Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.     

Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product

The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosome-targeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of trans-translation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.     

Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.     

Human macrophage migration inhibitory factor: a proven immunomodulatory cytokine?

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator with the ability to induce various immunomodulatory responses and override glucocorticoid-driven immunosuppression. Some of these functions have been linked to the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities. However, there are conflicting reports regarding the functional role of these enzymatic properties in normal physiological homeostasis and disease progression. Therefore, we have produced a highly pure, virtually endotoxin-free recombinant MIF preparation and fully characterized this using a variety of biochemical and biophysical approaches. The recombinant protein, with demonstrable enzymatic activity, was then used to systematically examine the biological activity of MIF. Surprisingly, treatment with MIF alone failed to induce cytokine expression, with the exception of IL-8. However, co-treatment of lipopolysaccharide (LPS) in conjunction with MIF produced synergistic secretion of tumor necrosis factor-alpha, interleukin (IL)-1, and IL-8 compared with LPS alone. The potentiating effect of MIF was seen at physiologically relevant concentrations. These data suggest that MIF has no conventional cytokine activity but, rather, acts to modulate and amplify the response to LPS.     

Cooperative DNA binding by the B-isoform of human progesterone receptor: thermodynamic analysis reveals strongly favorable and unfavorable contributions to assembly

Progesterone receptors (PR) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at multisite promoters are poorly understood. Here we present a thermodynamic analysis of the interactions of the PR B-isoform (PR-B) with promoters containing either one or two progesterone response elements (PREs). Utilizing quantitative footprinting, we have resolved the microscopic energetics of PR-B binding, including cooperativity terms. The results of this analysis challenge a number of assumptions found in traditional models of receptor function. First, PR-B interactions at a single PRE can be equally well described by mechanisms invoking either the receptor monomer or the dimer as the active DNA binding species. If, as is commonly accepted, PR-B interacts with response elements only as a preformed dimer, then its intrinsic binding affinity is not the typically observed nanomolar but is rather picomolar. This high affinity binding is opposed, however, by a large energetic penalty. The penalty presumably pays for costly structural rearrangements of the receptor dimer and/or response element that are needed to form the protein-DNA complex. If PR-B assembles at a single response element via successive monomer binding reactions, then this penalty minimizes cooperative interactions between adjacent monomers. When binding to two response elements, the receptor exhibits strong intersite cooperativity. Although this phenomenon has been observed before, the present work demonstrates that the energetics reach levels seen in highly cooperative systems such as lambda cI repressor. This first quantitative dissection of cooperative receptor-promoter interactions suggests that PR-B function is more complex than traditionally envisioned.     

Adenovirus type 5 intrinsic adsorption rates measured by surface plasmon resonance

Intrinsic adsorption rates of whole adenovirus type 5 (Ad5) onto a diethylaminoethyl (DEAE) anion exchange surface are measured for the first time by surface plasmon resonance (SPR). Fitting SPR sensorgrams to a two-compartment mass transport reaction model distinguishes intrinsic adsorption rates from slow diffusive Ad5 mass transport. Ad5 is a widely used viral vector for gene therapy that binds electrostatically to surfaces of cells and synthetics such as membranes, chromatographic resins, and glass. Increasing NaCl concentration from 4.8 to 14.4mM shifts binding of whole Ad5 from diffusion control to a regime where both sorption and diffusion affect binding. Intrinsic adsorption rates for Ad5-DEAE interaction are 16 times faster than intrinsic adsorption rates for Ad5 fiber knob interacting with soluble extracellular domain of coxsackievirus adenovirus receptors (s-CAR).     

A "holistic" kinesin phylogeny reveals new kinesin families and predicts protein functions

Kinesin superfamily proteins are ubiquitous to all eukaryotes and essential for several key cellular processes. With the establishment of genome sequence data for a substantial number of eukaryotes, it is now possible for the first time to analyze the complete kinesin repertoires of a diversity of organisms from most eukaryotic kingdoms. Such a "holistic" approach using 486 kinesin-like sequences from 19 eukaryotes and analyzed by Bayesian techniques, identifies three new kinesin families, two new phylum-specific groups, and unites two previously identified families. The paralogue distribution suggests that the eukaryotic cenancestor possessed nearly all kinesin families. However, multiple losses in individual lineages mean that no family is ubiquitous to all organisms and that the present day distribution reflects common biology more than it does common ancestry. In particular, the distribution of four families--Kinesin-2, -9, and the proposed new families Kinesin-16 and -17--correlates with the possession of cilia/flagella, and this can be used to predict a flagellar function for two new kinesin families. Finally, we present a set of hidden Markov models that can reliably place most new kinesin sequences into families, even when from an organism at a great evolutionary distance from those in the analysis.     

Evolution of Afrotropical freshwater crab lineages obscured by morphological convergence

We use sequence data derived from six DNA gene loci to examine evolutionary and biogeographic affinities among all freshwater crab families. With an emphasis on the Afrotropical fauna that includes Africa, Madagascar, and the Seychelles, we test the proposed Gondwanan cladogenesis of the group. Phylogenetic results demonstrate that contemporary distribution patterns of freshwater crab lineages are incongruent with the expected area cladogram of continental fragmentation. Instead, our phylogenetic estimate and divergence time estimation indicate a post-Gondwanan, early Cretaceous cladogenesis for freshwater crabs implying that the acquisition of a freshwater lifestyle was achieved more recently. A dispersal hypothesis as opposed to vicariance appears to best explain the contemporary distribution pattern of this group. However, our results do not explicitly disprove a Gondwanan origin for the Afrotropical freshwater crabs. Alarmingly, these results suggest that most of the currently recognized freshwater crab families are unreliable taxonomic groupings since virtually no Afrotropical freshwater crab families formed monophyletic units thus obscuring inferred biogeographic relationships. Convergence in characters associated with the terminal segment of the mandibular palp is clearly a pervasive obstacle in the taxonomy of this group.     

The pattern and timing of diversification of Philippine endemic rodents: evidence from mitochondrial and nuclear gene sequences

The 22 genera and 64 species of rodents (Muridae: Murinae) distributed in the Philippine Islands provide a unique opportunity to study patterns and processes of diversification in island systems. Over 90% of these rodent species are endemic to the archipelago, but the relative importance of dispersal from the mainland, dispersal within the archipelago, and in situ differentiation as explanations of this diversity remains unclear, as no phylogenetic hypothesis for these species and relevant mainland forms is currently available. Here we report the results of phylogenetic analyses of the endemic Philippine murines and a wide sampling of murine diversity from outside the archipelago, based on the mitochondrial cytochrome b gene and the nuclear-encoded IRBP exon 1. Analysis of our combined gene data set consistently identified five clades comprising endemic Philippine genera, suggesting multiple invasions of the archipelago. Molecular dating analyses using parametric and semiparametric methods suggest that colonization occurred in at least two stages, one ca. 15 Mya, and another 8 to 12 million years later, consistent with the previous recognition of "Old" and "New" endemic rodent faunas. Ancestral area analysis suggests that the Old Endemics invaded landmasses that are now part of the island of Luzon, whereas the three New Endemic clades may have colonized through either Mindanao, Luzon, or both. Further, our results suggest that most of the diversification of Philippine murines took place within the archipelago. Despite heterogeneity between nuclear and mitochondrial genes in most model parameters, combined analysis of the two data sets using both parsimony and likelihood increased phylogenetic resolution; however, the effect of data combination on support for resolved nodes was method dependent. In contrast, our results suggest that combination of mitochondrial and nuclear data to estimate relatively ancient divergence times can severely compromise those estimates, even when specific methods that account for rate heterogeneity among genes are employed. [Biogeography; divergence date estimation; mitochondrial DNA; molecular systematics; Murinae; nuclear exon; Philippines; phylogeny.].     

Apical-basal polarity, Wnt signaling and vertebrate organogenesis

Wnt proteins elicit several distinct signal transduction cascades and regulate multiple cellular processes that have proven essential for embryonic development in all metazoans investigated. During embryonic development, epithelial cells become polarized along two axes: apical/basal and within the plane of the tissue. Growing evidence suggests that polarization along each axis is essential for normal embryonic development and that this polarization is regulated in part by the different branches of the Wnt pathway. Here, we review the role of A/B cell polarity in vertebrate organogenesis with a focus on the involvement of canonical Wnt signaling in this process.