High Concentrations Of Galactose Research Articles

Fish oil prevents effect of high cholesterol diet on active intestinal transport of galactose.

We tested the hypothesis that diets containing fish oils prevent the effects of a high cholesterol diet on the morphology and nutrient uptake of the intestine. Isocaloric semisynthetic diets were supplemented with beef tallow or fish oil containing low or high amounts of cholesterol and were fed to growing female Wistar rats for 14 days, after which the in vitro jejunal and ileal uptake of glucose, galactose, long-chain fatty acids, and cholesterol was determined. Feeding cholesterol with beef tallow was associated with a 12% decrease in the jejunal mucosal surface area. Feeding fish oil decreased jejunal mucosal surface area by 24%, as compared with the beef tallow diet, but the reduction was increased to 42% when fish oil and cholesterol were fed together. Ileal surface area was unaffected by varying the major source of dietary lipid, or by adding cholesterol. Despite the effect of fish oil on the mucosal surface area, the jejunal and ileal uptake of saturated as well as unsaturated long-chain fatty acids and cholesterol was similar in the four diet groups. Cholesterol supplementation enhanced the jejunal uptake of high concentrations of galactose only when fed with beef tallow, i.e., feeding fish oil prevented the enhancing effect of cholesterol on galactose uptake observed when beef tallow was fed. Thus, (i) a fish oil diet prevents the enhancing effect of cholesterol on jejunal active transport of galactose, an effect not explained by the reduction in jejunal mucosal surface area observed with the fish oil diet; (ii) these dietary manipulations result in a clear dissociation of the morphological from the transport adaptation of the intestine; and (iii) substitution of fish oil for beef tallow as the major source of lipid in the diet prevents the influence of cholesterol on the active intestinal transport of galactose.

Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc.

This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin. This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.

EFFECT OF CHLORHEXIDINE GLUCONATE ON THE ADHERENCE OF CANDIDA SPECIES TO DENTURE ACRYLIC

The effect of pretreatment of denture acrylic with chlorhexidine gluconate on the subsequent adherence of Candida albicans GDH 2346 was measured in vitro. Adherence was significantly reduced by pretreatment with chlorhexidine; maximal inhibition was achieved by incubation at room temperature for 30 min in 2% chlorhexidine. Inhibition of adherence was greatest when the organisms were grown in conditions that enhanced adherence the most, i.e., growth to stationary phase in high concentrations of galactose and sucrose. Yeasts grown in high concentrations of galactose, which were the most adherent to acrylic, were also the most sensitive to the fungicidal action of chlorhexidine gluconate, whereas those grown in a low concentration of glucose were the least adherent and also the most resistant. Adherence to acrylic of seven strains of C. albicans isolated from active infections (I strains) and grown in medium containing 500 mM sucrose was significantly higher than that of four strains obtained from asymptomatic carriers (C strains). A spectrum of adherence values was obtained when various yeasts other than C. albicans were tested.

Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures

Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal organisms eventually took over the culture with all four strains examined. Gal cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-beta-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal-Gal cultures were repeatedly transferred in milk, the Gal cells became the dominant cell type. The Gal phenotype of stock cultures probably reflects their prolonged maintenance in milk.

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.

Solubility and selective crystallization of lactose from solutions of its hydrolysis products glucose and galactose

A high degree of conversion is desired when lactose is hydrolyzed to glucose and galactose. This produces, however, a high concentration of galactose, which is inhibitory for the enzyme catalyst (beta-galactosidase). The inhibition can be reduced by limiting the conversion per pass over the enzyme (e.g. to ca. 50%), separating unconverted lactose from the reactor effluent, and recycling it to the reactor inlet. (This allows the overall conversion to be raised to ca. 80-90%). The solubilities of lactose, glucose, and galactose have been determined at various temperatures and for sugar mixtures having different concentrations and degrees of hydrolysis. Various cooling crystallizations have defined convenient and simple processes for the selective separation of lactose from its hydrolysis products.

Hemagglutinating activity in extracts of mycelia from submerged mushroom cultures.

Extracts from mycelia of seven different mushrooms agglutinated erythrocytes of several species. More than one agglutinating factor was identified in the extracts of three different mycelia. Agglutination was partially inhibited nonspecifically by high concentrations of glucose, galactose, mannose, fucose, and rhamnose.

New typhoid fever vaccine a hit on international scene

The results of a recently completed field trial with a new live oral typhoid fever vaccine show promise that the disease, which is endemic in many parts of the world, eventually may be controlled. M. H. Wahdan, MD, and colleagues at the High Institute of Public Health, Alexandria, Egypt, the Pasteur Institute, France, and the Swiss Serum and Vaccine Institute, Bern, conducted a controlled trial in Alexandria with 32,388 first-grade schoolchildren. The results of the three-year study were published in the March issue of The Journal of Infectious Diseases . An attenuated mutant strain of Salmonella typhi was used as the immunogen in the study. Exposure to a high concentration of exogenous galactose (as occurs in the body) causes this strain to lyse within a day or two, but not before its cell wall lipopolysaccharides have provided the necessary antigenic stimuli. Although a number of S typhi strains exhibit this particular

Partial purification of galactinol synthase from leaves of Cucurbita pepo

An enzyme synthesizing galactinol, UDP-D-galactose:myo-inositol-1-α-D-galactosyl transferase (galactinol synthase), has been isolated and partially purified from mature leaves of Cucurbita pepo. The enzyme showed optimal activity between pH 7.5 and 8.0 and required Mn2+ and the presence throughout isolation, storage, and assay of a sulfhydryl protectant (β-mercaptoethanol). EDTA was completely inhibitory. From a range of metal ions only Mg2+ partially replaced Mn2+, while Co2+, Zn2+, Cu2+, and Ni2+ were inhibitory. The uridine nucleotides and UDP-glucose were from 40 to 80% inhibitory and probably constitute part of the in vivo control system. High concentrations of galactose, melibiose, and xylose were partially inhibitory. The enzyme appeared highly specific for myo-inositol and showed no ability for galactosyl transfer to any other naturally occurring sugar or sugar alcohol. Some reactivity was obtained with the isomeric scyllo-inositol but the product was not identified. A range of other sugar nucleotides were unreactive.

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium.

We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl beta-galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor IIIGlc of the PTS, lead to a low MglP activity, as measured by methyl beta-galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%-20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MGlP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MGlP synthesis can be relieved in two ways: by externally added cyclic adenosine 3',5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.

Biochemical studies on Bifidobacterium bifidum. V. Purification and properties of UDP-galactose 4-epimerase from Bifidobacterium bifidum.

UDP-galactose 4-epimerase (EC 5.1.3.2) was purified to a homogeneous state from Bifidobacterium bifidutn grown on a glucose medium. The molecular weight was estimated to be about 90,000. The purified enzyme was very stable and 60 % of its initial activity survived three months of storage at 4°C even at a low protein concentration (0.2 mg/ml). The optimum pH was 9.0, and the Km values for UDP-galactose and UDP-glucose were 5.4 × 10-4 M and 1.4×10 -3 M. UDP was a competitive inhibitor. The enzyme activity was stimulated by various sugar phosphates, but was slightly inhibited by fructose 1,6-diphosphate (FDP). A high concentration of galactose or glucose, which had no effect by itself, inhibited the activity in combination with UMP. The inhibition by FDP was also enhanced by combination with UMP.

Purification, composition, and serological characterization of histoplasmin-H and M antigens.

To obtain purified H and M antigens suitable as primary standards in the serological diagnosis of histoplasmosis by agar gel double-diffusion tests, H and M reactive components of histoplasmin were fractionated by column chromatography by using Sephadex G-100, Sephadex G-200, and diethylaminoethyl cellulose. Six fractions from diethylaminoethyl cellulose were reactive in agar gel double-diffusion, complement fixation, and capillary precipitin tests. When examined by electrophoresis on acrylamide gel, one M antigen (fraction 2, molecular weight greater than 200,000) and two H antigens (fractions 5 and 6, molecular weight greater than 200,000) each gave essentially a single protein band. In agar gel double-diffusion and complement fixation tests with sera from patients with proven cases of histoplasmosis, blastomycosis, or coccidioidomycosis, these two fractions of H antigen and the one of M antigen reacted only with sera from proven or suspect cases of histoplasmosis and showed reactivity with those sera known to contain only the anti-H or anti-M antibody, respectively. Fraction 2 (M antigen) and fractions 5 and 6 (H antigens) had carbohydrate-to-protein ratios of 1.60, 0.77 and 0.78, respectively. Both antigens contained galactose, glucose, mannose, and hexosamine, with mannose being the predominant sugar. Fraction 2 was characterized by a high proline and glucose content, whereas fractions 5 and 6 contained higher concentrations of galactose, mannose, glycine, and alanine. Each of these products appeared to separate into two active fractions, one of a molecular weight greater than 200,000 and one of a molecular weight less than 35,000. The M antigen component of fraction 2 was still characterized by a high proline content, whereas the H antigen components of fractions 5 and 6 had a high content of glutamic acid, serine, glycine, and proline.

Polyol accumulation in galactosemic and diabetic rats: control by an aldose reductase inhibitor.

An orally active inhibitor of aldose reductase, 1,3-dioxo-1H-benz[de]-isoquinoline-2(3H)acetic acid (AY-22,284), prevented cataractous changes in cultured lenses exposed to high concentrations of galactose. When given orally, AY-22,284 markedly decreased the accumulation of polyols in the lenses and sciatic nerves of galactosemic rats and rats with streptozotocin-induced diabetes. In addition, treatment of galactosemic rats with AY-22,284 effectively suppressed the formation of cataracts.

On the Uptake of Materials by the Intact Liver. THE TRANSPORT AND NET REMOVAL OF GALACTOSE

D-galactose, a monosaccharide rapidly phosphorylated within liver cells, is irreversibly removed from the portal circulation. We have studied the kinetic relations between the hepatic cell entry process and the metabolic sequestration process, by means of the multiple indicator dilution technique. Labeled red blood cells (a vascular indicator), labeled sucrose (an extracellular reference), and labeled galactose were rapidly injected into the portal vein, and from rapidly sampled hepatic venous blood, normalized outflow-time patterns were secured. The labeled red cell curve rises to the highest and earliest peak, and decays rapidly; and that for labeled sucrose rises to a later and lower peak. Its extrapolated recovery is equivalent to that of the labeled red cells. At low blood galactose concentrations, the labeled galactose appears at the outflow with labeled sucrose, but is much reduced in magnitude, and exhibits a long tailing. Its outflow recovery is much reduced. At high blood galactose concentrations, the initial part of the profile increases towards that for labeled sucrose, the tailing becomes much larger in magnitude, and the outflow recovery becomes virtually complete. We have modeled the uptake of labeled galactose, and find two parts to the predicted outflow pattern, corresponding to our experimental observations; throughput material, which sweeps past the cell surface in the extracellular space; and returning material, which has entered the cells but escaped the sequestration process. Analysis of the data by use of this model provides estimates of both transmembrane fluxes and rates of sequestration. The capacity of the process subserving cell entry is found to be 40 times that for phosphorylation; and, whereas the K(m) value for sequestration is less than 15 mg/100 ml, that for entry is approximately 500 mg/100 ml. Both processes are relatively stereospecific; the entry of the L-stereoisomer is very slow and it undergoes no significant amount of metabolic sequestration. The sequestration process produces a lobular intracellular concentration gradient; and this gradient, in turn, produces some uncertainty in the estimate of the true K(m) value for the sequestration process.

Transport Properties of the Galactose-binding Protein of Escherichia coli: SUBSTRATE-INDUCED CONFORMATIONAL CHANGE

Abstract The galactose-binding protein, a necessary component of a bacterical transport system, is shown by three independent methods to undergo a conformational change upon binding of substrate. 1. A Lineweaver-Burk plot of the binding data obtained by equilibrium dialysis at a protein concentration of 0.4 mg per ml shows heterogenous behavior. Extrapolation of the data at low galactose concentrations (10-8 to 3·10-7 m) yields an apparent Kdiss of 10-7 m and at higher galactose concentrations (3·10-7 to 10-5 m) an apparent Kdiss of 10-5 m. A Scatchard plot of the binding data extrapolated to high galactose concentrations indicates two binding sites per 36,000 molecular weight. 2. The presence of 10-4 m galactose during analytical polyacrylamide gel electrophoresis at pH 8.4 results in a changed electrophoretic mobility of the binding protein. This change is also observed in the presence of 10-4 m glucose and 10-4 m (d-glyceryl)-1-β-d-galactopyranoside substrates of the transport system. Methyl-1-O-β-d-galactopyranoside, methyl-1-thio-β-d-galactopyranoside, and isopropyl-1-thio-β-d-galactopyranoside at concentrations of 10-4 m do not cause the altered electrophoretic mobility. 3. The fluorescence of the protein is increased up to 13.5% in the presence of substrate between 310 to 350 nm when excited at 290 nm. The increase of fluorescence observed with galactose, glucose, and (d-glyceryl)-1-β-d-galactopyranoside occurs at half-maximal total sugar concentrations of 10-6, 10-6, and 10-5 m, respectively. The substrate-induced conformational change does not result in a change of the molecular weight of the protein as measured by high speed equilibrium centrifugation and sieve chromatography through Bio-Gel P-150. The conformational change of the protein cannot be observed by optical rotatory dispersion, circular dichroism, and infrared spectroscopy. These latter spectroscopic methods show, however, that the galactose-binding protein exists to a large extent in β conformation. Purified binding protein from a transport-negative mutant does not undergo the substrate-induced conformational changes measured by binding, electrophoretic mobility, and fluorescence.

Cooperative phenomena in the permeation of sugars through the lining epithelium of choroid plexus

The concentration of D-glucose alone or together with D-galactose, L-arabinose or D-fructose was measured enzymatically in new choroidal cerebrospinal fluid and in plasma, sampled simultaneously in rabbits. Plasma levels were systematically altered by the use of insulin or by the infusion of one or several sugars. The flow of glucose was approximately linear with plasma concentration to 14 m M while above 20 m M there was an interval of plasma concentrations in which there was a disproportionate increase in flow with increase in plasma level, suggesting increase in affinity of active sites or recruitment of new binding sites. For galactose, fructose and arabinose there was, with increasing plasma concentration, an inordinate increase in flow. Under the influence of high concentrations of galactose the behavior of glucose changed so that it was cooperative from the origin. The cooperative flow of fructose was greatly enhanced at levels of glucose at which recruitment of new glucose sites was greatest and cooperativity and flow fell off sharply at higher levels of glucose. The behavior and interactions are considered in terms of a mosaic of two carries, each a cooperative assembly, the state and thus the affinity for sugars being influenced by the local environment of sugars.

Response of the Mouse Lens to High Concentrations of Glucose or Galactose

In vitro studies show that the mouse lens exposed to elevated concentrations of glucose or galactose does not develop the changes which affect the rat lens under similar conditions. The young mouse lens (CFW strain) is resistant to the cataractogenic effect of a galactose diet or alloxan diabetes. This species difference appears to reside in the lens itself since both sugars can accumulate in the mouse lens to reachlevels comparable to those found in the rat lens exhibiting significant changes.

Effect of galactose toxicity on growth of the rat fetus and brain.

Extract: In pregnant albino rats fed a diet containing 40% galactose, sugar crossed the placenta and was found in the blood, urine, and amniotic fluid of the fetuses. Galactose-1-phosphate was also found in fetal brain tissue. The fetus of galactose-fed rats weighed 5 to 12% less than the fetus of control rats fed a diet of similar composition, but containing no galactose. Growth retardation was reversible. Fetuses of rats fed the galactose diet during the first half of pregnancy were less retarded than were those of rats fed galactose throughout pregnancy or during the second half of pregnancy only. Body weight of animals at 10 weeks of age did not differ significantly from that of control animals. In the galactosemic fetus, the reduction in brain weight was approximately equal to that in body weight. In galactosemic fetuses, the content of DNA in brain was less than that in control animals, indicating a reduction in cell number. In a group of preterm fetuses 18 days of gestation, total protein content in brain was also reduced when compared with that of controls, suggesting a reduction in cell size. Brains of galactosemic fetuses at term, however, contained normal amounts of protein. Offsprings of galactose-fed rats examined at three and ten weeks of age had smaller brains than offsprings of control animals, but total DNA content was normal, indicating normal cell number. These findings indicate that galactose toxicity is exhibited in the fetal rat by retardation in body growth. Brain growth is also affected, apparently as a result of interference with cell replication and, perhaps to some extent, with cell growth. Speculation: Although it is as yet unknown whether exposure of human fetuses to high concentrations of galactose affects growth of the brain, clinical observations suggest that it may do so. Mental performance of galactosemic children is at times subnormal despite early treatment with a galactose-free diet. Evidence is accumulating which suggests that mothers known to be heterozygous for the galactosemia gene should be treated with a diet low in lactose during pregnancy.

Role of lac Genes in Induction of β-Galactosidase Synthesis by Galactose

Strain BL1003, a lacO mutant, synthesizes beta-galactosidase constitutively at a low rate. The enzyme is further inducible by d-galactose to the same differential rate as is seen in the presence of an optimal concentration of thiomethylgalactoside. lacY Mutants derived from strain BL1003 are not inducible by galactose, although they synthesize beta-galactosidase at the low constitutive rate characteristic of the parent. Galactose is a weak inducer of beta-galactosidase synthesis in wild-type Escherichia coli K-12, but it is more effective when the wild type has been preinduced with isopropyl-beta-d-thiogalactoside. Nevertheless, the rise in the differential rate of synthesis in response to galactose in a preinduced wild-type culture is much lower than in strain BL1003. Thus, two factors are involved in the induction of strain BL1003 by galactose: the mutant operator and the constitutive permease. The operator has an altered sensitivity to the i product-galactose complex. The low constitutive level of permease enabled the cells, at the high concentrations of galactose used (5 x 10(-2)m), to maintain a sufficient internal concentration for further induction.

CORRECTION

In the Special Article, "Carbohydrate and Energy Metabolism in the Newborn" (Pediatrics, 39:582, 1967), Dr. John A. Davis is reported (p. 599) to have employed a 40% solution of galactose for the treatment of hypoglycemia. He writes: "This is not true, and indeed something I would not advocate. What I, in fact, said was that we use a 20% or 40% solution containing 5% galactose and 5% fructose with 10% glucose, (so that) the amount of galactose given is only marginally greater than the baby would get from being fed breast milk. Higher concentrations of fructose appear to produce lacticacidaemia; higher concentrations of glucose, thrombosis; and higher concentrations of galactose might produce a transient galactosaemia, with all the consequences thereof, especially in premature infants. I hope you will be able to print this correction in case readers might be tempted to follow incorrect precepts."