Tumoral microvesicle-activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma.

Metabolic reprogramming is a hallmark of cancer. Stromal cells could function as providers of energy metabolites for tumor cells by undergoing the "reverse Warburg effect," but the mechanism has not been fully elucidated. The interaction between the tumoral microvesicles (TMVs) and stroma in the tumor microenvironment plays a critical role in facilitating cancer progression. In this study, we demonstrated a novel mechanism for the TMV-mediated glycometabolic reprogramming of stromal cells. After being incubated with TMVs, normal human gingival fibroblasts exhibited a phenotype switch to cancer-associated fibroblasts and underwent a degradation of caveolin 1 (CAV1) through the ERK1/2-activation pathway. CAV1 degradation further induced the metabolic switch to aerobic glycolysis in the fibroblasts. The microvesicle-activated fibroblasts absorbed more glucose and produced more lactate. The migration and invasion of oral squamous cell carcinoma (OSCC) were promoted after being cocultured with the activated fibroblasts. Fibroblast-cancer cell glycometabolic coupling ring mediated by monocarboxylate transporter (MCT) 4 and MCT1 was then proved in the tumor microenvironment. Results indicated a mechanism for tumor progression by the crosstalk between tumor cells and stromal cells through the reverse Warburg effect via TMVs, thereby identifying potential targets for OSCC prevention and treatment.-Jiang, E., Xu, Z., Wang, M., Yan, T., Huang, C., Zhou, X., Liu, Q., Wang, L., Chen, Y., Wang, H., Liu, K., Shao, Z., Shang, Z. Tumoral microvesicle-activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma.

Comparative metabolic analysis in head and neck cancer and the normal gingiva.

OBJECTIVES: Chronic accumulation of lactate in malignant tumor tissue is associated with increased malignancy and radioresistance. For this study, biopsies of primary head and neck squamous cell carcinoma (HNSCC) and of the normal gingiva of the same patient were compared via metabolic profiling to the healthy gingiva from cancer-free patients. MATERIALS AND METHODS: Cryobiopsies of 140 HNSCC patients were used to determine ATP, lactate, and glucose concentrations of the tumor and normal gingiva via induced metabolic bioluminescence imaging (imBI). Additionally, these metabolites were quantified in a collective of 79 healthy (non-tumor-bearing) patients. Furthermore, tumor samples were analyzed via immunofluorescence imaging and quantitative real-time PCR for the expression of lactate and glucose transporters. RESULTS: There were significant differences in ATP concentrations detectable between the tumor, normal gingiva of tumor patients, and gingiva from healthy patients. Lactate concentrations were significantly increased in tumor tissue compared to the normal gingiva of tumor patients as well as the gingiva from healthy patients. Concerning glucose, there was a significant decrease in glucose concentrations detectable in the tumor biopsies compared to the normal gingiva of tumor patients. On the other hand, tumor samples from patients revealed significantly elevated relative expression levels of monocarboxylate transporters (MCT-1 and MCT-4), as well as glucose transporters (GLUT-1 and GLUT-3) compared to the corresponding normal gingiva of each patient. CONCLUSIONS: We could demonstrate that the lactate concentration in HNSCC correlates with primary tumor (T) stage. CLINICAL RELEVANCE: The aim of this study was to identify metabolic parameters to improve early cancer diagnosis, allow predictions on the degree of malignancy, and contribute to a personalized tumor therapy.

Nondigestible Fructans Alter Gastrointestinal Barrier Function, Gene Expression, Histomorphology, and the Microbiota Profiles of Diet-Induced Obese C57BL/6J Mice.

BACKGROUND: Obesity is associated with compromised intestinal barrier function and shifts in gastrointestinal microbiota that may contribute to inflammation. Fiber provides benefits, but impacts of fiber type are not understood. OBJECTIVE: We aimed to determine the impact of cellulose compared with fructans on the fecal microbiota and gastrointestinal physiology in obese mice. METHODS: Eighteen-wk-old male diet-induced obese C57BL/6J mice (n = 6/group; 40.5 g) were fed high-fat diets (45% kcal fat) containing 5% cellulose (control), 10% cellulose, 10% short-chain fructooligosaccharides (scFOS), or 10% inulin for 4 wk. Cecal and colon tissues were collected to assess barrier function, histomorphology, and gene expression. Fecal DNA extracts were subjected to 16S ribosomal RNA amplicon-based Illumina MiSeq sequencing to assess microbiota. RESULTS: Body weight gain was greater (P < 0.05) in scFOS-fed than in 10% cellulose-fed mice. Both groups of fructan-fed mice had greater (P < 0.05) cecal crypt depth (scFOS: 141 µm; inulin: 145 µm) than both groups of cellulose-fed mice (5% and 10%: 109 µm). Inulin-fed mice had greater (P < 0.05) cecal transmural resistance (101 Ω × cm(2)) than 5% cellulose-fed controls (45 Ω × cm(2)). Inulin-fed mice had lower (P < 0.05) colonic mRNA abundance of Ocln (0.41) and Mct1 (0.35) than those fed 10% cellulose (Ocln: 1.28; Mct1: 0.90). Fructan and cellulose groups had different UniFrac distances of fecal microbiota (P < 0.05) and α diversity, which demonstrated lower (P < 0.01) species richness in fructan-fed mice. Mice fed scFOS had greater (P < 0.05) Actinobacteria (15.9%) and Verrucomicrobia (Akkermansia) (17.0%) than 5% controls (Actinobacteria: 0.07%; Akkermansia: 0.08%). Relative abundance of Akkermansia was positively correlated (r = 0.56, P < 0.01) with cecal crypt depth. CONCLUSIONS: Fructans markedly shifted gut microbiota and improved intestinal physiology in obese mice, but the mechanisms by which they affect gut integrity and inflammation in the obese are still unknown.

HBsAg loss in patients treated with peginterferon alfa-2a and adefovir is associated with SLC16A9 gene variation and lower plasma carnitine levels.

BACKGROUND & AIMS: Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. METHODS: Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n = 9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro. RESULTS: One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 (p = 1.84 × 10(-8)). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence (p = 0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. CONCLUSIONS: In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome.

Lactate triggers KAT8-mediated LTBP1 lactylation at lysine 752 to promote skin rejuvenation by inducing collagen synthesis in fibroblasts.

Decreased collagen synthesis by fibroblasts is a key aspect of skin aging. Poly-L-Lactic Acid (PLLA) is a bioabsorbable material that can release lactate continuously, stimulating endogenous collagen synthesis in the skin. Herein, this study aimed to investigate the impact of PLLA-released lactate on collagen production in fibroblasts for skin rejuvenation. Human fibroblasts were exposed to varying concentrations of PLLA in vitro, while PLLA was injected into the back skin of aged mice in vivo. Safety and efficacy of PLLA on collagen synthesis and skin rejuvenation were evaluated through Calcein-AM/PI staining, EdU proliferation assay, and analysis of collagen I and collagen III expression in fibroblasts using western blotting and immunofluorescence. To elucidate the underlying mechanisms, lactate contents in cell-free supernatant and cell lysates from PLLA-treated fibroblasts, as well as total lysine lactylation (Pan Kla) levels were measured. Additionally, we found that fibroblasts can uptake extracellular lactate released from PLLA through monocarboxylate transporter-1 (MCT1) to facilitate latent-transforming growth factor beta-binding protein 1 (LTBP1) lactylation at lysine 752 (K752) via a KAT8-dependent mechanism, then increases the protein levels of collagen I and collagen III in fibroblasts. Overall, this study highlights a valuable insight into lactylation modification of non-histone protein for skin rejuvenation.

Genome-Wide Analysis of Dental Caries Variability Reveals Genotype-by-Environment Interactions.

Genotype-by-environment interactions (GEI) may influence dental caries, although their effects are difficult to detect. Variance quantitative trait loci (vQTL) may serve as an indicator of underlying GEI effects. The aim of this study was to investigate GEI effects on dental caries by prioritizing variants from genome-wide vQTL analysis. First, we identified vQTLs from ~4.3 M genome-wide variants in three cohorts of white children aged 3-5 (n = 396, n = 328, n = 773) using Levene's test. A total of 39 independent vQTLs with p < 1 × 10-6 were identified, some of which were located in or near genes with plausible biological roles in dental caries (IGFBP7, SLC5A8, and SHH involved in tooth development and enamel mineralization). Next, we used linear regression to test GEI effects on dental caries with the 39 prioritized variants and self-reported environmental factors (demographic, socioeconomic, behavioral, and dietary factors) in the three cohorts separately. We identified eight significant GEIs indicating that children with vQTL risk genotypes had higher caries experience if they had less educated parents, lower household/parental income, brushed their teeth less frequently, consumed sugar-sweetened beverages more frequently, were not breastfed, and were female. We reported the first genome-wide vQTL analysis of dental caries in children nominating several novel genes and GEI for further investigations.

Pyruvate-conjugation of PEGylated liposomes for targeted drug delivery to retinal photoreceptors.

Despite several promising candidates, there is a paucity of drug treatments available for patients suffering from retinal diseases. An important reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors. A promising and versatile method for drug delivery to specific cell types involves transporter-targeted liposomes, i.e., liposomes surface-coated with substrates for transporter proteins highly expressed on the target cell. We identified strong lactate transporter (monocarboxylate transporter, MCT) expression on photoreceptors as a potential target for drug delivery vehicles. To evaluate MCT suitability for drug targeting, we used PEG-coated liposomes and conjugated these with different monocarboxylates, including lactate, pyruvate, and cysteine. Monocarboxylate-conjugated and dye-loaded liposomes were tested on both human-derived cell-lines and murine retinal explant cultures. We found that liposomes conjugated with pyruvate consistently displayed higher cell uptake than unconjugated liposomes or liposomes conjugated with lactate or cysteine. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, suggesting an MCT-dependent uptake mechanism. Notably, pyruvate-conjugated liposomes loaded with the drug candidate CN04 reduced photoreceptor cell death in the murine rd1 retinal degeneration model while free drug solutions could not achieve the same therapeutic effect. Our study thus highlights pyruvate-conjugated liposomes as a promising system for drug delivery to retinal photoreceptors, as well as other neuronal cell types displaying high expression of MCT-type proteins.

Mitochondrial pyruvate carrier regulates the lignocellulosic decomposition rate through metabolism in Ganoderma lucidum.

The activity of mitochondrial pyruvate carrier (MPC) can be modulated to regulate intracellular metabolism under different culture conditions. In Ganoderma lucidum, the role of MPC in regulating carbon sources remains unknown. By knocking down MPC genes (MPC1 and MPC2), this research found that the loss of MPC increased the growth rate of G. lucidum by ~30% in a medium with wood chips as a carbon source. Then cellulase and laccase activities were tested. Endoglucanase and laccase activity increased by ~50% and ~35%, respectively, in MPC knockdown mutants compared with that in the wild type strain. Finally, the expression levels of genes related to glycolysis were assayed, and the transcription levels of these enzymes were found to be increased by ~250% compared with the wild type strain. In conclusion, the regulation of intracellular metabolism by MPC provides a new way to improve the use of nondominant carbon sources such as lignocellulose.

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.

Roles of monocarboxylate transporter subtypes in promotion and suppression of osteoclast differentiation and survival on bone.

Monocarboxylate transporters (MCTs) provide transmembrane transport of monocarboxylates such as lactate and pyruvate. The present results showed that α-cyano-4-hydroxycinnamic acid (CHC), an inhibitor of MCTs, promoted osteoclast differentiation from macrophages at lower concentrations (0.1-0.3 mM) and suppressed that at a higher concentration (1.0 mM). On the other hand, CHC reduced the number of mature osteoclasts on the surface of dentin in a concentration-dependent manner. Additionally, macrophages and osteoclasts were found to express the Mct1, Mct2, and Mct4 genes, with Mct1 and Mct4 expression higher in macrophages, and that of Mct2 higher in osteoclasts. Although Mct1 gene knockdown in macrophages enhanced osteoclast formation induced by RANKL, Mct2 gene knockdown suppressed that. Finally, Mct2 gene silencing in mature osteoclasts decreased their number and, thereby, bone resorption. These results suggest that MCT1 is a negative regulator and MCT2 a positive regulator of osteoclast differentiation, while MCT2 is required for bone resorption by osteoclasts.

[Significant role of poloxamer in drug transport across blood-brain barrier].

Poloxamers are found to be an efficient adjuvant with multiple effects and are applied generally in pharmaceutical field. In recent years, it is investigated that poloxamers can increase the permeability of a broad spectrum of drugs through blood-brain barrier (BBB) by means of manifold mechanisms included: (1) inhibiting P-glycoprotein and multidrug-resistance associated protein efflux systems on BBB; (2) adsorbing different apolipoproteins in plasma on the surface of poloxamer-coated nanoparticles, which could interact with BBB through different receptors and mechanisms; (3) connecting to specific ligands and monoclonal antibodies to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium. Significant roles of poloxamer in drug transport across BBB are considered in this review which provides for important guidance to the design of brain-targeted drug delivery system.

Glucose-dependent growth arrest of leukemia cells by MCT1 inhibition: Feeding Warburg's sweet tooth and blocking acid export as an anticancer strategy.

This study aims to investigate the utilization of The Warburg Effect, cancer's "sweet tooth" and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 µM) and the NHE1 inhibitor cariporide (10 µM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies.

Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.

Exercise training in normobaric hypoxia in endurance runners. III. Muscular adjustments of selected gene transcripts.

We hypothesized that specific muscular transcript level adaptations participate in the improvement of endurance performances following intermittent hypoxia training in endurance-trained subjects. Fifteen male high-level, long-distance runners integrated a modified living low-training high program comprising two weekly controlled training sessions performed at the second ventilatory threshold for 6 wk into their normal training schedule. The athletes were randomly assigned to either a normoxic (Nor) (inspired O2 fraction = 20.9%, n = 6) or a hypoxic group exercising under normobaric hypoxia (Hyp) (inspired O2 fraction = 14.5%, n = 9). Oxygen uptake and speed at second ventilatory threshold, maximal oxygen uptake (VO2 max), and time to exhaustion (Tlim) at constant load at VO2 max velocity in normoxia and muscular levels of selected mRNAs in biopsies were determined before and after training. VO2 max (+5%) and Tlim (+35%) increased specifically in the Hyp group. At the molecular level, mRNA concentrations of the hypoxia-inducible factor 1alpha (+104%), glucose transporter-4 (+32%), phosphofructokinase (+32%), peroxisome proliferator-activated receptor gamma coactivator 1alpha (+60%), citrate synthase (+28%), cytochrome oxidase 1 (+74%) and 4 (+36%), carbonic anhydrase-3 (+74%), and manganese superoxide dismutase (+44%) were significantly augmented in muscle after exercise training in Hyp only. Significant correlations were noted between muscular mRNA levels of monocarboxylate transporter-1, carbonic anhydrase-3, glucose transporter-4, and Tlim only in the group of athletes who trained in hypoxia (P < 0.05). Accordingly, the addition of short hypoxic stress to the regular endurance training protocol induces transcriptional adaptations in skeletal muscle of athletic subjects. Expressional adaptations involving redox regulation and glucose uptake are being recognized as a potential molecular pathway, resulting in improved endurance performance in hypoxia-trained subjects.

Tissue distribution of basigin and monocarboxylate transporter 1 in the adult male mouse: a study using the wild-type and basigin gene knockout mice.

Basigin (Bsg) is a transmembrane protein that is responsible for targeting of monocarboxylate transporters (MCTs) to the cell membrane. The present study was conducted to determine whether or not Bsg was required for the proper localization of MCT isoform 1 (MCT1) in a wide range of tissues in adult male mice. The tissue distributions of Bsg and MCT1 in wild-type (WT) mice, the tissue distribution of MCT1 in Bsg gene knockout (Bsg-KO) mice, and the protein and mRNA levels of MCT1 in both genotypes were studied. Immunohistochemistry demonstrated that Bsg colocalized with MCT1 in the cerebrum, retina, skeletal and cardiac muscle, duodenal epithelium, hepatic sinusoid, proximal uriniferous tubules, Leydig cells, and efferent ductule epithelium in WT mice. Bsg was absent but MCT1 was present in Sertoli cells, cauda epididymis, myoepithelial cells and duct of the mandibular gland, surface epithelium of the stomach and bronchioles. In Bsg-KO mice, with the exception of Leydig cells, MCT1 immunostaining was greatly reduced in intensity and its distribution was altered in tissues that expressed both Bsg and MCT1 in WT mice. Levels of the protein and mRNA for MCT1 in these tissues did not change significantly in Bsg-KO mice. On the other hand, immunostaining patterns in cells in which Bsg was absent but MCT1 was present in WT mice remained unchanged in Bsg-KO mice. These observations suggest that Bsg is required for the proper localization of MCT1 in a wide range of cells but not in every cell type.

Photodynamic Therapy Synergizes with Irinotecan to Overcome Compensatory Mechanisms and Improve Treatment Outcomes in Pancreatic Cancer.

The ability of tumor cells to adapt to therapeutic regimens by activating alternative survival and growth pathways remains a major challenge in cancer therapy. Therefore, the most effective treatments will involve interactive strategies that target multiple nonoverlapping pathways while eliciting synergistic outcomes and minimizing systemic toxicities. Nanoliposomal irinotecan is approved by the FDA for gemcitabine-refractory metastatic pancreatic cancer. However, the full potential of irinotecan treatment is hindered by several cancer cell survival mechanisms, including ATP-binding cassette G2 (ABCG2) transporter-mediated irinotecan efflux from cells. Here, we demonstrate that benzoporphyrin derivative-based photodynamic therapy (PDT), a photochemical cytotoxic modality that activates the apoptotic pathway, reduced ABCG2 expression to increase intracellular irinotecan levels in pancreatic cancer. Moreover, we show that PDT inhibited survivin expression. Although PDT potentiated irinotecan treatment, we also demonstrate that irinotecan reduced the tumoral expression of monocarboxylate transporter 4, which was upregulated by PDT. Notably, using orthotopic xenograft models, we demonstrate that combination of single low-dose PDT and a subclinical dose of nanoliposomal irinotecan synergistically inhibited tumor growth by 70% for 3 weeks compared with 25% reduction after either monotherapies. Our findings offer new opportunities for the clinical translation of PDT and irinotecan combination therapy for effective pancreatic cancer treatment.

Androgen Receptor Involvement in Rat Amelogenesis: An Additional Way for Endocrine-Disrupting Chemicals to Affect Enamel Synthesis.

Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERß remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.

Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).

Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nalecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.