The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732T

Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732^T) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Nippostrongylus-Induced Intestinal Hypercontractility Requires IL-4 Receptor Alpha-Responsiveness by T Cells in Mice

Gut-dwelling helminthes induce potent IL-4 and IL-13 dominated type 2 T helper cell (T_H2) immune responses, with IL-13 production being essential for Nippostrongylus brasiliensis expulsion. This T_H2 response results in intestinal inflammation associated with local infiltration by T cells and macrophages. The resulting increased IL-4/IL-13 intestinal milieu drives goblet cell hyperplasia, alternative macrophage activation and smooth muscle cell hypercontraction. In this study we investigated how IL-4-promoted T cells contributed to the parasite induced effects in the intestine. This was achieved using pan T cell-specific IL-4 receptor alpha-deficient mice (iLck^creIL-4Rα^−/lox) and IL-4Rα-responsive control mice. Global IL-4Rα^−/− mice showed, as expected, impaired type 2 immunity to N. brasiliensis. Infected T cell-specific IL-4Rα-deficient mice showed comparable worm expulsion, goblet cell hyperplasia and IgE responses to control mice. However, impaired IL-4-promoted T_H2 cells in T cell-specific IL-4Rα deficient mice led to strikingly reduced IL-4 production by mesenteric lymph node CD4⁺ T cells and reduced intestinal IL-4 and IL-13 levels, compared to control mice. This reduced IL-4/IL-13 response was associated with an impaired IL-4/IL-13-mediated smooth muscle cell hypercontractility, similar to that seen in global IL-4Rα^−/− mice. These results demonstrate that IL-4-promoted T cell responses are not required for the resolution of a primary N. brasiliensis infection. However, they do contribute significantly to an important physiological manifestation of helminth infection; namely intestinal smooth muscle cell-driven hypercontractility.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Temperature Dependence of Voltage-gated H+ Currents in Human Neutrophils,Rat Alveolar Epithelial Cells,and Mammalian Phagocytes

H⁺ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42°C. Effects of temperature on gating kinetics were distinguished from effects on the H⁺ current amplitude. The activation and deactivation of H⁺ currents were both highly temperature sensitive, with a Q ₁₀ of 6–9 (activation energy, E _a, ≈ 30–38 kcal/mol), greater than for most other ion channels. The similarity of E _a for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H⁺ currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant (τ_act) had similarly high Q ₁₀. These results could be explained if H⁺ channels were composed of several subunits, each of which undergoes a single rate-determining gating transition. H⁺ current gating in all mammalian cells studied had similarly strong temperature dependences. The H⁺ conductance increased markedly with temperature, with Q ₁₀ ≥ 2 in whole-cell experiments. In excised patches where depletion would affect the measurement less, the Q ₁₀ was 2.8 at >20°C and 5.3 at <20°C. This temperature sensitivity is much greater than for most other ion channels and for H⁺ conduction in aqueous solution, but is in the range reported for H⁺ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H⁺ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large E _a of permeation intrinsically limits the conductance of this channel, and appears inconsistent with the channel being a water-filled pore. At physiological temperature, H⁺ channels provide mammalian cells with an enormous capacity for proton extrusion.     

Growth and Nitrogen Uptake Characteristics Reveal Outbreak Mechanism of the Opportunistic Macroalga Gracilaria tenuistipitata

Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources'' (NH₄ ⁺, NO₃ ⁻) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH₄ ⁺ than that with NO₃ ⁻ addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH₄ ⁺ and NO₃ ⁻ uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH₄ ⁺ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO₃ ⁻ uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters V_max = 37.2 µM g⁻¹ DM h⁻¹ and K_s = 61.5 µM. Further, based on the isotope ¹⁵N tracer method, we found that ¹⁵N from NH₄ ⁺ accumulated faster and reached an atom% twice than that of ¹⁵N from NO₃ ⁻, suggesting when both NH₄ ⁺ and NO₃ ⁻ were available, NH₄ ⁺ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

A plasmamembrane redox system and proton transport in isolated mesophyll cells

Potassium ferricyanide (K₃Fe[CN]₆) was added to aerated and stirred nonbuffered suspensions of mechanically isolated photosynthetically competent Asparagus sprengeri Regel mesophyll cells. Rates of Fe(CN)₆³⁻ reduction and H⁺ efflux were measured with or without illumination. On the addition of 1 millimolar Fe(CN)₆³⁻ to nonilluminated cell suspensions acidification of the medium indicated an H⁺ efflux of 1.54 nanomoles H⁺/10⁶ cells per minute. Simultaneous Fe(CN)₆³⁻ reduction occurred at a rate of 1.55 nanomoles Fe(CN)₆³⁻/10⁶ cells per minute. Illumination stimulated these rates 14 to 17 times and corresponding values were 26.1 nanomoles H⁺/10⁶ cells per minute and 22.9 nanomoles Fe(CN)₆³⁻/10⁶ cells per minute. These two processes appeared to be tightly coupled and were rapidly inhibited when illuminated suspensions were transferred to darkness or treated with 1 micromolar 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Addition of 0.1 millimolar diethylstilbestrol eliminated ATP dependent H⁺ efflux in illuminated or nonilluminated cells but had no influence on Fe(CN)₆³⁻ dependent H⁺ efflux. Recent reports indicate that a transmembrane redox system spans the plasma membrane of root cells and is coupled to the efflux of H⁺. The present report extends these observations to photosynthetically competent mesophyll cells. The results indicate a transport process independent of ATP driven H⁺ efflux which operates with a H⁺/e⁻ stoichiometry of one.     

Rag2-deficient IL-1 Receptor Antagonist-deficient Mice Are a Novel Colitis Model in Which Innate Lymphoid Cell-derived IL-17 Is Involved in the Pathogenesis

Il1rn^−/− mice spontaneously develop arthritis and aortitis by an autoimmune mechanism and also develop dermatitis by an autoinflammatory mechanism. Here, we show that Rag2^−/−Il1rn^−/− mice develop spontaneous colitis with high mortality, making a contrast to the suppression of arthritis in these mice. Enhanced IL-17A expression in group 3 innate lymphoid cells (ILC3s) was observed in the colon of Rag2^−/−Il1rn^−/− mice. IL-17A-deficiency prolonged the survival of Rag2^−/−Il1rn^−/− mice, suggesting a pathogenic role of this cytokine in the development of intestinal inflammation. Although IL-17A-producing T cells were increased in Il1rn^−/− mice, these mice did not develop colitis, because CD4⁺Foxp3⁺ regulatory T cell population was also expanded. Thus, excess IL-1 signaling and IL-1-induced IL-17A from ILC3s cause colitis in Rag2^−/−Il1rn^−/− mice in which Treg cells are absent. These observations suggest that the balance between IL-17A-producing cells and Treg cells is important to keep the immune homeostasis of the colon.     

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K_M, V_max, K_D, and k_obs values for different sugar substrates. K_D and k_obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H⁺ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H⁺ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s⁻¹) and for sugar translocation (2 s⁻¹ − 30 s⁻¹ for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.     

NO3 ?-induced pH Changes in Mammalian Cells : Evidence for an NO3?-H+ Cotransporter

The effect of NO₃ ⁻ on intracellular pH (pH_i) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO₃ ⁻ induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO₃ ⁻- induced pH change was not due to modulation of Na⁺/H⁺ exchange, since it was also observed in Na⁺/H⁺ antiport-deficient mutants. Though NO₃ ⁻ is known to inhibit vacuolar-type (V) H⁺-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A₁. NO₃ ⁻/HCO₃ ⁻ exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO₃ ⁻, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B.S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448– 11454). Accumulation of intracellular NO₃ ⁻, measured by the Greiss method after reduction to NO₂ ⁻, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO₃ ⁻ with H⁺ (or the equivalent counter-transport of NO₃ ⁻ for OH⁻). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO₃ ⁻ and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO₃ ⁻ and was accentuated by acidification of the extracellular space. The putative NO₃ ⁻-H⁺ cotransport was inhibited markedly by ethacrynic acid and by α-cyano-4-hydroxycinnamate, but only marginally by 4,4′-diisothiocyanostilbene-2,2′ disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO₃ ⁻-induced pH changes in mammalian cells may be related, though not identical, to the NO₃ ⁻-H⁺ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO₃ ⁻ with H⁺ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Functional Characterization of Peroxiredoxins from the Human Protozoan Parasite Giardia intestinalis

The microaerophilic protozoan parasite Giardia intestinalis, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O₂ and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O₂, NO and superoxide anion (O₂ ^−•) have been recently reported for Giardia, but it is yet unknown how the parasite copes with H₂O₂ and peroxynitrite (ONOO⁻). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H₂O₂ and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO⁻ rapidly (k = 4×10⁵ M⁻¹ s⁻¹ and 2×10⁵ M⁻¹ s⁻¹ at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O₂ for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

The Retinoic Acid-Metabolizing Enzyme Cyp26b1 Regulates CD4 T Cell Differentiation and Function

The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4⁺ T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 ^−/− mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iT_reg) cell- and T_H17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iT_reg and T_H17 cells. Transfer of naïve Cyp26b1 ^−/− CD4⁺ T cells into Rag1 ^−/− mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.     

Proton Pump Activity of Mitochondria-rich Cells : The Interpretation of External Proton-concentration Gradients

We have hypothesized that a major role of the apical H⁺-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl⁻ via an apical Cl⁻/HCO₃ ⁻-exchanger. The activity of the H⁺ pump was studied by monitoring mucosal [H⁺]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO₂, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H⁺. The [H⁺] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H⁺]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H⁺ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace''s equation. Model calculations predicted that with a physiological density of MR cells, the [H⁺] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H⁺]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H⁺]-gradient depended on the mucosal anion such as to indicate that base (HCO₃ ⁻) is excreted in exchange not only for Cl ⁻, but also for Br⁻ and I⁻, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

T Cell Hypo-Responsiveness against Leishmania major in MAP Kinase Phosphatase (MKP) 2 Deficient C57BL/6 Mice Does Not Alter the Healer Disease Phenotype

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished T_H1 response. By contrast, in the present study footpad infection of MKP-2^−/− mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2^+/+ mice. Analysis of immune responses following infection demonstrated a reduced T_H1 response in MKP-2^−/− mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4⁺ and CD8⁺ T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2^−/− mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2^+/+ mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall T_H1/T_H2 balance was unaltered in MKP-2^−/− compared with wild-type mice. Although non-stimulated MKP-2^−/− macrophages were more permissive to L. major growth than macrophages from MKP-2^+/+ mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2^−/− and MKP-2^+/+ macrophages. Consequently, in the absence of any switch in the T_H1/T_H2 balance in MKP-2^−/− mice, no significant change in disease phenotype was observed.