Inhibitors of the Candida albicans Major Facilitator Superfamily Transporter Mdr1p Responsible for Fluconazole Resistance

ObjectiveTo identify a novel class of inhibitors of fungal transporters involved in drug resistance.MethodsA series of structurally-related low molecular mass compounds was synthesized using combinatorial chemistry of a cyclobutene-dione (squarile) core. These compounds were screened for their inhibition of plasma membrane Major Facilitator Superfamily (MFS) and ATP-binding cassette (ABC) transporters responsible for efflux pump-mediated drug resistance in the fungal pathogen Candida albicans. Strains of Saccharomyces cerevisiae that specifically overexpress the MFS pump CaMdr1p or the ABC transporter CaCdr1p were used in primary screens and counterscreens, respectively, and to detect inhibition of glucose-dependent Nile Red efflux. Efflux pump inhibition, activity as pump substrates and antifungal activity against yeast and clinical isolates expressing efflux pumps were determined using agarose diffusion susceptibility assays and checkerboard liquid chemosensitization assays with fluconazole.ResultsThe screen identified five structurally-related compounds which inhibited CaMdr1p. Two compounds, A and B, specifically chemosensitized AD/CaMDR1 to FLC in a pH-dependent fashion and acted synergistically with FLC in checkerboard liquid MIC assays but compound B had limited solubility. Compound A chemosensitized to FLC the azole-resistant C. albicans strain FR2, which over-expresses CaMdr1p, inhibited Nile Red efflux mediated by CaMdr1p but not CaCdr1p and was not toxic to cultured human cells. A minor growth-inhibitory effect of B on AD/CaMDR1, but not on AD/CaCDR1 and AD/CaCDR2, indicated that compound B may be a substrate of these transporters. The related compound F was found to have antifungal activity against the three pump over-expressing strains used in the study.ConclusionsCompound A is a ‘first in class’ small molecule inhibitor of MFS efflux pump CaMdr1p.     

The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.The S100 proteins comprise a multigene family of low-molecular-weight EF-hand calcium binding and zinc binding proteins (5, 13, 16, 24, 33). To date, 19 different S100 proteins have been assigned to this protein family, and they show different degrees of similarity, ranging from 25 to 56% identity at the amino acid level. With S100B, S100P, and S100Z being the exceptions, the majority of the S100 genes are clustered on human chromosome 1q21 (33). Most S100 proteins serve as calcium sensor proteins that, upon activation, regulate the function and/or subcellular distribution of specific target proteins (13, 33, 47), and they are characterized by common structural motifs, including two low-affinity (K_D [equilibrium dissociation constant] of ∼10 μM to 100 μM) helix-loop-helix calcium binding domains (EF hands) that are separated by a hinge region and flanked by amino- and carboxy-terminal domains. The carboxy-terminal domain is variable among S100 proteins, and it typically is the site that is responsible for the selective interaction of each individual S100 protein with specific target proteins (30). S100 proteins are often upregulated in cancers, in inflammation, and in response to cellular stress (14, 16), suggesting that they function in cell responses to stress situations. Consistent with this hypothesis, stress situations were necessary to reveal phenotypes associated with the S100 knockout in mice (11, 14, 33, 56). Moreover, recent observations revealed a new function for the S100 protein family that included their ability to assist and regulate multichaperone complex-ligand interactions (41, 50, 51).One member of the S100 protein family, S100B, has attracted much interest in the past few years because, like other proteins implicated in neurodegeneration (e.g., amyloid, superoxide dismutase, and dual-specificity tyrosine phosphorylation-regulated kinase 1A), its gene is located within a segment of chromosome 21, which is trisomic in Down''s syndrome (DS). Its expression in the brain of mammals coincides with defined periods of central nervous system (CNS) maturation and cell differentiation (43). In oligodendrocyte progenitor cells (OPC), S100B expression is associated with differentiation, and S100B contributes to OPC differentiation in response to demyelinating insult (11). To understand the contribution of S100B to OPC differentiation, we searched for high-affinity S100B target proteins in this cell type by using far-Western analysis. A major and highly specific S100B target protein was identified, the mitochondrial ATAD3A protein.ATAD3A belongs to a new family of eukaryote-specific mitochondrial AAA⁺ ATPase proteins (17). In the human genome, two genes, Atad3A and Atad3B, are located in tandem on chromosome 1p36.33. The Atad3A gene is ubiquitous among multicellular organisms but absent in yeast. The Atad3B gene is specific to the human genome (27). ATAD3A is a mitochondrial protein anchored into the mitochondrial inner membrane (IM) at contact sites with the outer membrane (OM). Thanks to its simultaneous interaction with the two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the inner and outer membranes and controls diverse cell responses ranging from mitochondrial metabolism, cell growth, and mitochondrial fission 20a, 25). The ATAD3A protein has also been identified as a mitochondrial DNA binding protein (23) and as a cell surface antigen in some human tumors (20, 21). The plasma membrane localization of ATAD3A in tumor cells is suggestive that ATAD3A mitochondrial routing can be compromised in pathological situations such as cancer. To understand the functional response resulting from the interaction between S100B and ATAD3A, we first characterized the minimal interaction domain on ATAD3A for S100B binding using thermodynamic studies of wild-type and ATAD3A variants as well as via nuclear magnetic resonance (NMR) spectroscopy techniques. These studies allowed us to further refine the consensus S100B binding motif, which is conserved in several other S100B target proteins, including the p53 protein and several newly discovered target proteins associated with the cell translational machinery. We next analyzed the cellular interaction of S100B with truncated ATAD3A mutants that harbor the S100B binding domain but that are deficient for mitochondrial import. These studies revealed that S100B could assist ATAD3A mutant proteins during cytoplasmic processing by preventing dysfunctional aggregation events. Our results are discussed in light of the possible function of S100B in assisting the cytoplasmic processing of proteins for proper folding and subcellular localization.     

Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panama: multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes

Colletotrichum interacts with numerous plant species overtly as symptomatic pathogens and cryptically as asymptomatic endophytes. It is not known whether these contrasting ecological modes are optional strategies expressed by individual Colletotrichum species or whether a species' ecology is explicitly pathogenic or endophytic. We explored this question by inferring relationships among 77 C. gloeosporioides s.l. strains isolated from asymptomatic leaves and from anthracnose lesions on leaves and fruits of Theobroma cacao (cacao) and other plants from Panamá. ITS and 5'-tef1 were used to assess diversity and to delineate operational taxonomic units for multilocus phylogenetic analysis. The ITS and 5'-tef1 screens concordantly resolved four strongly supported lineages, clades A-D: Clade A includes the ex type of C. gloeosporioides, clade B includes the ex type ITS sequence of C. boninense, and clades C and D are unidentified. The ITS yielded limited resolution and support within all clades, in particular the C. gloeosporioides clade (A), the focal lineage dealt with in this study. In contrast the 5'-tef1 screen differentiated nine distinctive haplotype subgroups within the C. gloeosporioides clade that were concordant with phylogenetic terminals resolved in a five-locus nuclear phylogeny. Among these were two phylogenetic species associated with symptomatic infections specific to either cacao or mango and five phylogenetic species isolated principally as asymptomatic infections from cacao and other plant hosts. We formally describe two new species, C. tropicale and C. ignotum, that are frequent asymptomatic associates of cacao and other Neotropical plant species, and epitypify C. theobromicola, which is associated with foliar and fruit anthracnose lesions of cacao. Asymptomatic Colletotrichum strains isolated from cacao plants grown in China included six distinct C. gloeosporioides clade taxa, only one of which is known to occur in the Neotropics.     

Fire as a selective force in a Bornean tropical everwet forest

Tree species rarely exposed to burning, like in everwet tropical forests, are unlikely to be fire adapted. Therefore, one could hypothesize that these species are affected equally by burning and that tree abundance changes are linked solely to fire behavior. Alternatively, if species do react differentially to burning, abundance changes should be linked to tree habitat preference and morphology. Using tree inventories from old-growth and adjacent burned Bornean forest in combination with a database on tree morphology and habitat preference, we test these alternative hypotheses by (1) determining whether species specific abundance changes after fire differ significantly from equal change, and (2) whether observed abundance changes are linked to species morphology and habitat preference. We found that of 196 species tested, 125 species showed an abundance change significantly different from that expected under our null model of equal change. These abundance changes were significantly linked to both tree morphology and habitat preference. Abundance declines were associated with slope or ridge preference, thin barks, and limited seed dormancy. Abundance increases were associated with high light preference, small adult stature, light wood, large leaves, small seeds and long seed dormancy. While species habitat preference and morphology explained observed abundance increases well, abundance declines were only weakly associated with them (R ² ~ 0.09). This suggests that most tree mortality was random and everwet tropical tree species are poorly fire adapted. As fire frequencies are increasing in the everwet tropics, this might eventually result in permanently altered species compositions and even species extinctions.     

A Na+ channel mutation linked to hypokalemic periodic paralysis exposes a proton-selective gating pore

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca(2+) channel) or NaV1.4 (the skeletal muscle voltage-gated Na(+) channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice ("gating-pore") in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K(+) channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K(+) and Cl(-) conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of V(REST) indirectly, for instance, by interfering with intracellular pH homeostasis.     

Identification of a novel risk locus for progressive supranuclear palsy by a pooled genomewide scan of 500,288 single-nucleotide polymorphisms

To date, only the H1 MAPT haplotype has been consistently associated with risk of developing the neurodegenerative disease progressive supranuclear palsy (PSP). We hypothesized that additional genetic loci may be involved in conferring risk of PSP that could be identified through a pooling-based genomewide association study of >500,000 SNPs. Candidate SNPs with large differences in allelic frequency were identified by ranking all SNPs by their probe-intensity difference between cohorts. The MAPT H1 haplotype was strongly detected by this methodology, as was a second major locus on chromosome 11p12-p11 that showed evidence of association at allelic (P<.001), genotypic (P<.001), and haplotypic (P<.001) levels and was narrowed to a single haplotype block containing the DNA damage-binding protein 2 (DDB2) and lysosomal acid phosphatase 2 (ACP2) genes. Since DNA damage and lysosomal dysfunction have been implicated in aging and neurodegenerative processes, both genes are viable candidates for conferring risk of disease.     

Adrenergic receptor density in brown adipose tissue of active and hibernating hamsters and ground squirrels

The ligand-binding characteristics (B(max) and K(D)) of alpha(1)- and beta(1)/beta(2)-adrenoceptors were investigated in membranes prepared from brown adipose tissue (BAT) of warm-acclimated, cold-acclimated, hibernating and arousing ground squirrels (Spermophillus undulatus) and hamsters (Mesocricetus auratus) by specific binding of [(3)H]prazosin and [(3)H]CGP-12177, respectively. The physiological state did not change the affinity for the adrenoceptors in the BAT of ground squirrels and hamsters. There was a significant decrease in alpha(1)-receptor density in arousing ground squirrels and a significant decrease in beta(1)/beta(2) density in hibernating ground squirrels. The level of alpha(1)-receptors was in all conditions higher than that of beta(1)/beta(2) receptors. The results indicate a possible change in balance of adrenoceptor density in the processes of cold acclimation, hibernation and arousal. The balance between the various adrenoceptor subtypes may be important for the final effect of catecholamines in BAT in different physiological states.     

Current trends in computational inference from mass spectrometry-based proteomics

Mass spectrometry offers a high-throughput approach to quantifying the proteome associated with a biological sample and hence has become the primary approach of proteomic analyses. Computation is tightly coupled to this advanced technological platform as a required component of not only peptide and protein identification, but quantification and functional inference, such as protein modifications and interactions. Proteomics faces several key computational challenges such as identification of proteins and peptides from tandem mass spectra as well as their quantitation. In addition, the application of proteomics to systems biology requires understanding the functional proteome, including how the dynamics of the cell change in response to protein modifications and complex interactions between biomolecules. This review presents an overview of recently developed methods and their impact on these core computational challenges currently facing proteomics.     

"Pseudosarcoma" in a pregnant woman

Background

As a result of improvements in diagnostic accuracy, the primary source of the tumour is identified in more than 99% of cases presenting with a malignancy. Whilst the axial skeleton is a common site of metastases, the sternum is rarely affected, especially by isolated metastases.

Case presentation

We report a case of a 68 year old male who was referred to the surgical outpatient clinic with a six month history of sternal pain. The patient was known to have essential thrombocythaemia, which had recently transformed into acute myeloid leukaemia but a sternal biospy showed mucinous adenocarcinoma. He had not localising symptoms and full evaluation failed to localise the primary tumour.

Conclusion

Solitary sternal metastases are rare and when found an underlying neoplasm is usually identified allowing targeted treatment. If however, there is no symptomatic tumour, the metastasis should simply be treated symptomatically.