Amino Acid Transport Mechanisms Research Articles

Types of Membrane Transporters and the Mechanisms of Interaction between Them and Reactive Oxygen Species in Plants.

Membrane transporters are proteins that mediate the entry and exit of substances through the plasma membrane and organellar membranes and are capable of recognizing and binding to specific substances, thereby facilitating substance transport. Membrane transporters are divided into different types, e.g., ion transporters, sugar transporters, amino acid transporters, and aquaporins, based on the substances they transport. These membrane transporters inhibit reactive oxygen species (ROS) generation through ion regulation, sugar and amino acid transport, hormone induction, and other mechanisms. They can also promote enzymatic and nonenzymatic reactions in plants, activate antioxidant enzyme activity, and promote ROS scavenging. Moreover, membrane transporters can transport plant growth regulators, solute proteins, redox potential regulators, and other substances involved in ROS metabolism through corresponding metabolic pathways, ultimately achieving ROS homeostasis in plants. In turn, ROS, as signaling molecules, can affect the activity of membrane transporters under abiotic stress through collaboration with ions and involvement in hormone metabolic pathways. The research described in this review provides a theoretical basis for improving plant stress resistance, promoting plant growth and development, and breeding high-quality plant varieties.

Integrated metabolomic and transcriptomic analyses reveal themolecular mechanism of amino acid transport between source and sink during tea shoot development.

The weighted gene co-expression network analysis and antisense oligonucleotide-mediated transient gene silencing revealed that CsAAP6 plays an important role in amino acid transport during tea shoot development. Nitrogen transport from source to sink is crucial for tea shoot growth and quality formation. Amino acid represents the major transport form of reduced nitrogen in the phloem between source and sink, but the molecular mechanism of amino acid transport from source leaves to new shoots is not yet clear. Therefore, the composition of metabolites in phloem exudates collected by the EDTA-facilitated method was analyzed through widely targeted metabolomics. A total of 326 metabolites were identified in the phloem exudates with the richest variety of amino acids and their derivatives (93), accounting for approximately 39.13% of the total metabolites. Moreover, through targeted metabolomics, it was found that the content of glutamine, glutamic acid, and theanine was the most abundant, and gradually increased with the development of new shoots. Meanwhile, transcriptome analysis suggested that the expression of amino acid transport genes changed significantly. The WGCNA analysis identified that the expression levels of CsAVT1, CsLHTL8, and CsAAP6 genes located in the MEterquoise module were positively correlated with the content of amino acids such as glutamine, glutamic acid, and theanine in phloem exudates. Reducing the CsAAP6 in mature leaves resulted in a significant decrease in the content of glutamic acid, aspartic acid, alanine, leucine, asparagine, glutamine, and arginine in the phloem exudates, indicating that CsAAP6 played an important role in the source to sink transport of amino acids in the phloem. The research results will provide the theoretical basis and genetic resources for the improvement of nitrogen use efficiency and tea quality.

Direct evidence for the electroconvective mechanism of neutral amino acid transport during electrodialysis

For the first time, direct evidence was obtained using laser interferometry and flicker-noise spectroscopy that the main cause of the increase in the transport of amino acids through a sulfonated cation-exchange membrane in intense current modes is electroconvection. The turbulent nature of electroconvective vortices at the solution/membrane interface, acidification of the solution in the demineralization compartment and a substantial intensification of amino acid fluxes under over-limiting current modes were revealed. This indicates the electroconvective destruction of the barrier effect of the solution layer with a high pH value near the cation-exchange membrane.

Evaluation of L-Alanine Metabolism in Bacteria and Whole-Body Distribution with Bacterial Infection Model Mice

The World Health Organization has cautioned that antimicrobial resistance (AMR) will be responsible for an estimated 10 million deaths annually by 2050. To facilitate prompt and accurate diagnosis and treatment of infectious disease, we investigated the potential of amino acids for use as indicators of bacterial growth activity by clarifying which amino acids are taken up by bacteria during the various growth phases. In addition, we examined the amino acid transport mechanisms that are employed by bacteria based on the accumulation of labeled amino acids, Na+ dependence, and inhibitory effects using a specific inhibitor of system A. We found that 3H-L-Ala accurately reflects the proliferative activity of Escherichia coli K-12 and pathogenic EC-14 in vitro. This accumulation in E. coli could be attributed to the amino acid transport systems being different from those found in human tumor cells. Moreover, biological distribution assessed in infection model mice with EC-14 using 3H-L-Ala showed that the ratio of 3H-L-Ala accumulated in infected muscle to that in control muscle was 1.20. By detecting the growth activity of bacteria in the body that occurs during the early stages of infection by nuclear imaging, such detection methods may result in expeditious diagnostic treatments for infectious diseases.

Chemical characterizations of neurotransmission receptors of human and plant to unfold the evolutionary relationships among them

Vertebrates have very well defined nervous systems. A group of researchers hypothesize that plant also has an alternative sort of sensitive nervous system. They find a close relationship of the neurotransmission mechanism of animal with that of the plant and suspect a close relationship in amino acid transport mechanism among both the organisms. Although the protein structure is conserved more than molecular sequences, but the 3D structure of protein is largely influenced by the amino acid residues in the interior part of it. The constituents of a primary protein sequence have a variety of biochemical information which control the structure, function and stability of the protein. Hence, in this present study it is tried for characterization and comparison of neurotransmission receptors associated with human and plant to unfold the evolutionary relationships among them in bio-molecular level based on the chemical properties of the amino acids. The protein sequences of ionotropic glutamate receptor and GABA receptor of human (from vertebrate) and Arabidopsis thaliana (from plant) are considered as datasets. The 20 standard amino acids are classified into 8 chemical groups and are identified by specific numeric values. Alignment-based methods are used to identify the identical and similar amino acids among the aligned sequences.The common pattern finding procedure finds some conserved regions in the receptor protein sequences of both the species. The proximity between the protein sequences are calculated based on the distribution of each chemical group (in percentage) in them and phylogenetic trees are constructed to show the evolutionary relationships of neurotransmission receptors of both the species. The conventional multiple sequence alignment (MSA) method is also applied on the datasets and the results are compared. The analysis is further extended to structural level to understand the extent to which the animal and plant proteins are similar.

Placental adaptation to maternal malnutrition.

Maternal malnutrition gives rise to both short- and long-term consequences for the survival and health of the offspring. As the intermediary between mother and fetus, the placenta has the potential to interpret environmental signals, such as nutrient availability, and adapt to support fetal growth and development. While this potential is present, it is clear that at times placental adaptation fails to occur resulting in poor pregnancy outcomes. This review will focus on placental responses to maternal undernutrition related to changes in placental vascularization and hemodynamics and placental nutrient transport systems across species. While much of the available literature describes placental responses that result in poor fetal outcomes, novel models have been developed to utilize the inherent variation in fetal weight when dams are nutrient restricted to identify placental adaptations that result in normal-weight offspring. Detailed analyses of the spectrum of placental responses to maternal malnutrition point to alternations in placental histoarchitectural and vascular development, amino acid and lipid transport mechanisms, and modulation of immune-related factors. Dietary supplementation with selected nutrients, such as arginine, has the potential to improve placental growth and function through a variety of mechanisms including stimulating cell proliferation, protein synthesis, angiogenesis, vasodilation, and gene regulation. Improved understanding of placental responses to environmental cues is necessary to develop diagnostic and intervention strategies to improve pregnancy outcomes.

Consensus mutagenesis approach improves the thermal stability of system xc - transporter, xCT, and enables cryo-EM analyses.

System xc − is an amino acid antiporter that imports L‐cystine into cells and exports intracellular L‐glutamate, at a 1:1 ratio. As L‐cystine is an essential precursor for glutathione synthesis, system xc − supports tumor cell growth through glutathione‐based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System xc − consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L‐type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single‐particle analyses of the xCT‐CD98hc complex. As wild‐type xCT‐CD98hc displayed poor stability and could not be purified to homogeneity, we applied a consensus mutagenesis approach to xCT. The consensus mutated construct exhibited increased stability as compared to the wild‐type, and enabled the cryoelectron microscopy (cryo‐EM) map to be obtained at 6.2 Å resolution by single‐particle analysis. The cryo‐EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our consensus‐based strategy could represent a good choice toward solving unstable protein structures.

Attractor Concepts to Evaluate the Transcriptome-wide Dynamics Guiding Anaerobic to Aerobic State Transition in Escherichia coli

For any dynamical system, like living organisms, an attractor state is a set of variables or mechanisms that converge towards a stable system behavior despite a wide variety of initial conditions. Here, using multi-dimensional statistics, we investigate the global gene expression attractor mechanisms shaping anaerobic to aerobic state transition (AAT) of Escherichia coli in a bioreactor at early times. Out of 3,389 RNA-Seq expression changes over time, we identified 100 sharply changing genes that are key for guiding 1700 genes into the AAT attractor basin. Collectively, these genes were named as attractor genes constituting of 6 dynamic clusters. Apart from the expected anaerobic (glycolysis), aerobic (TCA cycle) and fermentation (succinate pathways) processes, sulphur metabolism, ribosome assembly and amino acid transport mechanisms together with 332 uncharacterised genes are also key for AAT. Overall, our work highlights the importance of multi-dimensional statistical analyses for revealing novel processes shaping AAT.

Proteomic response of marine-derived Staphylococcus cohnii #NIOSBK35 to varying Cr(vi) concentrations.

Chromium in its hexavalent state is a water-soluble and toxic element to living organisms present in the environment. However, some organisms are resistant and reduce the toxic forms of Cr(vi) to less toxic or non-toxic forms. A global proteomic analysis of Staphylococcus sp. #NIOSBK35 under different chromate concentrations (0, 100, 200 and 300 mg L-1) at different time points in its growth stages (6, 9, 12, 18, 24 and 36 h) resulted in the identification of 878 proteins. Of all the proteins expressed, 13 proteins [23 rDNA (uracil-5-) methyltransferase RumA, multidrug ABC transporter ATP binding protein, dihydroxy acid dehydratase, polysaccharide biosynthesis protein, etc.] were expressed only in the presence of chromium. 14 proteins were up-regulated in response to chromium(vi), namely, alkyl hydroperoxide reductase, ATP-dependent Zn metallopeptidase, hsp90- like protein, NAD (P)-dependent oxidoreductase, etc. Most of the proteins involved in normal cell functioning like 1-pyrroline-5-carboxylate dehydrogenase, ribosomal proteins (30S ribosomal protein S11, 30S ribosomal protein S2, and 50S ribosomal protein L32), aconitate hydratase, DNA primase, serine-tRNA ligase, phosphoenolpyruvate-protein phosphotransferase, enolase, sulfur transferase FdhD, etc. were found to be down-regulated. On grouping these proteins into their COG (cluster of orthologous groups) functional categories, they were found to be involved in translation, carbohydrate metabolism, stress proteins, amino acid transport and membrane transport mechanisms. The proteomic response given by Staphylococcus sp. #NIOSBK35 did not show expression of Cr-specific proteins, indicating a different mechanism of Cr-tolerance as the organism was able to survive and grow at high concentrations of Cr(vi).

Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc.

The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.

The Dose‐Related Effects of Doxorubicin Chemotherapy on Interstitial Amino Acid Concentrations in Skeletal Muscle

Doxorubicin (DOX) is a frontline anticancer therapeutic which has proven effective in the treatment of various cancers. However, very little is known regarding the effect that DOX has on SM metabolism, specifically its influence on interstitial amino acid (AA) concentrations. In SM, the interstitial space represents an important active compartment located between the vasculature and the tissue and it has been shown to play a functional role in the regulation and integration of various metabolic substances. The purpose of the current study was to determine the effect of DOX on interstitial AA concentrations in SM. Rats received a 1.5 or 4.5 mg/kg dose of DOX i.p. and grouped into endpoints every 24 hrs up to 192 hrs. A control group was administered a sham i.p. injection of saline. Total amino acids (TAA), essential amino acids (EAA) and branched‐chain amino acids (BCAA) were quantified in the interstitial space. The administration of DOX resulted in a dose‐related difference in interstitial AA. The 1.5 mg/kg dose resulted in a transient increase (P<0.05) in TAA, EAA and BCAA from 24–96 hrs as compared to control. Contrarily, TAA, EAA and BCAA decreased (P<0.05) 24–192 hours following the administration of 4.5 mg/kg DOX as compared to control. This study by way of the microdialysis technique, represents the first time that amino acid concentrations have been quantified in the interstitial space following the administration of a chemotherapeutic drug. In addition, the possibility of a dose‐related effect of DOX on amino acid transport mechanisms from the skeletal muscle compartment into the interstitium/vasculature is of particular interest as this may represent a regulatory mechanism that has not previously been considered.Support or Funding InformationSupported by NOSMFAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Amino Acid Metabolism and Transport Mechanisms as Potential Antifungal Targets.

Discovering new drugs for treatment of invasive fungal infections is an enduring challenge. There are only three major classes of antifungal agents, and no new class has been introduced into clinical practice in more than a decade. However, recent advances in our understanding of the fungal life cycle, functional genomics, proteomics, and gene mapping have enabled the identification of new drug targets to treat these potentially deadly infections. In this paper, we examine amino acid transport mechanisms and metabolism as potential drug targets to treat invasive fungal infections, including pathogenic yeasts, such as species of Candida and Cryptococcus, as well as molds, such as Aspergillus fumigatus. We also explore the mechanisms by which amino acids may be exploited to identify novel drug targets and review potential hurdles to bringing this approach into clinical practice.

Synthesis and biological properties of radiohalogenated α,α-disubstituted amino acids for PET and SPECT imaging of amino acid transporters (AATs).

Fluorine-18 and iodine-123 labeled nonnatural alicyclic and methyl branched disubstituted α,α-amino acids are a diverse and useful class of tumor imaging agents suitable for positron emission tomography and single photon emission computed tomography. These tracers target the increased expression of the cell membrane amino acid transporter systems L, ASC, and A exhibited by many human tumor cells. The most established clinical use for these radiolabeled amino acids is imaging primary and recurrent gliomas and primary, recurrent, and metastatic prostate cancer. This review focuses on the synthesis, radiolabeling, and amino acid transport mechanism of a series of nonnatural fluorine-18 and iodine-123 labeled analogs of 1-aminocyclobutane-1-carboxylic acid, 1-aminocyclopentane-1-carboxylic acid, α-aminoisobutyric acid, and α-methylaminoisobutyric acid.

Identification and characterization of cationic amino acid transporters (CATs) in tea plant (Camellia sinensis)

Amino acids are constituents of proteins, precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. However, the amino acid transport in tea plant is rarely reported. In this study, six cationic amino acid transporter (CAT) family genes were cloned. Phylogenetic analysis categorized these CsCATs into four subgroups. These CsCATs all contain the 12–14 transmembrane domains and the conserved CAT motifs. Their expression was tissue-specific, with higher expression levels in root and stem and correlated to the abundances of key free amino acids such as Theanine. Some CsCATs expression responded to some abiotic stress conditions and to the exogenous application of theanine (Thea), glutamine or ethylamine hydrochloride, an ethylamine precursor for Thea biosynthesis. Our results indicated that the CsCATs expression is regulated by amino acid contents and is sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants.

Characterization of the Inward- and Outward-Facing Substrate Binding Sites of the Prokaryotic Aspartate Transporter, GltPh.

Crystal structures of the prokaryotic aspartate transporter, GltPh, have provided important insights into the mechanism of amino acid transport by GltPh and related eukaryotic members of the glutamate transporter family (SLC1A family). Identification of inhibitors of GltPh can provide valuable tools for understanding the molecular basis for substrate and inhibitor specificity and selectivity of SLC1A members, but at present, few inhibitors of GltPh have been identified. We have screened a collection of commercially available aspartate analogues and identified new transportable and nontransportable GltPh inhibitors. We have explored the inhibition profile of GltPh by utilizing a thiol modification assay that isolates sided populations of the transporters reconstituted in liposomes to determine if any aspartate analogues display a preference for either the inwardly or outwardly directed binding sites. Here, we have characterized several new inhibitors of GltPh and identified three β-carbon-substituted molecules that display a strong preference for the outwardly directed binding site of GltPh.

In vitro Characterization of Uptake Mechanism of l-[methyl-3H]-methionine in Hepatocellular Carcinoma

Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-(11)C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-(11)C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-(11)C]-Met in HCC cells. A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-(3)H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively. Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na(+) dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-(3)H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of MAT2A expression (the duration of SAM treatment). The incorporated (3)H was mostly distributed in the protein phase and, to a lesser degree, in the lipid phase via PE methylation pathway in HCC cells with SAM treatment. The downregulated MAT2A expression led to the decreased uptake in protein and water-soluble phases. In addition, an increased uptake in the lipid phase was observed in WCH17 cells transfected with PEMT2 expression vector. The amino acid transport processes may be responsible for the rapid accumulation of radiolabeled Met after the intravenous injection of tracers for the imaging of HCC. Upregulated MAT2A expression and impaired PEMT2 activities in HCC are associated with the specific metabolic pattern of L-[methyl-(11)C]-Met detected by PET.

A de novo‐designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 μM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.

Renal imino acid and glycine transport system ontogeny and involvement in developmental iminoglycinuria

Renal maturation occurs post-natally in many species and reabsorption capacity at birth can vary substantially from the mature kidney. However, little is known regarding the maturation of amino acid transport mechanisms, despite the well-known physiological state of developmental iminoglycinuria. Commonly seen during early infancy, developmental iminoglycinuria is a transient version of the persistent inherited form of the disorder, referred to as iminoglycinuria, and manifests as a urinary hyperexcretion of proline, hydroxyproline and glycine. The transporters involved in developmental iminoglycinuria and their involvement in the improvement of renal reabsorption capacity remain unknown. qPCR (quantitative real-time PCR) and Western blot analysis in developing mouse kidney revealed that the expression of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 was lower at birth (approx. 3.4-, 5.0-, 2.4- and 3.0-fold less than adult kidney by qPCR respectively) and increased during development. Furthermore, immunofluorescence confocal microscopy demonstrated the absence of apical expression of Slc6a18, Slc6a19, Slc6a20a and the auxiliary protein collectrin in kidneys of mice at birth. This correlated with the detection of iminoglycinuria during the first week of life. Iminoglycinuria subsided (proline reduction preceded glycine) in the second week of life, which correlated with an increase in the expression of Slc6a19 and Slc6a20a. Mice achieved an adult imino acid and glycine excretion profile by the fourth week, at which time the expression level of all transporters was comparable with adult mice. In conclusion, these results demonstrate the delayed expression and maturation of Slc6a18, Slc6a19, Slc6a20a and Slc36a2 in neonatal mice and thus the molecular mechanism of developmental iminoglycinuria.

Global transcriptional profiles of Trichophyton rubrum in response to Flucytosine

Trichophyton rubrum (T.rubrum) is one of the most common human fungal pathogens that cause chronic infections of the skin and nails. To identify antifungal responsive genes, cDNA microarray analysis was performed for T. rubrum to reveal global transcriptional profiles of drug-specific responses to 5-Flucytosine (5-FC). cDNA microarray was constructed from the T. rubrum expressed sequence tag (ESTs) database, the minimum inhibitory concentration (MIC) of 5-FC was determined, and microarray hybridization and data analysis were applied. The expression pattern of 7 genes observed by microarray was confirmed by the quantitative real-time reverser transcription polymerase chain reaction (RT-PCR). Data analysis indicated that a total of 474 genes were found differentially expressed, 196 showed an increase in expression and 278 showed a decrease in expression. Marked down-regulation of genes involved in nucleotide metabolism (such as CDC21), transcription (such as E2F1), and RNA processing (such as SGN1, RIM4 and NOP1) was observed. Other genes involved in signal transduction, chaperones, inorganic ion transport, secondary metabolite biosynthesis, amino acid transport, lipid transport and potential drug resistance mechanism were also affected by 5-FC. Quantitative real-time RT-PCR of the selected genes confirmed the reliability of the microarray results. This is the first analysis of transcriptional profiles in response to 5-FC for T. rubrum. The findings may be valuable for the identification of genes involved in mechanisms of action and mechanisms of antifungal drug resistance of 5-FC.

Amino Acid Transport Mechanisms in Mouse Oocytes During Growth and Meiotic Maturation1

Amino acids are transported into cells by a number of different transport systems, each with their own specific range of substrates. The amino acid transport systems active in preimplantation embryos and the amino acids required by embryos for optimal development have been extensively investigated. Much less is known about amino acid transport systems active in growing and meiotically maturing oocytes or about developmental changes in their activity. As a first step in determining the array of amino acid transporters active in oocytes, the transport characteristics of nine amino acids were measured in small, medium, and large growing oocytes; in fully grown germinal vesicle (GV)-stage oocytes; in metaphase I oocytes; and in metaphase II eggs. Whether each of 11 classically defined amino acid transport systems was likely active in oocytes at each stage was determined using assays based on measuring the transport of radiolabeled amino acids into oocytes and the effect of a limited set of potential competitive inhibitors. Six amino acid transport systems were found to be active during oocyte growth or maturation. L, b(0,+), and ASC/asc were active throughout oocyte growth and maturation, increasing during growth. In contrast, GLY, beta, and x(c)(-) had little or no activity during growth but became activated during meiotic maturation. Surprisingly, the presence of follicular cells surrounding medium growing oocytes or cumulus cells surrounding GV oocytes did not confer amino acid transport by additional transport systems not present in the oocyte. In some cases, however, follicular cells coupled to the oocyte enhanced uptake of amino acids by the same systems present in the oocyte.