an autosomal recessive Mendelian entity linked to the SFTPB gene (MIM 1786640). The disease usually generally entails neonatal respiratory distress with rapid fatal outcome, although partial or transient deficiencies have also been observed. The second is alveolar proteinosis, characterized by the storage of a mixed protein and lipid material, which constitutes a relatively heterogeneous clinical and biological syndrome, especially with regard to age at onset (from the neonate through to adulthood) as well as the severity of associated signs. Murine models, with a targeted mutation of the gene encoding granulocyte macrophage colony-stimulating factor (GM-CSF) (Csfgm) or the beta subunit of its receptor (II3rb1) support the hypothesis of an abnormality of surfactant turnover in which the alveolar macrophage is a key player. Apart from SP-B deficiency, in which a near-consensus diagnostic chart can be designed, the ascertainment of other abnormalities of surfactant metabolism is not straightforward. The disentanglement of this disease cluster is however essential to propose specific therapeutic procedures: repeated broncho-alveolar ravages, GM-CSF replacement, bone marrow grafting or lung transplantation

Discovery and optimization of a selective ligand for the switch/sucrose nonfermenting-related bromodomains of polybromo protein-1 by the use of virtual screening and hydration analysis

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein−ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5)

Maternal Pregnancy Levels of trans-Nonachlor and Oxychlordane and Prevalence of Cryptorchidism and Hypospadias in Boys

Background: The etiologies of the male urogenital anomalies—cryptorchidism and hypospadias—are poorly understood. Given positive associations between chlordane isomers and testicular germ cell tumors, it is reasonable to assume that chlordanes might also be associated with other testicular dysgenesis syndrome disorders, namely cryptorchidism and hypospadias