We are coming to appreciate that at fertilization human spermatozoa deliver the paternal genome alongside a suite of structures, proteins and RNAs. Although the role of some of the structures and ...proteins as requisite elements for early human development has been established, the function of the sperm-delivered RNAs remains a point for discussion. The presence of RNAs in transcriptionally quiescent spermatozoa can only be derived from transcription that precedes late spermiogenesis. A cross-platform microarray strategy was used to assess the profile of human spermatozoal transcripts from fertile males who had fathered at least one child compared to teratozoospermic individuals. Unsupervised clustering of the data followed by pathway and ontological analysis revealed the transcriptional perturbation common to the affected individuals. Transcripts encoding components of various cellular remodeling pathways, such as the ubiquitin–proteosome pathway, were severely disrupted. The origin of the perturbation could be traced as far back as the pachytene stage of spermatogenesis. It is anticipated that this diagnostic strategy will prove valuable for understanding male factor infertility.
Down syndrome cell adhesion molecule (
) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod
is massive alternative splicing generating thousands of ...different isoforms from three variable clusters of alternative exons.
expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of
is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention.
C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are ...important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with Kd values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd = 110 nM) and fumarate (Kd = 400 nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.
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•Only secondary transporters are known to move C4-dicarboxylates across biological membranes.•We identify MatBAC, a novel C4-dicarboxylate transporter with a periplasmic binding protein, MatC.•MatBAC is the first known tripartite tricarboxylate transporter for C4-dicarboxylates.•MatC binds to d- and l-malate with very high affinity and to succinate and fumarate.•A 2.1-Å crystal structure of MatC bound to malate shows participation of two conserved water molecules.
The tripartite tricarboxylate transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate‐binding protein (SBP) ...for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP–ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with KD values in the μm range. The highest affinity was found for the C6‐dicarboxylate adipate (1,6‐hexanedioate). Crystal structures of AdpC, either adipate or 2‐oxoadipate bound, revealed a lack of positively charged amino acids in the binding pocket and showed that water molecules are involved in bridging hydrogen bonds to the substrate, a conserved feature in the TTT SBP family that is distinct from other types of SBP. In AdpC, both of the ligand carboxylate groups and a linear chain conformation are needed for coordination in the binding pocket. RT‐PCR showed that adpC expression is upregulated by low environmental adipate concentrations, suggesting adipate is a physiologically relevant substrate but as adpC is not genetically linked to any TTT membrane transport genes, the role of AdpC may be in signalling rather than transport. Our data expand the known ligands for TTT systems and identify a novel high‐affinity binding protein for adipate, an important industrial chemical intermediate and food additive.
Databases
Protein structure co‐ordinates are available in the PDB under the accession numbers 5OEI and 5OKU.
Tripartite tricarboxylate transporters (TTT) are a family of poorly characterised bacterial solute‐binding protein (SBP)‐dependent high‐affinity uptake systems that may be a new source of transporters for biotechnological processes. We have identified a TTT SBP (AdpC) from Rhodopseudomonas palustris that binds the industrial C6‐dicarboxylate adipate. Here, we present the crystal structure of AdpC and elucidate the mode of adipate binding.
Toxicogenomic analysis of five environmental chemicals was performed to investigate the ability of genomics to predict toxicity, categorize chemicals, and elucidate mechanisms of toxicity. Three ...triazole antifungals (myclobutanil, propiconazole, and triadimefon) and two perfluorinated chemicals perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) were administered daily via oral gavage for one, three, or five consecutive days to male Sprague-Dawley rats at single doses of 300, 300, 175, 20, or 10 mg/kg/day, respectively. Clinical chemistry, hematology, and histopathology were measured at all time points. Gene expression profiling of livers from three rats per treatment group at all time points was performed on the CodeLink Uniset Rat I Expression array. Data were analyzed in the context of a large reference toxicogenomic database containing gene expression profiles for over 630 chemicals. Genomic signatures predicting hepatomegaly and hepatic injury preceded those results for all five chemicals, and further analysis segregated chemicals into two distinct classes. The triazoles caused similar gene expression changes as other azole antifungals, particularly the induction of pregnane X receptor (PXR)-regulated xenobiotic metabolism and oxidative stress genes. In contrast, PFOA and PFOS exhibited peroxisome proliferator–activated receptor α agonist-like effects on genes associated with fatty acid homeostasis. PFOA and PFOS also resulted in downregulation of cholesterol biosynthesis genes, matching an in vivo decrease in serum cholesterol, and perturbation of thyroid hormone metabolism genes matched by serum thyroid hormone depletion in vivo. The concordance of in vivo observations and gene expression findings demonstrated the ability of genomics to accurately categorize chemicals, identify toxic mechanisms of action, and predict subsequent pathological responses.
To establish the stability of spermatozoal RNAs as a means to validate their use as a male fertility marker.
Semen samples were randomly selected for 1 of 3 cryopreservation treatments.
An academic ...research environment.
Men aged 19 to 55 years who had fathered a child by natural conception within the past 6 months.
Ejaculates were collected by masturbation and total spermatozoan RNA was isolated from two semen samples of ideal quality; one sample of medium quality, having been subjected to an additional freeze-thaw cycle, and two samples of poor quality, having been subjected to a third freeze-thaw cycle.
Labeled cDNAs were generated and then used to interrogate Atlas Nylon Human Toxicology 1.2 microarrays. The spermatozoan transcriptomes were compared using a binomial approach.
The analysis identified a total of 228 unique spermatozoal transcripts among all samples. The medium quality sample shared 98% and 39% of its RNAs with the ideal and poor quality samples, respectively. A set of 36 RNAs resistant to insult were observed, some of which have been implicated in regulating male fertility, when all individuals were compared.
These results support the view that a population of spermatozoal RNAs is rapidly degraded in response to insult, whereas another population appears protected from such damage. Because spermatozoal RNAs echo the gene expression of spermatogenesis, the latter is likely to prove useful as a clinical maker of fertility status.
Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is
relevant because heat shock proteins (HSPs) are required for spermatogenesis ...and also protect cells from environmental hazards
such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat
shock (43°C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that
spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated
with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression
networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and
thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated
after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of
HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional
non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was
recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes;
5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further
analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.
Multigeneration reproduction studies are used to characterize parental and offspring systemic toxicity, as well as reproductive toxicity of pesticides, industrial chemicals and pharmaceuticals. ...Results from 329 multigeneration studies on 316 chemicals have been digitized into standardized and structured toxicity data within the Toxicity Reference Database (ToxRefDB). An initial assessment of data quality and consistency was performed prior to profiling these environmental chemicals based on reproductive toxicity and associated toxicity endpoints. The pattern of toxicity across 75 effects for all 316 chemicals provided sets of chemicals with similar in vivo toxicity for future predictive modeling. Comparative analysis across the 329 studies identified chemicals with sensitive reproductive effects, based on comparisons to chronic and subchronic toxicity studies, as did the cross-generational comparisons within the multigeneration study. The general pattern of toxicity across all chemicals and the more focused comparative analyses identified 19 parental, offspring and reproductive effects with a high enough incidence to serve as targets for predictive modeling that will eventually serve as a chemical prioritization tool spanning reproductive toxicities. These toxicity endpoints included specific reproductive performance indices, male and female reproductive organ pathologies, offspring viability, growth and maturation, and parental systemic toxicities. Capturing this reproductive toxicity data in ToxRefDB supports ongoing retrospective analyses, test guideline revisions, and computational toxicology research.
Abstract Understanding the potential health risks posed by environmental chemicals is a significant challenge elevated by the large number of diverse chemicals with generally uncharacterized ...exposures, mechanisms, and toxicities. The present study is a performance evaluation and critical analysis of assay results for an array of 292 high-throughput cell-free assays aimed at preliminary toxicity evaluation of 320 environmental chemicals in EPA's ToxCast™ project (Phase I). The chemicals (309 unique, 11 replicates) were mainly precursors or the active agent of commercial pesticides, for which a wealth of in vivo toxicity data is available. Biochemical HTS (high-throughput screening) profiled cell and tissue extracts using semi-automated biochemical and pharmacological methodologies to evaluate a subset of G-protein coupled receptors (GPCRs), CYP450 enzymes (CYPs), kinases, phosphatases, proteases, HDACs, nuclear receptors, ion channels, and transporters. The primary screen tested all chemicals at a relatively high concentration 25 μM concentration (or 10 μM for CYP assays), and a secondary screen re-tested 9132 chemical-assay pairs in 8-point concentration series from 0.023 to 50 μM (or 0.009–20 μM for CYPs). Mapping relationships across 93,440 chemical-assay pairs based on half-maximal activity concentration (AC50) revealed both known and novel targets in signaling and metabolic pathways. The primary dataset, summary data and details on quality control checks are available for download at http://www.epa.gov/ncct/toxcast/.
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