Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth ≈8 mM but with Vmax for generation time ≈1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme IIGlc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG+ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax ≈7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme IIMan (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl α-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Transport of sugars by the fat body of the silkworm, Bombyx mori

The transport of glucose and α-methyl glucoside into the fat body of the silkworm, Bombyx mori L., has been studied. Glucose is transported into the tissue by a mechanism similar to facilitated diffusion and α-methyl glucoside by a diffusion process. The uptake of these sugars is neither energy dependent nor coupled to a phosphotransferase system.

Accumulation of glycine in the fat body of the silkworm, bombyx mori L. [31]

INVESTIGATIONS of intestinal transport of amino-acids in the locust1,2 and silkworm3,4 have shown no evidence for active accumulation in a transport from the insect gut of amino-acids. When glycine-2-14C was administered in vivo to fifth instar larvae of the silkworm, 96 per cent of the radioactivity was incorporated into various tissues within 1 h whereas in vitro only 19 per cent of the activity was transported by the mid-gut of silkworm (unpublished work). These results suggested that continued absorption of glycine by the intestine could be aided by a facilitated diffusion mechanism in which amino-acids are rapidly removed from the site of absorption either by accumulation into other tissues or by degradation. Although the insect fat body has been assigned both accumulatory and dissimilatory roles5, the mechanism of accumulation of amino-acids has not been investigated. Our present experiments show that the silkworm fat body possesses an efficient mechanism for accumulating glycine and that both the accumulation and the release of glycine are metabolically controlled.

Facilitative glucose transporter Glut1 is actively excluded from rod outer segments.

Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.

Glucosamine resistance in the yeast, Saccharomyces cerivisae [sic]

By using glucosamine resistant mutants of Saccharomyces ceriv~sa~ an attempt was made to discover the mechanisms which cause glucose repression and/or the Crabtree effect. The strains used are 4B2, GR6, lOP3r, GR8l and GRI08. 4B2 is a wild type yeast while the others are its mutants. To characterize the biochemical reactions which made these mutants resistant to glucosamine poisoning the following experiments were done~ 1. growth and respiration; 2. transport of sugars; 3. effect of inorganic phosphate (Pi): 4. Hexokinase; 5. In yivo phosphorylation. From the above experiments the following conclusions may be drawn: (i) GR6 and lOP3r have normal respiratory and fermentative pathways. These mutants are resistant to glucosamine poisoning due to a slow rate of sugar transport which is due to change in the cell membrane. (ii) GR8l has a normal respiratory pathway. The slow growth on fermentable carbon sourCEE indicates that in GR8l the lesion is in or associated with the glycolytic pathway. The lower rate of sugar transport may be due to a change in energy metabolism. The invivo phosphorylation rate indicates that in GR81 facilitated diffusion is the dominant transport mechanism. (iii) GR108 msa normal glycolytic pathway but the respiratory pathway is abnormal. The slow rate of sugar transport is due to a change in energy metabolism. The lower percentage of in vivo phosphorylation is probably due to a lowered availability of ATP because of the mitochondrial lesion. In all mutants resistance to glucosamine poisoning is due to a lower rate of utilization of ATP. which is caused by various mechanisms (see above), making less ADP available for phosphorylation via ATP synthase which utilizes inorganic phosphate. Because of the lower utilization of Pi, the concentration of intra-mitochondrial Pi does not go down thus protecting mutants from glucosamine poisoning.

Mecanismos de absorção de aminoácidos e oligopeptídios. Controle e implicações na dietoterapia humana

The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion) or active (Na + or H + co-transporters) pathways. Active Na +-dependent system occurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H + co-transporter). Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na + dependency whereas the later is active H +-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H +-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.

Analysis of fluoride concentration in mother's milk substitutes

The aim of the present study was to determine the fluoride concentration in some brands of mother's milk substitutes and evaluate the possibility of developing dental fluorosis by consuming these products. The products, all powdered, were divided into 3 groups: infant formulae (group I, n = 7), milk-based (group M, n = 8) and soy-based (group 8, n = 3). Samples from 3 cans of different batches of each brand were reconstituted in deionized water and analyzed using the specific electrode method, after hexamethyldisiloxane (HMDS) facilitated diffusion. The fluoride content (mg F/L) of the products ranged from 0.044 to 0.326 (I), 0.014 to 0.045 (M) and 0.253 to 0.702 (S). There was significant difference in the fluoride content of cans from distinct batches (p < 0.05) in most of the brands. The reconstitution of all products in water with optimal fluoride concentration for consumption during the mineralization phase of the primary teeth could result in daily fluoride intake above 0.07 mg F/kg body weight/day. Therefore, the consumption of these products, especially when reconstituted with optimally fluoridated water, could increase the risk of developing dental fluorosis.

Fluoride concentrations in industrialized beverages consumed by children in the City of Bauru, Brazil

The increasing consumption of juices, soft drinks and teas among children has increased significantly fluoride ingestion at the age range of risk for development of dental fluorosis. Objective: The purpose of this study was to evaluate fluoride concentrations in some brands of industrialized beverages consumed by children in the city of Bauru, SP, Brazil. Material and Methods: 98 brands of beverages were analyzed, divided into 3 lots, comprising 36, 32 and 30 brands, respectively, for the first, second and third lots. Fluoride concentrations were determined by HMDS-facilitated diffusion, using a fluoride ion-specific electrode (Orion 9409). Results: Fluoride concentrations ranged between 0.04 and 1.76 μg F/mL. It was observed a wide variation in fluoride concentrations among the different brands, as well as the different lots of the same brand. There was no information on fluoride concentrations on the labels of any product. Conclusions: Some of the products analyzed could contribute significantly to the total fluoride intake and, thus, be important risk factors for development of dental fluorosis, which indicates the need of controlling the production of these beverages with respect to fluoride concentration.

Evaluation of some properties of fermented milk beverages that affect the demineralization of dental enamel

The aim of this in vitro study was to evaluate the erosive capacity of fermented milk beverages, as well as some of their properties that affect the demineralization of dental enamel (pH, buffering capacity, fluoride, calcium and phosphorus contents). Three different batches of 6 commercial brands of fermented milk beverages were analyzed. pH evaluation was accomplished using a potentiometer. The buffering capacity was measured by adding 1 mol L -1 NaOH. Fluoride concentration was assessed by an ion specific electrode after hexamethyldisiloxane-facilitated diffusion, and calcium and phosphorus concentrations were assessed by a colorimetric test using a spectrophotometer. Sixty specimens of bovine enamel were randomly assigned to 6 groups (n = 10). They were exposed to 4 cycles of demineralization in the fermented milk and remineralization in artificial saliva. Enamel mineral loss was determined by surface microhardness (%SMHC) and profilometric tests. The samples' pH ranged from 3.51 to 3.87; the buffering capacity ranged from 470.8 to 804.2 μl of 1 mol L -1 NaOH; the fluoride concentration ranged from 0.027 to 0.958 μgF/g; the calcium concentration ranged from 0.4788 to 0.8175 mgCa/g; and the phosphorus concentration ranged from 0.2662 to 0.5043 mgP/g. The %SMHC ranged from-41.0 to -29.4. The enamel wear ranged from 0.15 μm to 0.18 μm. In this in vitro study, the fermented milk beverages did not promote erosion of the dental enamel, but rather only a superficial mineral loss.

Análise da concentração de íon flúor em leite em pó

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito de dentifrícios com concentração reduzida de fluoreto suplementados com polifosfatos na incidência de lesões de cárie dentária e na ingestão de fluoreto: estudo clínico longitudinal em crianças

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Longitudinal evaluation of fluoride levels in nails of 18-30-month-old children that were using toothpastes with 500 and 1100 mu g F/g

ObjectivesThis study aimed to evaluate the fluoride concentration in the fingernails and toenails of children aged 18-30months during use of fluoride-containing toothpastes supplemented with calcium glycerophosphate (CaGP) or sodium trimetaphosphate (TMP).MethodsAccording to the toothpaste used, children (n=56) were randomly assigned into three groups: 500gF/g with 1% TMP, 500gF/g with 0.25% CaGP, and 1100gF/g. Fingernails and toenails were collected monthly over a period of 330days, from the beginning of toothpaste use. Fluoride concentration in the water consumed by the volunteers and fluoride intake from diet and toothpaste were also determined. Fluoride analyses were performed with the electrode after hexamethyldisiloxane-facilitated diffusion or by the direct method, according to the samples. Data passed normality and homoscedasticity tests and were analyzed by 2-way analysis of variance (anova) and 1-way anova followed by Student-Newman-Keuls test (P<0.05).ResultsFluoride levels in the fingernails and toenails as well as fluoride intake from toothpaste were similar for the groups treated with 500gF/g with 1% TMP and 500gF/g with 0.25% CaGP toothpastes, but significantly lower than the 1100 gF/g group (P<0.05). No significant differences were noted among the groups regarding fluoride intake from diet and that by water consumed by the volunteers (P>0.05).ConclusionThe results of the longitudinal study suggest that the level of fluoride present in nails was lower with the use of toothpastes with a low fluoride concentration.

Intraoral fluoride levels after use of conventional and high-fluoride dentifrices

This study aimed to evaluate saliva and plaque as indicators of intraoral fluoride (F) levels after the use of conventional and high-fluoride dentifrices.Subjects were randomly assigned to brush their teeth with conventional (1000 ppm F), high-fluoride (5000 ppm F), and placebo dentifrices (fluoride free) for 10 days, following a double-blind, crossover protocol. Saliva and plaque samples were collected on the morning of the 5(th) and 10th days, respectively at 1 and 12 h after brushing, and analyzed with an ion-selective electrode after HMDS-facilitated diffusion. Data were analyzed by two-way repeated measures ANOVA, Tukey's test and Spearman's correlation coefficient (p < 0.05).Plaque and salivary F levels were significantly increased after the use of conventional and high-fluoride dentifrices when compared to values obtained for placebo, except plaque 12 h after the use of conventional dentifrice. A positive and significant correlation was found between fluoride concentrations in plaque and saliva for both times of sample collection.Both indicators assessed were able to detect significant differences among treatments and between times after brushing. The use of a high-fluoride dentifrice is able to significantly increase intraoral fluoride levels throughout the day, being therefore a useful therapy for patients at high caries risk.A dentifrice with high fluoride concentration could be regarded as a useful therapy of F delivery for high caries-risk patients, since intraoral F levels were sustained throughout most of the day after using this formulation.

Fluoride concentrations of milk, infant formulae, and soy-based products commercially available in Brazil

To assess the fluoride (F) content in commercially available milk formulae in Brazil and to estimate the F intake in children from this source in the first year of life. Samples of cow's milk (n = 51), infant formulae (n = 15), powdered milk (n = 13), and soy-based products (n = 4) purchased in Araçatuba (Brazil) had their F content measured using an ion-specific electrode, after hexamethyldisiloxane-facilitated diffusion. Powdered milk and infant formulae were reconstituted with deionized water, while ready-to-drink products were analyzed without any dilution. Using average infant body masses and suggested volumes of formula consumption for infants 1-12 months of age, possible F ingestion per body mass was estimated. Data were analyzed by descriptive analysis. Mean F content ranged from 0.02 to 2.52 mg/L in all samples. None of the cow's milk provided F intake higher than 0.07 mg/kg. However, two infant formulae, one powdered milk, and one soy-milk led to a daily F intake above the suggested threshold for fluorosis when reconstituted with deionized water. Assuming reconstitution of products with tap water at 0.7 ppm F, two infant formulae, five powdered milk, and four soymilks led to daily F intake ranging from 0.108 to 0.851 mg/kg. The results suggest that the consumption of some brands of infant formulae, powdered milk, and soy-based milk in the first year of age could increase the risk of dental fluorosis, reinforcing the need for periodic surveillance of the F content of foods and beverages typically consumed by young children.

Zuckertransport und Regulation des Hexosestoffwechsels bei Oenococcus oeni

Das Milchsäurebakterium Oenococcus oeni, welches für den biologischen Säureabbau im Wein eingesetzt wird, verstoffwechselt Hexosen über den Phosphoketolaseweg. Dabei können beträchtliche Mengen Acetat entstehen. Die Ursachen dafür wurden untersucht, insbesondere der Fructosestoffwechsel. Außerdem wurde der Hexosetransport untersucht, über den bei O. oeni noch nichts bekannt war. Die Aufnahme von Hexosen in die Zelle erfolgt mit hoher Affinität (KM=10 µM) über einen Symport mit H+, aber mit sehr niedriger spezifischer Aktivität (Vmax=9 U / g TG). Zusätzlich werden Hexosen mit ausreichender Aktivität über (vermutlich erleichterte) Diffusion in die Zelle transportiert, allerdings nur bei hohen Hexosekonzentrationen. Es wurden Gene gefunden, die für ein Hexose- Phosphotransferasesystem kodieren, welches in O. oeni keine bedeutende Rolle beim Transport spielt, aber vermutlich eine regulative Funktion hat. Zur Bildung von Essigsäure tragen verschiedene Faktoren bei: Der Ethanolweg, der in der heterofermentativen Milchsäuregärung die Reoxidation von NAD(P)H bewerkstelligt, ist durch die niedrige spezifische Aktivität der Acetaldehyddehydrogenase limitiert. Diese Limitierung wird noch verstärkt, wenn die zellulären Gehalte von Coenzym A aufgrund von Pantothensäuremangel niedrig sind. O. oeni umgeht durch Bildung von Erythrit die Limitierung, und Acetylphosphat wird nicht zu Ethanol reduziert, sondern als Acetat ausgeschieden. Bei Cofermentation von Hexosen mit externen Elektronenakzeptoren, wie Fructose, Pyruvat oder Sauerstoff, werden letztere zur Reoxidation von NAD(P)H genutzt, und als Folge wird Acetat ausgeschieden. Der Fluss von Fructose in den Phosphoketolaseweg wird durch das Enzym Phosphoglucoseisomerase verhindert, wenn dieses durch 6-Phosphogluconat gehemmt wird. Als Konsequenz wird Fructose im Mannitweg reduziert, was die Bildung von Essigsäure im Phosphoketolaseweg fördert. Bei niedrigen Wachstums- und Stoffwechselraten, z.B. bei C-Limitierung, ist der Ethanolweg nicht limitierend für den Stoffwechsel, und Hexosen werden über heterofermentative Milchsäuregärung umgesetzt, ohne daß Acetat entsteht. Pyruvat kann gleichzeitig als Elektronenakzeptor und als Energiequelle dienen: O. oeni ist in der Lage, Pyruvat mittels Disproportionierung zu Lactat und Acetat+CO2 zu fermentieren, und dabei Energie zu konservieren (0,5 ATP / Pyruvat).