Mannitol Transport Research Articles

The transport of mannitol in Sinorhizobium meliloti is carried out by a broad-substrate polyol transporter SmoEFGK and is affected by the ability to transport and metabolize fructose.

The smo locus (sorbitol mannitol oxidation) is found on the chromosome of S. meliloti's tripartite genome. Mutations at the smo locus reduce or abolish the ability of the bacterium to grow on several carbon sources, including sorbitol, mannitol, galactitol, d-arabitol and maltitol. The contribution of the smo locus to the metabolism of these compounds has not been previously investigated. Genetic complementation of mutant strains revealed that smoS is responsible for growth on sorbitol and galactitol, while mtlK restores growth on mannitol and d-arabitol. Dehydrogenase assays demonstrate that SmoS and MtlK are NAD+-dependent dehydrogenases catalysing the oxidation of their specific substrates. Transport experiments using a radiolabeled substrate indicate that sorbitol, mannitol and d-arabitol are primarily transported into the cell by the ABC transporter encoded by smoEFGK. Additionally, it was found that a mutation in either frcK, which is found in an operon that encodes the fructose ABC transporter, or a mutation in frk, which encodes fructose kinase, leads to the induction of mannitol transport.

Silencing of a mannitol transport gene in Phelipanche aegyptiaca by the tobacco rattle virus system reduces the parasite germination on the host root

ABSTRACT Root parasitic weed Phelipanche aegyptiaca is an obligate plant parasite that causes severe damage to host crops. Agriculture crops mainly belong to the Brassicaceae, Leguminosae, Cruciferae, and Solanaceae plant families affected by this parasitic weed, leading to the devastating loss of crop yield and economic growth. This root-specific parasitic plant is not able to complete its life cycle without a suitable host and is dependent on the host plant for nutrient uptake and germination. Therefore, selected parasitic genes of P. aegyptiaca which were known to be upregulated upon interaction with the host were chosen. These genes are essential for parasitism, and reduced activity of these genes could affect host-parasitic interaction and provide resistance to the host against these parasitic weeds. To check and examine the role of these parasitic genes which can affect the development of host resistance, we silenced selected genes in the P. aegyptiaca using the tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS) method. Our results demonstrated that the total number of P. aegyptiaca parasite tubercles attached to the root of the host plant Nicotiana benthamiana was substantially decreased in all the silenced plants. However, silencing of the P. aegyptiaca MNT1 gene which encodes the mannitol transporter showed a significantly reduced number of germinated shoots and tubercles. Thus, our study indicates that the mannitol transport gene of P. aegyptiaca plays a crucial role in parasitic germination, and silencing of the PaMNT1 gene abolishes the germination of parasites on the host roots.

P-glycoprotein-mediated transport in a mucus-supplemented Caco-2 cell model in the presence of different surfactants

The aim of the present study was to investigate if mucus applied to Caco-2 cell monolayers protects cells from high concentrations of surfactants, while still allowing for an identification of the surfactant’s inhibitory effects on P-glycoprotein (P-gp). Two types of porcine mucin and six surfactants (Polysorbate 20 (PS20) and 80 (PS80), Kolliphor EL (Kol. EL) and RH40 (Kol. RH40), Labrafil M 2125 CS (L.fil) and Labrasol (L.sol)) were applied to Caco-2 cells, and TEER, paracellular transport and P-gp mediated digoxin transport was measured. The results showed that 15% porcine mucin type II was incompatible with Caco-2 cell monolayer integrity, resulting in a dramatic drop in monolayer TEER and increased mannitol transport. In contrast, mucin type III was compatible with Caco-2 cell monolayers in the concentration range of 2.5–15% without substantially disturbing barrier properties. The highest concentration of mucin type III impaired the ability of all six surfactants to decrease P-gp mediated digoxin transport. Subsequently lowering the mucin concentration to 5% facilitated adequate protection of cells and enabled e.g., 5% PS20 to inhibit P-gp mediated digoxin transport. Overall, the present work is useful for early-stage permeability investigations on how mucus affects P-gp mediated transport in the presence of formulation excipients.

Regulation of Mannitol Metabolism in Enterococcus faecalis and Association with parEF0409 Toxin-Antitoxin Locus Function.

The parEF0409 type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address the possibility that parEF0409 function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons: the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon harbors a gene similar to mtlR, the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for the regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual-operon organization of mannitol utilization genes is uncommon in enterococci and that the association with a toxin-antitoxin system is unique to Enterococcus faecalis. Finally, we consider possible links between parEF0409 function and mannitol utilization. IMPORTANCE Enterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility, in particular its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remain undetermined. Here, we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of the association with toxin-antitoxin systems.

Regulation of the Mannitol Locus by MtlR in Vibrio cholerae

The intake of mannitol, a sugar produced by plants and algae, by Vibrio cholerae increases biofilm formation and thereby persistence of the pathogenic bacterium. The process of mannitol uptake is mediated by the mannitol phosphotransferase system (PTS). Within this system, the protein MtlA is directly responsible for the intracellular transport of mannitol, and is encoded by the mtlA gene located within a group of genes (mtlA, D, and R) on the mannitol (mtl) locus. While it is known that protein MtlR represses the expression of mtlA at the transcriptional level, the mechanism of this repression is unknown. The aim of this project is to better understand the dynamics between MtlR and other protein regulators of the mtl locus, such as the CRP activator protein, to gain insight into the mechanism of MtlR regulation of mtlA expression. This was accomplished through assessment of MtlR and CRP levels across V. cholerae via Western Blots, as well as through measuring mtlA expression in the absence of CRP and/or MtlR via LacZ assays. Previous work has shown that MtlR levels fluctuate depending on available carbon source, but we found CRP levels to be relatively unaffected. We also found that while deletion of the mtlR gene increases mtlA expression, this is not observed when the crp gene is also deleted, suggesting that MtlR repression is secondary to CRP activation of mtlA expression. This work helps shed light on possible mechanisms by which MtlR might be negatively regulating the expression of mtlA and overall intracellular mannitol intake by V. cholerae.Support or Funding InformationNational Institutes of Health; The Camille and Henry Dreyfus Foundation

Structural insight into glucose repression of the mannitol operon

Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within the Enterobacteriaceae family.

Transcription of cis Antisense Small RNA MtlS in Vibrio cholerae Is Regulated by Transcription of Its Target Gene, mtlA.

Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA's target gene.IMPORTANCEVibrio cholerae is a bacterial pathogen that relies on genetic tools, such as regulatory RNAs, to adapt to changing extracellular conditions. While many studies have focused on how these regulatory RNAs function, fewer have focused on how they are themselves modulated. V. cholerae expresses the noncoding RNA MtlS, which can regulate mannitol transport and use, and here we demonstrate that MtlS levels are controlled by the level of transcription occurring in the antisense direction. Our findings provide a model of regulation describing how bacteria like V. cholerae can modulate the levels of an important regulatory RNA. Our work contributes to knowledge of how bacteria deploy regulatory RNAs as an adaptive mechanism to buffer against environmental flux.

The impact of chlorophyllin on deoxynivalenol transport across jejunum mucosa explants obtained from adult pigs

Regardless of the efforts put into preventing or reducing fungal growth, extensive mycotoxin contamination has been reported in animal feeds. In the case of pigs, one of the mycotoxins of major concern is deoxynivalenol (DON). The use of adsorbents as feed additives represents one of the strategies to control mycotoxins’ contamination in feedstuff. Therefore, the aim of the study was to verify the ability of chlorophyllin (CHL) to reduce the absorption rate of DON in swine mucosa explants. Intestine was obtained from routinely slaughtered adult pigs. The mucosa explants were studied by means of Ussing chamber technique. The effect of DON (10 and 30 μg/ml) on mucosa viability and permeability and CHL (100 μg/ml) impact on DON (30 μg/ml) absorption was verified. The results revealed that mucosa explants isolated from adult animals remained unaffected for 90 min in the presence of DON in the lower concentration (10 μg/ml). Mycotoxin in the higher dose (30 μg/ml) increased mucosa permeability (decreased transepithelial electrical resistance value) and enhanced paracellular transport of lucifer yellow and mannitol but did not affect lactate dehydrogenase leakage. The introduction of CHL neither diminished the absorption rate of DON across swine mucosa explants nor prevented the toxic effects of DON on intestine. In conclusion, the results confirm the negative effect of DON on pig jejunum mucosa. However, the toxic effect of DON was observed only when it was used in relatively high doses. A promising adsorbent agent, CHL, failed to reduce the intensity of DON transport across intestine under in vitro conditions.

Buccal delivery of small molecules – Impact of levulinic acid, oleic acid, sodium dodecyl sulfate and hypotonicity on ex vivo permeability and spatial distribution in mucosa

Studies of drug permeability rate and localization in buccal mucosa are essential to gain new knowledge of means such as chemical enhancers or osmolality to enhance buccal drug transport in the development of new buccal drug products. The transport of caffeine, diazepam and mannitol across porcine buccal mucosa was studied in modified Ussing chambers with a hypotonic donor solution, in the presence of levulinic acid (LA), oleic acid (OA), propylene glycol (PG) as well as sodium dodecyl sulfate (SDS). Subsequently, matrix-assisted laser desorption ionization – mass spectrometry imaging (MALDI-MSI) was applied to image the spatial distribution of caffeine, mannitol and SDS in cross-sections of porcine buccal mucosa. The results revealed that none of the permeation enhancing strategies improved the permeability of caffeine or diazepam, despite impact on the tissue integrity by OA and SDS, as seen by an increased permeability of mannitol. Further studies are needed with OA since PG solvent may have concealed the possible impact of OA. SDS decreased the permeability of caffeine and diazepam, a decrease which can be explained by micellar lipid extraction and encapsulation in micelles. MALDI-MSI showed that SDS permeated into approximately one-third of the epithelium, and it therefore appears that the main permeability barrier for mannitol is located in the outer epithelium. MALDI-MSI was shown to be a useful method for imaging spatial distribution of drugs and permeations enhancers in buccal mucosa.

The influence of fibroblast on the arachnoid leptomeningeal cells in vitro

ObjectiveFibroblast is pervasive in the setting of injury. Its invasion into the arachnoid tissue causes scarring, cortical adhesion of the brain, and obstruction of cerebrospinal fluid outflow. The purpose of this study is to determine the phenotypic and physiologic effects of fibroblasts on arachnoid in culture. MethodsWe studied the effects of fibroblast on the arachnoid cell growth, motility, phenotypic changes, and transport properties. Immortalized rat (Rattus norvegicus, Sprague Dawley breed) arachnoid cells were grown with fibroblast on opposite sides of polyethylene membranes or co-cultured in plastic wells. Arachnoid cell growth rate and DNA content, morphology, transport physiology, and extracellular matriceal content were determined in the presence of normal and irradiated fibroblast cells. ResultsWhen arachnoid cells were grown in the presence of fibroblasts, mannitol permeability increased and transepithelial electrical resistance (TEER) decreased. Arachnoid cell growth rate also significantly decreased. When arachnoid cells were grown in close proximity (i.e. on the same monolayer) with fibroblasts, the arachnoid cells were overrun by day 2, yet when physically separated, no significant change was seen in growth. Apoptosis increased markedly in arachnoid cultures in the presence of fibroblast. Fibroblast caused arachnoid cell to exhibit avoidance behavior, and irradiated fibroblast induced arachnoidal cells to move faster and exhibited greater directional changes. Subcellular glycosaminoglycan (GAG) content was significantly altered by fibroblast. InterpretationFibroblasts influence arachnoid cell’s mannitol transport likely via soluble factors. While the arachnoid cells did not change morphologically, cell growth was influenced. Over time, the cells had profound changes in transport and motility. The immortalized arachnoid cell/fibroblast culture system provides a unique model mimicking the pathologic event of leptomeningeal scarring.

Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells

1. Effect of IL-6, a pro-inflammatory cytokine, on efflux transport of rebamipide, an antiulcer drug, was investigated in Caco-2 cells.2. Rebamipide had a greater basal-to-apical than apical-to-basal transport rate. Efflux transport of rebamipide was inhibited by cyclosporine A, a P-gp inhibitor, and probenecid, which is a general MRP inhibitor, but not by Ko143, a BCRP inhibitor.3. By the addition of IL-6, mannitol transport was slightly increased in a concentration-dependent manner in both directions of absorption and efflux. The addition of IL-6 did not change efflux transport of rebamipide even though efflux transport of digoxin, a typical substrate of P-gp, was significantly decreased by the addition of IL-6, indicating decrease of the function of P-gp.4. Therefore, it was suggested that increase of MRP(s)-mediated transport compensates for the decrease of P-gp mediated transport of rebamipide. These findings suggested that rebamipide absorption is unlikely to be changed in IBD patients.

Characterization of cornified oral mucosa for iontophoretically enhanced delivery of chlorhexidine

Topical administration of chlorhexidine for periodontal disease can provide advantages over systemic delivery, but is limited by the permeability of the cornified oral mucosal tissue. In the present study, passive and iontophoretic transport of tetraethylammonium, salicylate, mannitol, dexamethasone, fluoride, and chlorhexidine across bovine palate was investigated to (a) determine the intrinsic barrier properties of bovine palate for its eventual use as a model of human cornified oral mucosa, (b) examine the feasibility of iontophoretically enhanced transport of chlorhexidine into and across bovine palate, and (c) identify the transport mechanisms involved in iontophoretic transport across the palate. The histology study suggests that bovine and human palates have similar cornified epithelium structures; bovine palate could be a model tissue of human hard palate for drug delivery studies. Transport study of tetraethylammonium, salicylate, and mannitol suggests that bovine palate was net negatively charged and the cornified epithelial layer was the rate-determining barrier. The direct-field effect (electrophoresis) was shown to be the dominant flux-enhancing mechanism in iontophoretic transport of ionic compounds. Electroosmosis also contributed to the iontophoretic transport of both neutral and ionic permeants. Anodal iontophoresis enhanced the delivery of chlorhexidine into and across the palate, reduced the transport lag time, and provided tissue concentration above the drug minimum inhibitory concentration, and therefore could be a promising method to assist in the treatment of periodontal disease.

Ex Vivo Correlation of the Permeability of Metoprolol Across Human and Porcine Buccal Mucosa

The pH partition theory proposes a correlation between fraction of unionized drug substance and permeability. The aim of this study was to compare the permeability of metoprolol and mannitol in ex vivo human and porcine buccal mucosa models at varying pH to validate whether the porcine permeability model is predictive for human buccal absorption. Human (n = 9-10) and porcine (n = 6-7) buccal mucosa were mounted in a modified Ussing chamber, and the kinetics of metoprolol and mannitol transport was assessed for a period of 5.5 h with the pH values of donor medium set at 7.4, 8.5, and 9.0. In addition, hematoxylin-eosin and Alcian blue-van Gieson were used as tissue stains to evaluate the histology and the presence of acidic polysaccharides (e.g., mucins), respectively. The permeability of metoprolol was decreased in human buccal mucosa by almost twofold when compared with porcine buccal mucosa with a positive correlation (r(2) = 0.96) between the permeability assessed in porcine and human buccal mucosa. There was no change in the degree of either epithelial swelling or desquamation when treating with the pH 9.0 donor medium for 5.5 h. These data suggest that buccal mucosa from pigs can be used to predict human buccal absorption.

Characterization of the respiration‐induced yeast mitochondrial permeability transition pore

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity.

Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae

ObjectiveThe complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype. MethodsThe crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl. ResultsIn this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. ConclusionThe five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.

Poly(amido amine) dendrimers as absorption enhancers for oral delivery of camptothecin

Oral delivery of camptothecin has a treatment advantage but is limited by low bioavailability and gastrointestinal toxicity. Poly(amido amine) or PAMAM dendrimers have shown promise as intestinal penetration enhancers, drug solubilizers and drug carriers for oral delivery in vitro and in situ. There have been very limited studies in vivo to evaluate PAMAM dendrimers for oral drug delivery. In this study, camptothecin (5 mg/kg) was formulated and co-delivered with cationic, amine-terminated PAMAM dendrimer generation 4.0 (G4.0) (100 and 300 mg/kg) and anionic, carboxylate-terminated PAMAM generation 3.5 (G3.5) (300 and 1000 mg/kg) in CD-1 mice. Camptothecin associated to a higher extent with G4.0 than G3.5 in the formulation, attributed to an electrostatic interaction on the surface of G4.0. Both PAMAM G4.0 and G3.5 increased camptothecin solubilization in simulated gastric fluid and caused a 2-3 fold increase in oral absorption of camptothecin when delivered at 2 h. PAMAM G4.0 and G3.5 did not increase mannitol transport suggesting that the oral absorption of camptothecin was not due to tight junction modulation. Histologic observations of the epithelial layer of small intestinal segments of the gastrointestinal tract (GIT) at 4 h post dosing supported no evidence of toxicity at the evaluated doses of PAMAM dendrimers. This study demonstrates that both cationic (G.4) and anionic (G3.5) PAMAM dendrimers were effective in enhancing the oral absorption of camptothecin. Results suggest that drug inclusion in PAMAM interior controlled solubilization in simulated gastric and intestinal fluids, and increased oral bioavailability.

Mannitol and the Mannitol-Specific Enzyme IIB Subunit Activate Vibrio cholerae Biofilm Formation

Vibrio cholerae is a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera. V. cholerae use a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport by V. cholerae requires the mannitol-specific EII (EII(Mtl)), which is expressed only in the presence of mannitol. Here we show that mannitol activates V. cholerae biofilm formation and transcription of the vps biofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EII(Mtl) is sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability of V. cholerae to respond to environmental mannitol by forming a biofilm may play an important role in habitat selection.

Evidence for insulin-mediated control of AQP9 expression in human placenta

The AQP9 gene contains a negative insulin response element, suggesting that it may be modulated by insulin. Previously, we reported AQP9 overexpression in preeclamptic placentas but a lack of functionality of AQP9 in water and mannitol transport. We also observed high serum levels of insulin and TNF-α in preeclamptic women. ObjectiveTo evaluate whether AQP9 expression is regulated by insulin in the human placenta, and whether the dysregulation of AQP9 observed in preeclamptic placentas may be related to the inability to respond to insulin stimuli. MethodsExplants from normal and preeclamptic placentas were cultured at different concentrations of insulin. Treatment with TNF-α was used to induce phosphorylation of insulin receptor substrate (IRS), which may desensitize insulin action. AQP9 molecular expression and water uptake was determined. ResultsInsulin decreased the molecular expression of AQP9 exclusively in explants from normal placentas in a concentration-dependent manner. Treatment with TNF-α previous to insulin addition prevented these changes. Moreover, insulin treatment did not modify water uptake neither its sensitivity to HgCl2. ConclusionAQP9 water permeability seems to be independent of its molecular expression, strongly suggesting that AQP9 might not have a key role in water transport in human placenta. We also propose another mechanism of down-regulation of AQP9 molecular expression mediated by insulin in a concentration-dependent manner in human placenta and provide new evidence that in preeclamptic placentas the mechanisms of insulin signaling may be altered, producing an overexpression of AQP9 that does not correlate with an increase in its functionality.

Mannitol Transport and Mannitol Dehydrogenase Activities are Coordinated in Olea europaea Under Salt and Osmotic Stresses

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

Characterization of the mannitol catabolic operon of Corynebacterium glutamicum

Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5'-TCTAACA-3' being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene.