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Sample records for galactose

  1. Galactose metabolism by Streptococcus mutans.

    PubMed

    Abranches, Jacqueline; Chen, Yi-Ywan M; Burne, Robert A

    2004-10-01

    The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons. PMID:15466549

  2. Endogenous galactose formation in galactose-1-phosphate uridyltransferase deficiency.

    PubMed

    Schadewaldt, Peter; Kamalanathan, Loganathan; Hammen, Hans-Werner; Kotzka, Jorg; Wendel, Udo

    2014-12-01

    Patients with classical galactosaemia (galactose-1-phosphate uridyltransferase (GALT) deficiency) manifest clinical complications despite strict dietary galactose restriction. Therefore the significance of endogenous galactose production has been assessed. Previous in vivo studies primarily focused on patients homozygous for the most common genetic variant Q188R but little is known about other genetic variants. In the present study the endogenous galactose release in a group of non-Q188R homozygous galactosaemic patients (n = 17; 4-34 years) exhibiting comparably low residual GALT activity in red blood cells was investigated. Primed continuous infusion studies with D-[1-(13)C]galactose as substrate were conducted under post-absorptive conditions and in good metabolic control. The results demonstrate that all patients exhibiting residual GALT activity of <1.5% of control showed a comparable pathological pattern of increased endogenous galactose release irrespective of the underlying genetic variations. Possible implications of the findings towards a more differentiated dietary regimen in galactosaemia are discussed. PMID:25268296

  3. Genetics Home Reference: glucose-galactose malabsorption

    MedlinePlus

    ... mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects. Biochim Biophys Acta. 1999 Feb 24;1453( ... Accessibility FOIA Viewers & Players U.S. Department of Health & Human Services National Institutes of Health National Library of ...

  4. Lactose uptake driven by galactose efflux in Streptococcus thermophilus: Evidence for a galactose-lactose antiporter

    SciTech Connect

    Hutkins, R.W.; Ponne, C. )

    1991-04-01

    Galactose-nonfermenting (Gal{sup {minus}}) Streptococcus thermophilus TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal{sup {minus}} cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated ({sup 14}C)lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force ({Delta}p) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a {Delta}p of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in Gal{sup {minus}} S. thermophilus is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system.

  5. Glucose and galactose transport in Bifidobacterium bifidum DSM 20082.

    PubMed

    Krzewinski, F; Brassart, C; Gavini, F; Bouquelet, S

    1997-09-01

    Sugar uptake was measured with 3H-galactose and 14C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters KM and Vmax were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K+ ions. PMID:9236301

  6. Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

    PubMed

    Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su

    2016-07-10

    In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without

  7. 21 CFR 862.1310 - Galactose test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Galactose test system. 862.1310 Section 862.1310 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... hereditary disease galactosemia (a disorder of galactose metabolism) in infants. (b) Classification. Class I....

  8. Galactose oxidation using (13)C in healthy and galactosemic children.

    PubMed

    Resende-Campanholi, D R; Porta, G; Ferrioli, E; Pfrimer, K; Ciampo, L A Del; Junior, J S Camelo

    2015-03-01

    Galactosemia is an inborn error of galactose metabolism that occurs mainly as the outcome of galactose-1-phosphate uridyltransferase (GALT) deficiency. The ability to assess galactose oxidation following administration of a galactose-labeled isotope (1-(13)C-galactose) allows the determination of galactose metabolism in a practical manner. We aimed to assess the level of galactose oxidation in both healthy and galactosemic Brazilian children. Twenty-one healthy children and seven children with galactosemia ranging from 1 to 7 years of age were studied. A breath test was used to quantitate (13)CO2 enrichment in exhaled air before and at 30, 60, and 120 min after the oral administration of 7 mg/kg of an aqueous solution of 1-(13)C-galactose to all children. The molar ratios of (13)CO2 and (12)CO2 were quantified by the mass/charge ratio (m/z) of stable isotopes in each air sample by gas-isotope-ratio mass spectrometry. In sick children, the cumulative percentage of (13)C from labeled galactose (CUMPCD) in the exhaled air ranged from 0.03% at 30 min to 1.67% at 120 min. In contrast, healthy subjects showed a much broader range in CUMPCD, with values from 0.4% at 30 min to 5.58% at 120 min. The study found a significant difference in galactose oxidation between children with and without galactosemia, demonstrating that the breath test is useful in discriminating children with GALT deficiencies. PMID:25608239

  9. Galactose effects on enterocyte differentiation in the mouse jejunum.

    PubMed

    Smith, M W; James, P S; Peacock, M A

    1991-07-10

    The present work investigates the ability of galactose to affect enterocyte differentiation during normal development in vivo. Energy intake has also been varied to take account of the fact that galactose is poorly metabolized in mice. Brush-border lactase, alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N, alkaline phosphatase and microvillus length were measured as markers of enterocyte differentiation in mice fed diets containing galactose (G diet), corn oil (E diet) or galactose + corn oil (G + E diet). Maintaining mice on a G instead of E diet reduced brush-border lactase activity and enterocyte migration rates; alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N and microvillus length expression increased and alkaline phosphatase activity remained unchanged. Feeding the G + E diet restored enterocyte migration rates, lactase, aminopeptidase N and dipeptidylpeptidase-IV activities to values found in mice fed the E diet. Galactose stimulation of alpha-glucosidase and microvillus length expression was, however, fully maintained in mice fed the G + E diet. Present results show that enterocyte differentiation is affected independently by varying dietary galactose and energy levels; that galactose effects always increase and energy effects usually decrease expression of enterocyte components and that energy stimulation of lactase activity is exceptional. PMID:1907492

  10. Quantification of extracellular UDP-galactose

    PubMed Central

    Lazarowski, Eduardo R.

    2009-01-01

    The human P2Y14 receptor is potently activated by UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid. Recently, cellular release of UDP-Glc and UDP-GlcNAc has been reported, but whether additional UDP-sugars are endogenous agonists for the P2Y14 receptor remains poorly defined. In the present study, we describe an assay for the quantification of UDP-Gal with sub-nanomolar sensitivity. This assay is based on the enzymatic conversion of UDP-Gal to UDP, using 1–4-β-galactosyltransferase. UDP is subsequently phosphorylated by nucleoside diphosphokinase in the presence of [γ32P]ATP and the formation of [γ32P]UTP is monitored by high performance liquid chromatography. The overall conversion of UDP-Gal to [γ32P]UTP was linear between 0.5 and 30 nM UDP-Gal. Extracellular UDP-Gal was detected on resting cultures of various cell types, and increased release of UDP-Gal was observed in 1321N1 human astrocytoma cells stimulated with the protease-activated receptor agonist thrombin. Occurrence of regulated release of UDP-Gal suggests that, in addition to its role in glycosylation reactions, UDP-Gal is an important extracellular signaling molecule. PMID:19699703

  11. Signal integration in the galactose network of Escherichia coli.

    PubMed

    Semsey, Szabolcs; Krishna, Sandeep; Sneppen, Kim; Adhya, Sankar

    2007-07-01

    The gal regulon of Escherichia coli contains genes involved in galactose transport and metabolism. Transcription of the gal regulon genes is regulated in different ways by two iso-regulatory proteins, Gal repressor (GalR) and Gal isorepressor (GalS), which recognize the same binding sites in the absence of d-galactose. DNA binding by both GalR and GalS is inhibited in the presence of d-galactose. Many of the gal regulon genes are activated in the presence of the adenosine cyclic-3',5'-monophosphate (cAMP)-cAMP receptor protein (CRP) complex. We studied transcriptional regulation of the gal regulon promoters simultaneously in a purified system and attempted to integrate the two small molecule signals, d-galactose and cAMP, that modulate the isoregulators and CRP respectively, at each promoter, using Boolean logic. Results show that similarly organized promoters can have different input functions. We also found that in some cases the activity of the promoter and the cognate gene can be described by different logic gates. We combined the transcriptional network of the galactose regulon, obtained from our experiments, with literature data to construct an integrated map of the galactose network. Structural analysis of the network shows that at the interface of the genetic and metabolic network, feedback loops are by far the most common motif. PMID:17630975

  12. Porcine Endogenous Retrovirus Transmission Characteristics of Galactose α1-3 Galactose-Deficient Pig Cells

    PubMed Central

    Quinn, Gary; Wood, James C.; Ryan, David J. J.; Suling, Kristen M.; Moran, Kathleen M.; Kolber-Simonds, Donna L.; Greenstein, Julia L.; Schuurman, Henk-Jan; Hawley, Robert J.; Patience, Clive

    2004-01-01

    Galactose α1-3 galactose (Gal) trisaccharides are present on the surface of wild-type pig cells, as well as on viruses particles produced from such cells. The recognition of Gal sugars by natural anti-Gal antibodies (NAb) in human and Old World primate serum can cause the lysis of the particles via complement-dependent mechanisms and has therefore been proposed as an important antiviral mechanism. Recently, pigs have been generated that possess disrupted galactosyl-transferase (GGTA1) genes. The cells of these pigs do not express Gal sugars on their surface, i.e., are Gal null. Concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of the Gal sugars. We investigated the sensitivity of porcine endogenous retrovirus (PERV) produced from Gal-null and Gal-positive pig cells to inactivation by purified NAb and human serum. PERV produced in Gal-null pig cells was resistant to inactivation by either NAb or human serum. In contrast, although Gal-positive PERV particles were sensitive to inactivation by NAb and human serum, they required markedly higher concentrations of NAb for inactivation compared to the Gal-positive cells from which they were produced. Complete inactivation of Gal-positive PERV particles was not achievable despite the use of high levels of NAb, indicating that NAb-mediated inactivation of cell-free PERV particles is an inefficient process. PMID:15140978

  13. Mathematical model of galactose regulation and metabolic consumption in yeast.

    PubMed

    Mitre, Tina M; Mackey, Michael C; Khadra, Anmar

    2016-10-21

    The galactose network has been extensively studied at the unicellular level to broaden our understanding of the regulatory mechanisms governing galactose metabolism in multicellular organisms. Although the key molecular players involved in the metabolic and regulatory processes of this system have been known for decades, their interactions and chemical kinetics remain incompletely understood. Mathematical models can provide an alternative method to study the dynamics of this network from a quantitative and a qualitative perspective. Here, we employ this approach to unravel the main properties of the galactose network, including equilibrium binary and temporal responses, as a way to decipher its adaptation to actively-changing inputs. We combine its two main components: the genetic branch, which allows for bistable responses, and a metabolic branch, encompassing the relevant metabolic processes that can be repressed by glucose. We use both computational tools to estimate model parameters based on published experimental data, as well as bifurcation analysis to decipher the properties of the system in various parameter regimes. Our model analysis reveals that the interplay between the inducer (galactose) and the repressor (glucose) creates a bistable regime which dictates the temporal responses of the system. Based on the same bifurcation techniques, we explain why the system is robust to genetic mutations and molecular instabilities. These findings may provide experimentalists with a theoretical framework with which they can determine how the galactose network functions under various conditions. PMID:27395401

  14. Drug allergens and food—the cetuximab and galactose-α-1,3-galactose story

    PubMed Central

    Berg, Emily A.; Platts-Mills, Thomas A.E.; Commins, Scott P.

    2014-01-01

    Objective A novel form of food allergy has been described that initially became apparent from IgE reactivity with the drug cetuximab. Ongoing work regarding the etiology, distribution, clinical management, and cellular mechanisms of the IgE response to the oligosaccharide galactose-α-1,3-galactose (α-gal) is reviewed. Data Sources Brief review of the relevant literature in peer-reviewed journals. Study Selection Studies on the clinical and immunologic features, pathogenesis, epidemiology, laboratory evaluation, and management of IgE to α-gal are included in this review. Results Recent work has identified a novel IgE antibody response to the mammalian oligosaccharide epitope, α-gal, that has been associated with 2 distinct forms of anaphylaxis: (1) immediate-onset anaphylaxis during first exposure to intravenous cetuximab and (2) delayed-onset anaphylaxis 3 to 6 hours after ingestion of mammalian food products (eg, beef and pork). Study results have suggested that tick bites are a cause of IgE antibody responses to α-gal in the United States. Patients with IgE antibody to α-gal continue to emerge, and, increasingly, these cases involve children. Nevertheless, this IgE antibody response does not appear to pose a risk for asthma but may impair diagnostic testing in some situations. Conclusion The practicing physician should understand the symptoms, evaluation, and management when diagnosing delayed allergic reactions to mammalian meat from IgE to α-gal or when initiating treatment with cetuximab in patients who have developed an IgE antibody response to α-gal. PMID:24468247

  15. Galactose-α-1,3-galactose and Delayed Anaphylaxis, Angioedema, and Urticaria in Children

    PubMed Central

    Kennedy, Joshua L.; Stallings, Amy P.; Platts-Mills, Thomas A.E.; Oliveira, Walter M.; Workman, Lisa; James, Haley R.; Tripathi, Anubha; Lane, Charles J.; Matos, Luis; Heymann, Peter W.

    2013-01-01

    BACKGROUND AND OBJECTIVE: Despite a thorough history and comprehensive testing, many children who present with recurrent symptoms consistent with allergic reactions elude diagnosis. Recent research has identified a novel cause for “idiopathic” allergic reactions; immunoglobulin E (IgE) antibody specific for the carbohydrate galactose-α-1,3-galactose (α-Gal) has been associated with delayed urticaria and anaphylaxis that occurs 3 to 6 hours after eating beef, pork, or lamb. We sought to determine whether IgE antibody to α-Gal was present in sera of pediatric patients who reported idiopathic anaphylaxis or urticaria. METHODS: Patients aged 4 to 17 were enrolled in an institutional review board–approved protocol at the University of Virginia and private practice allergy offices in Lynchburg, VA. Sera was obtained and analyzed by ImmunoCAP for total IgE and specific IgE to α-Gal, beef, pork, cat epithelium and dander, Fel d 1, dog dander, and milk. RESULTS: Forty-five pediatric patients were identified who had both clinical histories supporting delayed anaphylaxis or urticaria to mammalian meat and IgE antibody specific for α-Gal. In addition, most of these cases had a history of tick bites within the past year, which itched and persisted. CONCLUSIONS: A novel form of anaphylaxis and urticaria that occurs 3 to 6 hours after eating mammalian meat is not uncommon among children in our area. Identification of these cases may not be straightforward and diagnosis is best confirmed by specific testing, which should certainly be considered for children living in the area where the Lone Star tick is common. PMID:23569097

  16. Galactose promotes fat mobilization in obese lactating and nonlactating women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Galactose consumption as the only carbohydrate source, results in little increase in plasma glucose and insulin concentrations when compared with fasting. Lower insulin might promote endogenous lipolysis during meal absorption, which may facilitate fat loss. The objective was to test the hypothesis ...

  17. 21 CFR 862.1310 - Galactose test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Galactose test system. 862.1310 Section 862.1310 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  18. 21 CFR 862.1310 - Galactose test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Galactose test system. 862.1310 Section 862.1310 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  19. 21 CFR 862.1310 - Galactose test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Galactose test system. 862.1310 Section 862.1310 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  20. Contribution of galactose and fructose to glucose homeostasis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine the contributions of galactose and fructose to glucose formation, 6 subjects (26 +/- 2 years old; body mass index, 22.4 +/-0.2 kg/m2) (mean +/- SE) were studied during fasting conditions. Three subjects received a primed constant intravenous infusion of[6,6-2H2] glucose for 3 hours foll...

  1. 21 CFR 862.1310 - Galactose test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Galactose test system. 862.1310 Section 862.1310 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  2. Elemental and structural studies of the rat galactose cataract

    SciTech Connect

    Harding, C.V.; Unakar, N.J.; Bagchi, M.; Chylack, L.T. Jr.; Jampel, R.S.; Bobrowski, W.F.; Dang, L.; Tsui, J.Y.; Harding, D. )

    1989-01-01

    A series of rat galactose lenses, from 1 to 20 days on the 50% galactose diet, were frozen in the whole eye, and fractured from pole to pole in the frozen state. Lyophilized half-lenses were prepared for analysis by energy dispersive spectrometry (EDS). Following elemental analysis, some specimens were embedded and sectioned for histological studies. Elemental X-ray maps, and/or profiles, were made for K, Na, Cl, Ca, P, and S. As early as two days on the galactose diet, a crescent-shaped region (streak) of Cl, Na, and Ca gain, and K loss develops near the equatorial surface. Between this region and the equatorial surface are the nucleated differentiating fiber cells which maintain low Cl, Na, and Ca, and high K (viable equatorial zone, VEZ). With time the streak expands anteriorly, centrally and posteriorly, eventually (by 20 days) including most of the lens. The VEZ, including the epithelium, however, is non-reactive to the galactose diet, which is deleterious to the fully differentiated fiber cells. Eventually, the VEZ undergoes a characteristic morphological change, apparently due in part to changes in its physical environment.

  3. Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides*

    PubMed Central

    Lee, Mark J.; Gravelat, Fabrice N.; Cerone, Robert P.; Baptista, Stefanie D.; Campoli, Paolo V.; Choe, Se-In; Kravtsov, Ilia; Vinogradov, Evgeny; Creuzenet, Carole; Liu, Hong; Berghuis, Albert M.; Latgé, Jean-Paul; Filler, Scott G.; Fontaine, Thierry; Sheppard, Donald C.

    2014-01-01

    The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis. PMID:24257745

  4. Five Arab children with glucose-galactose malabsorption.

    PubMed

    Assiri, Asaad; Saeed, Anjum; Alnimri, Abdulrehman; Ahmad, Sarfaraz; Saeed, Elshazalay; Jameel, Sajjad

    2013-05-01

    Five children with glucose-galactose malabsorption (GGM) are presented. Two infants from Saudi Arabia were first-degree relatives, the third infant was unrelated and the other two were of Yemeni and Syrian origin, respectively. All the infants had chronic diarrhoea and four had failed to thrive since early infancy. All had stools positive for reducing substances, and sugar chromatography showed glucose and galactose malabsorption. Small bowel biopsies were normal in all. One infant developed gangrene of both legs as a complication of hypernatraemia and dehydration, necessitating bilateral amputation. Two infants had nephrolithiasis. All the infants responded well to fructose-based formulae. GGM should be considered in the differential diagnosis of chronic diarrhoea in infants breastfed or artificially fed from early life. PMID:23925285

  5. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy

    PubMed Central

    Ma, Yuxiang; Chen, Haiyan; Su, Shanyuhan; Wang, Tong; Zhang, Congying; Fida, Guissi; Cui, Sisi; Zhao, Juan; Gu, Yueqing

    2015-01-01

    Galactose residues could be specifically recognized by the asialoglycoprotein receptor (ASGPR) which is highly exhibited on liver tissues. However, ASGPR has not been widely investigated on different tumor cell lines except for hepatoma carcinoma cells, which motivates us to investigate the possibility of galactose serving as a board tumor ligand. In this study, a galactose (Gal)-based probe conjugated with fluorescence dye MPA (Gal-MPA) was constructed for the evaluation of tumor affinities/targeted ability on different tumor cell lines. In the vitro cell study, it was indicated that the fluorescence probe Gal-MPA displayed higher cell affinity to tumor cells (HepG2, MCF-7 and A549) than that of the normal liver cells l02. In the vivo dynamic study of Gal-MPA in tumor-bearing mice (HepG2, MCF-7, A549, HCT116, U87, MDA-MB-231 and S180), it was shown that its high tumor targeted ability with the maximal tumor/normal tissue ratio reached up to 6.8. Meanwhile, the fast tumor-targeted ability within 2 hours and long retention on tumor site up to 120 hours were observed. Our results demonstrated that galactose should be a promising broad ligand for multiple tumor imaging and targeted therapy. Subsequently, Gal was covalently conjugated to doxorubicin (DOX) to form prodrug Gal-DOX for tumor targeted therapy. The therapeutic results of Gal-DOX than DOX being better suggested that galactosylated prodrugs might have the prospective potential in tumor targeted therapy. PMID:26078797

  6. Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii*

    PubMed Central

    Marchant, Jan; Cowper, Ben; Liu, Yan; Lai, Livia; Pinzan, Camila; Marq, Jean Baptiste; Friedrich, Nikolas; Sawmynaden, Kovilen; Liew, Lloyd; Chai, Wengang; Childs, Robert A.; Saouros, Savvas; Simpson, Peter; Roque Barreira, Maria Cristina; Feizi, Ten; Soldati-Favre, Dominique; Matthews, Stephen

    2012-01-01

    Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system. PMID:22399295

  7. Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis

    PubMed Central

    Damerow, Sebastian; Hoppe, Carolin; Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk F.R.; Ferguson, Michael A.J.; Routier, Françoise H.

    2015-01-01

    Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection. PMID

  8. Galactose inhibits auxin-induced growth of Avena coleoptiles by two mechanisms

    NASA Technical Reports Server (NTRS)

    Cheung, S. P.; Cleland, R. E.

    1991-01-01

    Galactose inhibits auxin-induced growth of Avena coleoptiles by at least two mechanisms. First, it inhibits auxin-induced H(+)-excretion needed for the initiation of rapid elongation. Galactose cannot be doing so by directly interfering with the ATPase since fusicoccin-induced H(+)-excretion is not affected. Secondly, galactose inhibits long-term auxin-induced growth, even in an acidic (pH 4.5) solution. This may be due to an inhibition of cell wall synthesis. However, galactose does not reduce the capacity of walls to be loosened by H+, given exogenously or excreted in response to fusicoccin.

  9. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  10. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  11. Galactose uncovers face recognition and mental images in congenital prosopagnosia: the first case report.

    PubMed

    Esins, Janina; Schultz, Johannes; Bülthoff, Isabelle; Kennerknecht, Ingo

    2014-09-01

    A woman in her early 40s with congenital prosopagnosia and attention deficit hyperactivity disorder observed for the first time sudden and extensive improvement of her face recognition abilities, mental imagery, and sense of navigation after galactose intake. This effect of galactose on prosopagnosia has never been reported before. Even if this effect is restricted to a subform of congenital prosopagnosia, galactose might improve the condition of other prosopagnosics. Congenital prosopagnosia, the inability to recognize other people by their face, has extensive negative impact on everyday life. It has a high prevalence of about 2.5%. Monosaccharides are known to have a positive impact on cognitive performance. Here, we report the case of a prosopagnosic woman for whom the daily intake of 5 g of galactose resulted in a remarkable improvement of her lifelong face blindness, along with improved sense of orientation and more vivid mental imagery. All these improvements vanished after discontinuing galactose intake. The self-reported effects of galactose were wide-ranging and remarkably strong but could not be reproduced for 16 other prosopagnosics tested. Indications about heterogeneity within prosopagnosia have been reported; this could explain the difficulty to find similar effects in other prosopagnosics. Detailed analyses of the effects of galactose in prosopagnosia might give more insight into the effects of galactose on human cognition in general. Galactose is cheap and easy to obtain, therefore, a systematic test of its positive effects on other cases of congenital prosopagnosia may be warranted. PMID:24164936

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

    PubMed Central

    Thomas, Terence D.; Crow, Vaughan L.

    1984-01-01

    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-β-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. PMID:16346586

  13. Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

    PubMed Central

    Nwamba, Charles O.; Chilaka, Ferdinand C.

    2012-01-01

    Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞ − [P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0 and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0 and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed. PMID:23008759

  14. Galactose promotes fat mobilization in obese lactating and non-lactating women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Galactose consumption results in a lower rise in plasma glucose and insulin concentrations when compared to glucose. The lower insulin might promote lipolysis during meal absorption. An isocaloric galactose drink, when compared to glucose, will sustain fat mobilization during meal consumption while...

  15. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  16. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  17. Electrochemical biosensing of galactose based on carbon materials: graphene versus multi-walled carbon nanotubes.

    PubMed

    Dalkıran, Berna; Erden, Pınar Esra; Kılıç, Esma

    2016-06-01

    In this study, two enzyme electrodes based on graphene (GR), Co3O4 nanoparticles and chitosan (CS) or multi-walled carbon nanotubes (MWCNTs), Co3O4 nanoparticles, and CS, were fabricated as novel biosensing platforms for galactose determination, and their performances were compared. Galactose oxidase (GaOx) was immobilized onto the electrode surfaces by crosslinking with glutaraldehyde. Optimum working conditions of the biosensors were investigated and the analytical performance of the biosensors was compared with respect to detection limit, linearity, repeatability, and stability. The MWCNTs-based galactose biosensor provided about 1.6-fold higher sensitivity than its graphene counterpart. Moreover, the linear working range and detection limit of the MWCNTs-based galactose biosensor was superior to the graphene-modified biosensor. The successful application of the purposed biosensors for galactose biosensing in human serum samples was also investigated. PMID:27074783

  18. Mutations in GAL2 or GAL4 alleviate catabolite repression produced by galactose in Saccharomyces cerevisiae.

    PubMed

    Rodríguez; Flores

    2000-06-01

    Galactose does not allow growth of pyruvate carboxylase mutants in media with ammonium as a nitrogen source, and inhibits growth of strains defective in phosphoglyceromutase in ethanol-glycerol mixtures. Starting with pyc1, pyc2, and gpm1 strains, we isolated mutants that eliminated those galactose effects. The mutations were recessive and were named dgr1-1 and dgr2-1. Strains bearing those mutations in an otherwise wild-type background grew slower than the wild type in rich galactose media, and their growth was dependent on respiration. Galactose repression of several enzymes was relieved in the mutants. Biochemical and genetic evidence showed that dgr1-1 was allelic with GAL2 and dgr2-1 with GAL4. The results indicate that the rate of galactose consumption is critical to cause catabolite repression. PMID:10862881

  19. Timing and Variability of Galactose Metabolic Gene Activation Depend on the Rate of Environmental Change

    PubMed Central

    Ma, Bo; Ott, William; Josić, Krešimir; Bennett, Matthew R.

    2015-01-01

    Modulation of gene network activity allows cells to respond to changes in environmental conditions. For example, the galactose utilization network in Saccharomyces cerevisiae is activated by the presence of galactose but repressed by glucose. If both sugars are present, the yeast will first metabolize glucose, depleting it from the extracellular environment. Upon depletion of glucose, the genes encoding galactose metabolic proteins will activate. Here, we show that the rate at which glucose levels are depleted determines the timing and variability of galactose gene activation. Paradoxically, we find that Gal1p, an enzyme needed for galactose metabolism, accumulates more quickly if glucose is depleted slowly rather than taken away quickly. Furthermore, the variability of induction times in individual cells depends non-monotonically on the rate of glucose depletion and exhibits a minimum at intermediate depletion rates. Our mathematical modeling suggests that the dynamics of the metabolic transition from glucose to galactose are responsible for the variability in galactose gene activation. These findings demonstrate that environmental dynamics can determine the phenotypic outcome at both the single-cell and population levels. PMID:26200924

  20. Lithium Induces ER Stress and N-Glycan Modification in Galactose-Grown Jurkat Cells

    PubMed Central

    Kátai, Emese; Yahiro, Rikki K. K.; Poór, Viktor S.; Montskó, Gergely; Zrínyi, Zita; Kovács, Gábor L.; Miseta, Attila

    2013-01-01

    We previously reported that lithium had a significant impact on Ca2+ regulation and induced unfolded protein response (UPR) in yeast cells grown on galactose due to inhibition of phosphoglucomutase (PGM), however the exact mechanism has not been established yet. In this study, we analysed lithium's effect in galactose-fed cells to clarify whether these ER-related changes are the result of a relative hypoglycemic state. Furthermore, we investigated whether the alterations in galactose metabolism impact protein post-translational modifications. Thus, Jurkat cells were incubated in glucose or galactose containing media with or without lithium treatment. We found that galactose-fed and lithium treated cells showed better survivability than fasting cells. We also found higher UDP-Hexose and glycogen levels in these cells compared to fasting cells. On the other hand, the UPR (X-box binding protein 1 mRNA levels) of galactose-fed and lithium treated cells was even greater than in fasting cells. We also found increased amount of proteins that contained N-linked N-acetyl-glucosamine, similar to what was reported in fasting cells by a recent study. Our results demonstrate that lithium treatment of galactose-fed cells can induce stress responses similar to hypoglycemia, however cell survival is still secured by alternative pathways. We propose that clarifying this process might be an important addition toward the better understanding of the molecular mechanisms that regulate ER-associated stress response. PMID:23894652

  1. Effects of IMOD™ and Angipars™ on mouse D-galactose-induced model of aging

    PubMed Central

    2012-01-01

    The aim of this study was to evaluate the effects of two registered herbal drugs called IMOD and Angipars on mouse model. Aging was induced by D-galactose (500 mg/kg) administered to animals for 6 weeks through drinking water. Male BALB/c mice were randomly divided into 5 groups receiving D-galactose (D-galactose, 500 mg/kg) for 6 weeks; positive control (D-galactose [500 mg/kg] for 6 weeks + Vitamin E [200 mg/kg/day] intraperitoneally for 4 weeks); IMOD (D-galactose [500 mg/kg] for 6 weeks + IMOD [20 mg/kg/day] intraperitoneally for 4 weeks), Angipars (D-galactose [500 mg/kg] for 6 weeks + Angipars [2.1 mg/kg/day] by gavage for 4 weeks); and the fifth group that was sham and not given D-galactose. At the end of treatment, pro-inflammatory markers including tumor necrosis factor-α (TNF-α), interlukine-1β (IL-β), interlukine-6 (IL-6), Nuclear Factor-kappaB (NF-κb), total antioxidant power (TAP), lipid peroxides (LPO) and male sex hormones i.e. testosterone and dehydroepiandrosterone-sulfate (DHEA-S) were measured in the blood. Results showed that D-Galactose induces a significant oxidative stress and proinflammatory cascade of aging while both IMOD and Angipars recovered all of them. Interestingly, IMOD and Angipars were better than Vitamin E in improving male sex hormones in aged mice. This effect is so important and should be considered as an advantage although it cannot be explained with current knowledge. The conclusion is that IMOD and Angipars have marked anti-aging effect on D-galactose-induced model of aging. PMID:23351487

  2. Effects of Exendin-4 on Male Reproductive Parameters of D-Galactose Induced Aging Mouse Model

    PubMed Central

    Ahangarpour, Akram; Heidari, Hamid

    2014-01-01

    Purpose The purpose of this study was to evaluate the role of exendin-4 on reproductive alteration in a D-galactose-induced aging mouse model. Materials and Methods In this experimental study, 72 male Naval Medical Research Institute mice (20~25 g) were randomly divided into six groups: control, exendin-4 (1 nmol/kg), exendin-4 (10 nmol/kg), D-galactose (500 mg/kg), D-galactose+exendin-4 (1 nmol/kg), and D-galactose+exendin-4 (10 nmol/kg). The aging model animals were gavaged with D-galactose for six weeks, and exendin-4 was injected intraperitoneally in the last 10 days. At the end of treatment serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone levels were evaluated and the cauda epididymis and testis were removed to analyze the sperm count and testis morphology. Results The testis weight and volume decreased in the D-galactose group (p<0.01 and p<0.05) respectively. Exendin-4 (1, 10 nmol/kg) increased these parameters in the normal and aging mouse models. Serum LH and FSH levels increased and the sperm count decreased in the D-galactose group (p<0.05). Further, exendin-4 (1 nmol/kg) decreased LH and FSH levels and increased the serum testosterone level and sperm count in both normal and aging animals. Conclusions D-galactose can induce aging alternations in the male reproductive system such as decreased sperm count and increased serum LH and FSH levels through reactive oxygen species over production and reduced antioxidant enzyme activity. Further, co-administration of exendin-4 reduced reproductive complications of D-galactose in an aging mouse model. PMID:25606567

  3. Effects of IMOD™ and Angipars™ on mouse D-galactose-induced model of aging.

    PubMed

    Ghanbari, Samane; Yonessi, Mahsa; Mohammadirad, Azadeh; Gholami, Mahdi; Baeeri, Maryam; Khorram-Khorshid, Hamid Reza; Gharibdoost, Farhad; Abdollahi, Mohammad

    2012-01-01

    The aim of this study was to evaluate the effects of two registered herbal drugs called IMOD and Angipars on mouse model. Aging was induced by D-galactose (500 mg/kg) administered to animals for 6 weeks through drinking water. Male BALB/c mice were randomly divided into 5 groups receiving D-galactose (D-galactose, 500 mg/kg) for 6 weeks; positive control (D-galactose [500 mg/kg] for 6 weeks + Vitamin E [200 mg/kg/day] intraperitoneally for 4 weeks); IMOD (D-galactose [500 mg/kg] for 6 weeks + IMOD [20 mg/kg/day] intraperitoneally for 4 weeks), Angipars (D-galactose [500 mg/kg] for 6 weeks + Angipars [2.1 mg/kg/day] by gavage for 4 weeks); and the fifth group that was sham and not given D-galactose. At the end of treatment, pro-inflammatory markers including tumor necrosis factor-α (TNF-α), interlukine-1β (IL-β), interlukine-6 (IL-6), Nuclear Factor-kappaB (NF-κb), total antioxidant power (TAP), lipid peroxides (LPO) and male sex hormones i.e. testosterone and dehydroepiandrosterone-sulfate (DHEA-S) were measured in the blood.Results showed that D-Galactose induces a significant oxidative stress and proinflammatory cascade of aging while both IMOD and Angipars recovered all of them. Interestingly, IMOD and Angipars were better than Vitamin E in improving male sex hormones in aged mice. This effect is so important and should be considered as an advantage although it cannot be explained with current knowledge. The conclusion is that IMOD and Angipars have marked anti-aging effect on D-galactose-induced model of aging. PMID:23351487

  4. Impairments of astrocytes are involved in the D-galactose-induced brain aging

    SciTech Connect

    Lei Ming; Hua Xiangdong; Xiao Ming Ding Jiong; Han Qunying Hu Gang

    2008-05-16

    Astrocyte dysfunction is implicated in course of various age-related neurodegenerative diseases. Chronic injection of D-galactose can cause a progressive deterioration in learning and memory capacity and serve as an animal model of aging. To investigate the involvement of astrocytes in this model, oxidative stress biomarkers, biochemical and pathological changes of astrocytes were examined in the hippocampus of the rats with six weeks of D-galactose injection. D-galactose-injected rats displayed impaired antioxidant systems, an increase in nitric oxide levels, and a decrease in reduced glutathione levels. Consistently, western blotting and immunostaining of glial fibrillary acidic protein showed extensive activation of astrocytes. Double-immunofluorescent staining further showed activated astrocytes highly expressed inducible nitric oxide synthase. Electron microscopy demonstrated the degeneration of astrocytes, especially in the aggregated area of synapse and brain microvessels. These findings indicate that impairments of astrocytes are involved in oxidative stress-induced brain aging by chronic injection of D-galactose.

  5. Polyvinylamine-G-galactose is a route to bioactivated silica surfaces.

    PubMed

    Mokhtari, Hajir; Pelton, Robert; Jin, Liqiang

    2014-01-01

    Polyvinylamine (PVAm) was derivatized with lactobionic acid to give PVAm-GAL with pendant galactose groups along the PVAm chain. The galactose substituents were shown to undergo two types of specific interactions: (1) condensation with phenylboronic acid moieties on polymers and on surfaces; and, (2) the specific binding of RCA120, a galactose-specific lectin. Surface binding and assembly was monitored with Quartz Crystal Microbalance (QCM-D) measurements. Multilayer assemblies based on boronic ester formation were destroyed by lowering the pH, or by introducing sorbitol. We propose that the physical adsorption of PVAm-GAL onto silica or other negatively charged support surfaces is a simple route to galactose-rich interfaces, possibly useful for affinity separations, cell targeting and cell culturing. PMID:24183434

  6. Insights into the role of the aromatic residue in galactose-binding sites: MP2/6-311G++** study on galactose- and glucose-aromatic residue analogue complexes.

    PubMed

    Sujatha, Mannargudi S; Sasidhar, Yellamraju U; Balaji, Petety V

    2005-06-14

    The presence of an aromatic residue (Trp, Phe, Tyr) facing the nonpolar face of galactose is a common feature of galactose-specific lectins. The interactions such as those between the C-H groups of galactose and the pi-electron cloud of aromatic residues have been characterized as weak hydrogen bonds between soft acids and soft bases, largely governed by dispersive and charge transfer interactions. An analysis of the binding sites of several galactose-specific lectins revealed that the spatial position-orientation of galactose relative to the binding site aromatic residue varies substantially. The effect of variations in position-orientations of galactose on the interaction energies of galactose-aromatic residue complexes has not been determined so far. In view of this, MP2/6-311G++** calculations were performed on galactose- and glucose-aromatic residue analogue complexes in eight position-orientations. The results show that the strength of the C-H...pi interactions in galactose-aromatic residue complexes is comparable to that of a hydrogen bond. Rather than the type of aromatic residue, the position-orientation of the saccharide appears to be more critical in determining the strength of their interactions. Earlier studies have found the binding site aromatic residue to be critical, but its role was not clear. This study shows that the aromatic residue is important for discriminating galactose from glucose, in addition to its contribution to binding energy. PMID:15938646

  7. Gene Transcriptional and Metabolic Profile Changes in Mimetic Aging Mice Induced by D-Galactose

    PubMed Central

    Zhou, Yue-Yue; Zhu, Xiao-Juan; Li, Rong-Hua; Mu, Chang-Kao; Wang, Chun-Lin; Song, Wei-Wei

    2015-01-01

    D-galactose injection has been shown to induce many changes in mice that represent accelerated aging. This mouse model has been widely used for pharmacological studies of anti-aging agents. The underlying mechanism of D-galactose induced aging remains unclear, however, it appears to relate to glucose and 1ipid metabolic disorders. Currently, there has yet to be a study that focuses on investigating gene expression changes in D-galactose aging mice. In this study, integrated analysis of gas chromatography/mass spectrometry-based metabonomics and gene expression profiles was used to investigate the changes in transcriptional and metabolic profiles in mimetic aging mice injected with D-galactose. Our findings demonstrated that 48 mRNAs were differentially expressed between control and D-galactose mice, and 51 potential biomarkers were identified at the metabolic level. The effects of D-galactose on aging could be attributed to glucose and 1ipid metabolic disorders, oxidative damage, accumulation of advanced glycation end products (AGEs), reduction in abnormal substance elimination, cell apoptosis, and insulin resistance. PMID:26176541

  8. Galactose content of legumes, caseinates, and some hard cheeses: implications for diet treatment of classic galactosemia.

    PubMed

    Van Calcar, Sandra C; Bernstein, Laurie E; Rohr, Frances J; Yannicelli, Steven; Berry, Gerard T; Scaman, Christine H

    2014-02-12

    There are inconsistent reports on the lactose and/or galactose content of some foods traditionally restricted from the diet for classic galactosemia. Therefore, samples of cheeses, caseinates, and canned black, pinto, kidney, and garbanzo beans were analyzed for free galactose content using HPLC with refractive index or pulsed amperometric detection. Galactose concentrations in several hard and aged cheeses and three mild/medium Cheddars, produced by smaller local dairies, was <10 mg/100 g sample compared to 55.4 mg/100 g sample in four sharp Cheddars produced by a multinational producer. Galactose in sodium and calcium caseinate ranged from undetectable to 95.5 mg/100 g sample. Free galactose level in garbanzo beans was lower than previously reported at 24.6 mg/100 g sample; black beans contained 5.3 mg/100 g, and free galactose was not detected in red kidney or pinto beans. These data provide a basis for recommending inclusion of legumes, caseinate-containing foods, and some aged hard cheeses that had been previously restricted in the diet for individuals with galactosemia. PMID:24456566

  9. Preparation of low galactose yogurt using cultures of Gal(+) Streptococcus thermophilus in combination with Lactobacillus delbrueckii ssp. bulgaricus.

    PubMed

    Anbukkarasi, Kaliyaperumal; UmaMaheswari, Thiyagamoorthy; Hemalatha, Thiagarajan; Nanda, Dhiraj Kumar; Singh, Prashant; Singh, Rameshwar

    2014-09-01

    Streptococcus thermophilus is an important lactic starter used in the production of yogurt. Most strains of S. thermophilus are galactose negative (Gal(-)) and are able to metabolize only glucose portion of lactose and expel galactose into the medium. This metabolic defect leads to the accumulation of free galactose in yogurt, resulting in galactosemia among consumers. Hence there is an absolute need to develop low galactose yogurt. Therefore, in this study, three galactose positive (Gal(+)) S. thermophilus strains from National Collection of Dairy Cultures (NCDC) viz. NCDC 659 (AJM), NCDC 660 (JM1), NCDC 661 (KM3) and a reference galactose negative (Gal(-)) S. thermophilus NCDC 218 were used for preparation of low galactose yogurt. In milk fermented using S. thermophilus isolates alone, NCDC 659 released less galactose (0.27 %) followed by NCDC 661 (0.3 %) and NCDC 660 (0.45 %) after 10 h at 42 °C. Milk was fermented in combination with Gal(-) L. delbrueckii subsp. bulgaricus NCDC 04, in which NCDC 659 released least galactose upto 0.49 % followed by NCDC 661 (0.51 %) and NCDC 660 (0.60 %) than reference Gal(-) NCDC 218(0.79 %). Low galactose yogurt was prepared following standard procedure using Gal(+) S. thermophilus isolates and Gal(-) L. delbrueckii subsp. bulgaricus NCDC 04 in 1:1 ratio. Among which low galactose yogurt by NCDC 659 combination contained less galactose 0.37 % followed by NCDC 661 (0.51 %), NCDC 660 (0.65 %) and reference Gal(-) NCDC 218 (0.98 %) after 4 h of fermentation. This study clearly reveals that Gal(+) S. thermophilus isolates can be paired with Gal(-) L. delbrueckii subsp. bulgaricus for developing low galactose yogurt. PMID:25190881

  10. Highly swelling hydrogels from ordered galactose-based polyacrylates.

    PubMed

    Martin, B D; Linhardt, R J; Dordick, J S

    1998-01-01

    High swelling galactose-based hydrogels have been prepared using a chemoenzymatic procedure. Regioselective acylation of beta-O-methyl-galactopyranoside in nearly anhydrous pyridine with lipase from Pseudomonas cepacia yields the 6-acryloyl derivative (Compound I). Further lipase-catalysed acylation of the monoacrylate derivative in nearly anhydrous acetone yielded 2,6-diacryloyl-beta-O-methyl galactopyranoside (Compound II) that can act as a cross-linker with a structure similar to that of the sugar-based monomer. The high selectivity of enzyme catalysis yielded apparently highly regular hydrogel networks with swelling ratios at equilibrium ranging from 170 to 1100. elastic moduli ranging from 0.005 to 0.088 MPa and calculated mesh sizes ranging from 1160 to 6600 A. These values are far higher than conventional uncharged or lightly charged hydrogels at similar elastic moduli. Gel swelling was fast, with 75% of the equilibrium swelling value reached in a fractional time of 0.17. Non-selective chemical acryloylation of beta-O-methyl galactopyranoside followed by polymerization yielded a far lower-swelling hydrogel than that obtained using selective enzyme catalysis. These results indicate that the highly regular polymer structure achieved by regioselective enzyme-catalysed acylation yields relatively strong and highly swellable materials. Sugar-based hydrogels, such as those described herein, may find particular use as biomaterials because of their high water content, homogeneity, stability and expected non-toxicity. A wide range of pore sizes can be attained, suggesting that they may also be especially useful as matrices for enzyme immobilization and controlled delivery of biological macromolecules. PMID:9678852

  11. Utilization of lactose and galactose by Streptococcus mutans: transport, toxicity, and carbon catabolite repression.

    PubMed

    Zeng, Lin; Das, Satarupa; Burne, Robert A

    2010-05-01

    Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by Streptococcus mutans, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways was performed in strain UA159. Deletion of each gene in the lac operon caused various alterations in expression of a P(lacA)-cat promoter fusion and defects in growth on either lactose (lacA, lacB, lacF, lacE, and lacG), galactose (lacA, lacB, lacD, and lacG) or both sugars (lacA, lacB, and lacG). Failure to grow in the presence of galactose or lactose by certain lac mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EII(Man) and EII(Lac), respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIAB(Man), led to increased resistance to glucose-mediated CCR when lactose was used to induce the lac operon, but resulted in reduced lac gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism. PMID:20190045

  12. D-Galactose as an autoinducer 2 inhibitor to control the biofilm formation of periodontopathogens.

    PubMed

    Ryu, Eun-Ju; Sim, Jaehyun; Sim, Jun; Lee, Julian; Choi, Bong-Kyu

    2016-09-01

    Autoinducer 2 (AI-2) is a quorum sensing molecule to which bacteria respond to regulate various phenotypes, including virulence and biofilm formation. AI-2 plays an important role in the formation of a subgingival biofilm composed mostly of Gram-negative anaerobes, by which periodontitis is initiated. The aim of this study was to evaluate D-galactose as an inhibitor of AI-2 activity and thus of the biofilm formation of periodontopathogens. In a search for an AI-2 receptor of Fusobacterium nucleatum, D-galactose binding protein (Gbp, Gene ID FN1165) showed high sequence similarity with the ribose binding protein (RbsB), a known AI-2 receptor of Aggregatibacter actinomycetemcomitans. D-Galactose was evaluated for its inhibitory effect on the AI-2 activity of Vibrio harveyi BB152 and F. nucleatum, the major coaggregation bridge organism, which connects early colonizing commensals and late pathogenic colonizers in dental biofilms. The inhibitory effect of D-galactose on the biofilm formation of periodontopathogens was assessed by crystal violet staining and confocal laser scanning microscopy in the absence or presence of AI-2 and secreted molecules of F. nucleatum. D-Galactose significantly inhibited the AI-2 activity of V. harveyi and F. nucleatum. In addition, D-galactose markedly inhibited the biofilm formation of F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia induced by the AI-2 of F. nucleatum without affecting bacterial growth. Our results demonstrate that the Gbp may function as an AI-2 receptor and that galactose may be used for prevention of the biofilm formation of periodontopathogens by targeting AI-2 activity. PMID:27572513

  13. A phylogenetic study on galactose-containing Candida species based on 18S ribosomal DNA sequences.

    PubMed

    Suzuki, Motofumi; Suh, Sung-Oui; Sugita, Takashi; Nakase, Takashi

    1999-10-01

    Phylogenetic relationships of 33 Candida species containing galactose in the cells were investigated by using 18S ribosomal DNA sequence analysis. Galactose-containing Candida species and galactose-containing species from nine ascomycetous genera were a heterogeneous assemblage. They were divided into three clusters (II, III, and IV) which were phylogenetically distant from cluster I, comprising 9 galactose-lacking Candida species, C. glabrata, C. holmii, C. krusei, C. tropicalis (the type species of Candida), C. albicans, C. viswanathii, C. maltosa, C. parapsilosis, C. guilliermondii, and C. lusitaniae, and 17 related ascomycetous yeasts. These three clusters were also phylogenetically distant from Schizosaccharomyces pombe, which contains galactomannan in its cell wall. Cluster II comprised C. magnoliae, C. vaccinii, C. apis, C. gropengiesseri, C. etchellsii, C. floricola, C. lactiscondensi, Wickerhamiella domercqiae, C. versatilis, C. azyma, C. vanderwaltii, C. pararugosa, C. sorbophila, C. spandovensis, C. galacta, C. ingens, C. incommunis, Yarrowia lipolytica, Galactomyces geotrichum, and Dipodascus albidus. Cluster III comprised C. tepae, C. antillancae and its synonym C. bondarzewiae, C. ancudensis, C. petrohuensis, C. santjacobensis, C. ciferrii (anamorph of Stephanoascus ciferrii), Arxula terrestris, C. castrensis, C. valdiviana, C. paludigena, C. blankii, C. salmanticensis, C. auringiensis, C. bertae, and its synonym C. bertae var. chiloensis, C. edax (anamorph of Stephanoascus smithiae), Arxula adeninivorans, and C. steatolytica (synonym of Zygoascus hellenicus). Cluster IV comprised C. cantarellii, C. vinaria, Dipodascopsis uninucleata, and Lipomyces lipofer. Two galactose-lacking and Q-8-forming species, C. stellata and Pichia pastoris, and 5 galactose-lacking and Q-9-forming species, C. apicola, C. bombi, C. bombicola, C. geochares, and C. insectalens, were included in Cluster II. Two galactose-lacking and Q-9-forming species, C. drimydis and C

  14. In utero and lactational β-carotene supplementation attenuates D-galactose-induced hearing loss in newborn rats.

    PubMed

    Yu, Fei; Hao, Shuai; Zhao, Yue; Yang, Hui; Fan, Xiao-Lan; Yang, Jun

    2011-08-01

    D-Galactose could give rise to free radical damage by disturbing the some maternal antioxidants. The oxidative stress induced by D-galactose is a potent inducer of apoptosis, which is accompanied by the activation of protein-splitting enzymes called caspases. Apoptosis is a crucial physiological determinant of embryonic and neonatal development, and play an essential role in the development of the inner ear structures. Recently the increasing of D-galactose exposure is due to high consumption of dairy foods or reduced galactose metabolism. An overwhelming presence of D-galactose is known to become highly ototoxicity to humans. The purpose of this study was to investigate whether supplementation of pregnant and lactational mothers with β-carotene could attenuate cochlear function damage and hair cells apoptosis induced by d-galactose in newborn rats. Pregnant rats were supplemented with D-galactose, or D-galactose and β-carotene from gestational day (GD) 7 until postnatal day (PND) 21. On PND 22, offspring were examined in the distortion product otoacoustic emission (DPOAE) task, cochleae were then harvested for assessment of apoptosis by immunohistochemical stain for cysteine-aspartic acid proteases 3 (caspase-3) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Maternal and offspring blood samples were then collected by direct cardiac puncture in heparin tubes, blood levels of D-galactose and β-carotene were measured, plasma was separated for malondialdehyde (MDA) analysis, erythrocytes were left for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH). D-Galactose could significantly disturb the balance between maternal antioxidants and free radicals, and induce hearing loss in the offspring and cochlear hair cell apoptosis. In contrast, β-carotene supplementation, coincidentally with D-galactose exposure, ameliorated these changes. Our data offer a conceptual framework for designing

  15. Long-term oral galactose treatment prevents cognitive deficits in male Wistar rats treated intracerebroventricularly with streptozotocin.

    PubMed

    Salkovic-Petrisic, Melita; Osmanovic-Barilar, Jelena; Knezovic, Ana; Hoyer, Siegfried; Mosetter, Kurt; Reutter, Werner

    2014-02-01

    Basic and clinical research has demonstrated that dementia of sporadic Alzheimer's disease (sAD) type is associated with dysfunction of the insulin-receptor (IR) system followed by decreased glucose transport via glucose transporter GLUT4 and decreased glucose metabolism in brain cells. An alternative source of energy is d-galactose (the C-4-epimer of d-glucose) which is transported into the brain by insulin-independent GLUT3 transporter where it might be metabolized to glucose via the Leloir pathway. Exclusively parenteral daily injections of galactose induce memory deterioration in rodents and are used to generate animal aging model, but the effects of oral galactose treatment on cognitive functions have never been tested. We have investigated the effects of continuous daily oral galactose (200 mg/kg/day) treatment on cognitive deficits in streptozotocin-induced (STZ-icv) rat model of sAD, tested by Morris Water Maze and Passive Avoidance test, respectively. One month of oral galactose treatment initiated immediately after the STZ-icv administration, successfully prevented development of the STZ-icv-induced cognitive deficits. Beneficial effect of oral galactose was independent of the rat age and of the galactose dose ranging from 100 to 300 mg/kg/day. Additionally, oral galactose administration led to the appearance of galactose in the blood. The increase of galactose concentration in the cerebrospinal fluid was several times lower after oral than after parenteral administration of the same galactose dose. Oral galactose exposure might have beneficial effects on learning and memory ability and could be worth investigating for improvement of cognitive deficits associated with glucose hypometabolism in AD. PMID:24055495

  16. Galactose and Lactose Genes from the Galactose-Positive Bacterium Streptococcus salivarius and the Phylogenetically Related Galactose-Negative Bacterium Streptococcus thermophilus: Organization, Sequence, Transcription, and Activity of the gal Gene Products

    PubMed Central

    Vaillancourt, Katy; Moineau, Sylvain; Frenette, Michel; Lessard, Christian; Vadeboncoeur, Christian

    2002-01-01

    Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (β-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3′ end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities. PMID:11790749

  17. Serum galactose-deficient IgA1 levels in children with IgA nephropathy.

    PubMed

    Jiang, Mengjie; Jiang, Xiaoyun; Rong, Liping; Xu, Yuanyuan; Chen, Lizhi; Qiu, Zeting; Mo, Ying

    2015-01-01

    Immunoglobulin A nephropathy (IgAN) is an immunopathologic diagnosis based on a renal biopsy, it is characterized by deposits of IgA-containing immune complexes in the mesangium. Adults with IgAN have a galactose-deficient IgA1 in the circulation and glomerular deposition. There are few studies on the glycosylation of serum IgA1 in children with IgAN. To measure the serum levels of galactose-deficient IgA1 in pediatric patients with IgAN, 72 biopsy-proven IgAN children were divided into 3 groups based on the clinical features: isolated hematuria group (24 patients), hematuria and proteinuria group (22 patients), and nephritic syndrome group (26 patients). They were also divided into 3 groups according to pathologic grading: grade I + II group (25 patients), grade III group (33 patients) and grade IV + V group (14 patients). 30 healthy children were recruited as a control group. We used vicia villosa lectin binding enzyme-linked immunosorbent assay to measure the serum levels of galactose-deficient IgA1 in all groups and controls. Serum levels of galactose-deficient IgA1 in children with IgAN were higher than controls (P < 0.01). There were no significant differences in serum levels of galactose-deficient IgA1 among the different clinical and pathologic grading groups. The values of the area under the curve for galactose-deficient IgA1 levels were 0.976 (95% CI, 0.953-1.000). The cutoff point for galactose-deficient IgA1 levels was 0.125, with a sensitivity of 87.5% and a specificity of 83.3%, with a positive predictive value of 92.6% and a negative predictive value of 73.5% (P < 0.01). Children with IgAN presented serum galactose-deficient IgA1, which has shown no relationship with the clinical manifestations and pathologic grading of the disease. Detection of serum galactose-deficient IgA1 levels by vicia villosa lectin binding enzyme-linked immunosorbent assay has a certain clinical value in diagnosis of children with IgAN. PMID:26221341

  18. Serum galactose-deficient IgA1 levels in children with IgA nephropathy

    PubMed Central

    Jiang, Mengjie; Jiang, Xiaoyun; Rong, Liping; Xu, Yuanyuan; Chen, Lizhi; Qiu, Zeting; Mo, Ying

    2015-01-01

    Immunoglobulin A nephropathy (IgAN) is an immunopathologic diagnosis based on a renal biopsy, it is characterized by deposits of IgA-containing immune complexes in the mesangium. Adults with IgAN have a galactose-deficient IgA1 in the circulation and glomerular deposition. There are few studies on the glycosylation of serum IgA1 in children with IgAN. To measure the serum levels of galactose-deficient IgA1 in pediatric patients with IgAN, 72 biopsy-proven IgAN children were divided into 3 groups based on the clinical features: isolated hematuria group (24 patients), hematuria and proteinuria group (22 patients), and nephritic syndrome group (26 patients). They were also divided into 3 groups according to pathologic grading: grade I + II group (25 patients), grade III group (33 patients) and grade IV + V group (14 patients). 30 healthy children were recruited as a control group. We used vicia villosa lectin binding enzyme-linked immunosorbent assay to measure the serum levels of galactose-deficient IgA1 in all groups and controls. Serum levels of galactose-deficient IgA1 in children with IgAN were higher than controls (P < 0.01). There were no significant differences in serum levels of galactose-deficient IgA1 among the different clinical and pathologic grading groups. The values of the area under the curve for galactose-deficient IgA1 levels were 0.976 (95% CI, 0.953-1.000). The cutoff point for galactose-deficient IgA1 levels was 0.125, with a sensitivity of 87.5% and a specificity of 83.3%, with a positive predictive value of 92.6% and a negative predictive value of 73.5% (P < 0.01). Children with IgAN presented serum galactose-deficient IgA1, which has shown no relationship with the clinical manifestations and pathologic grading of the disease. Detection of serum galactose-deficient IgA1 levels by vicia villosa lectin binding enzyme-linked immunosorbent assay has a certain clinical value in diagnosis of children with IgAN. PMID:26221341

  19. Transport of galactose, glucose and their molecular analogues by Escherichia coli K12.

    PubMed Central

    Henderson, P J; Giddens, R A; Jones-Mortimer, M C

    1977-01-01

    1. Strains of Escherichia coli K12 were made that are unable to assimilate glucose by the phosphotransferase system, since they lack the glucose-specific components specified by the genes ptsG and ptsM. 2. Derivative organisms lacking the methyl galactoside or galactose-specific transport system were examined for their ability to transport galactose, d-fucose, methyl beta-D-galactoside, glucose, 2-deoxy-D-glucose and methyl alpha-D-glucoside. 3. Galactose, glucose and to a lesser extent fucose are substrates for both transport systems. 4. 2-Deoxyglucose is transported on the galactose-specific but not the methyl galactoside system. 5. The ability of sugars to elicit anaerobic proton transport is associated with the galactose-specific, but not with the methyl galactoside transport activity. Hence a chemiosmotic mechanism of energization is likely to apply to the former but not to the latter. Alternatively the methyl galactoside system may be switched off under certain conditions, which would indicate a novel regulatory mechanism. 6. Details of the procedure for the derivation of strains may be obtained from the authors, and have been deposited as Supplementary Publication SUP 50074 (8 pages at the) British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977), 161,1. PMID:15558

  20. Transport of d-galactose by the gastrointestinal tract of the locust, Locusta migratoria.

    PubMed

    Pascual, I; Berjón, A; Lostao, M P; Barber, A

    2006-01-01

    Due to exoskeleton, the absorption of nutrients in adult insects takes place across the gastrointestinal tract epithelium. In most physiological studies, sugar intestinal absorption has been described as a diffusional process and to date no sugar transporter has been cloned from the digestive tract of insects. In the present work, the existence of a saturable transport system for galactose in the gastric caeca of Locusta migratoria is clearly demonstrated. This transport shows a relatively high affinity for galactose (apparent K0.5=2-3 mM) and is inhibited by glucose, 2-deoxyglucose and with less potency by fructose and alpha-methyl-d-glucoside. The absence of sodium or the presence of phloridzin hardly affects galactose absorption, indicating that it is not mediated by a SGLT1-like transporter. The absence of K+, Cl-, Mg2+ and Ca2+ or changes in the pH do not modify galactose absorption either. Nevertheless, phloretin, cytochalasin B and theophylline (inhibitors of facilitative transporters) decrease sugar uptake around 50%. Xenopus laevis oocytes microinjected with poly A+ RNA isolated from gastric caeca show sodium-independent galactose uptake that is three times higher than in non-injected oocytes, further supporting the existence of a mRNA coding for at least one equilibrative sugar transporter in L. migratoria gastric caeca. PMID:16314134

  1. Balamuthia mandrillaris: role of galactose in encystment and identification of potential inhibitory targets.

    PubMed

    Siddiqui, Ruqaiyyah; Jarroll, Edward L; Khan, Naveed Ahmed

    2010-09-01

    Balamuthia mandrillaris is a causative agent of granulomatous encephalitis that almost always proves fatal. A major concern during the course of therapy is that B. mandrillaris can transform into cysts. Cysts are highly resistant to physical and chemical conditions and present a problem in successful antimicrobial chemotherapy. However, the underlying mechanisms of B. mandrillaris transformation into cysts are not known. In this study, we examined the effects of exogenous sugars on B. mandrillaris encystment. The findings revealed that free exogenous galactose, but not other sugars, enhanced parasite differentiation into cysts, and apparently a galactose-binding protein is involved in B. mandrillaris encystment. Cytoskeletal re-arrangements and phosphatidylinositol 3-kinase (PI3K)-mediated pathways are involved in B. mandrillaris encystment based on inhibitor studies. Dual functionality of galactose-binding protein in B. mandrillaris pathogenesis and encystment is discussed further. PMID:19766634

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

    PubMed

    Bourne, J R; Hegglin, M; Prenosil, J E

    1983-06-01

    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. PMID:18551445

  3. Accumulation of myoinositol and rubidium ions in galactose-exposed rat lens

    SciTech Connect

    Kawaba, T.; Cheng, H.M.; Kinoshita, J.H.

    1986-10-01

    When rat lens is incubated in 30 mM galactose overnight, the extent of accumulation of rubidium ions (Rb) and myoinositol (MI) are affected, as well as the Na-K ATPase activity. Rb accumulation and Na-K ATPase activity are only slightly affected compared to the dramatic drop in MI accumulation. These changes are completely abolished by sorbinil, which blocks polyol formation, or by rendering the galactose medium hypertonic to offset the osmotic effect of polyol formation. On the other hand, the addition of excess MI to the galactose medium had no effect on correcting these changes. The results obtained are consistent with the polyol-osmotic theory of sugar cataract formation.

  4. Targeted allylation and propargylation of galactose-containing polysaccharides in water.

    PubMed

    Leppänen, Ann-Sofie; Xu, Chunlin; Parikka, Kirsti; Eklund, Patrik; Sjöholm, Rainer; Brumer, Harry; Tenkanen, Maija; Willför, Stefan

    2014-01-16

    Galactose units of spruce galactoglucomannan (GGM), guar galactomannan (GM), and tamarind (galacto)xyloglucan (XG) were selectively allylated. Firstly aldehyde functionalities were formed at the C-6 position via enzymatic oxidation by galactose oxidase. The formed aldehydes were further derivatized by an indium mediated Barbier-Grignard type reaction, resulting in the formation of homoallylic alcohols. In addition to allylic halides, the same reaction procedure was also applicable for GGM, when using propargyl bromide as halide. All reaction steps were done in water, thus the polysaccharides were modified in a one-pot reaction. The formation of the allylated, or propargylated, product was identified by MALDI-TOF-MS. All polysaccharide products were isolated and further characterized by GC-MS or NMR spectroscopy. By this chemo-enzymatic process, we have demonstrated a novel method for derivatization of GGM and other galactose-containing polysaccharides. The derivatized polysaccharides are potential platforms for further functionalizations. PMID:24188837

  5. Substitution of L-fucose by L-galactose in cell walls of arabidopsis mur1

    SciTech Connect

    Zablackis, E.; York, W.S.; Pauly, M.

    1996-06-21

    An Arabidopsis thaliana mutant (mur1) has less than 2 percent of the normal amounts of L-fucose in the primary cell walls of aerial portions of the plant. The survival of mur1 plants challenged the hypothesis that fucose is a required component of biologically active oligosaccharides derived from cell wall xyloglucan. However, the replacement of L-fucose (that is, 6-deoxyl-L-galactose) by L-galactose does not detectably alter the biological activity of the oligosaccharides derived from xyloglucan. Thus, essential structural and conformational features of xyloglucan and xyloglucan-derived oligosaccharides are retained when L-galactose replaces L-fucose. 29 refs., 2 figs., 2 tabs.

  6. A Second GDP-l-galactose Phosphorylase in Arabidopsis en Route to Vitamin C

    PubMed Central

    Linster, Carole L.; Adler, Lital N.; Webb, Kristofor; Christensen, Kathryn C.; Brenner, Charles; Clarke, Steven G.

    2008-01-01

    The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-l-galactose to l-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and l-galactose 1-phosphate from GDP-l-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-l-galactose, forming l-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP. PMID:18463094

  7. Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model

    PubMed Central

    Tang, Manshu; Siddiqi, Anwer; Witt, Benjamin; Yuzyuk, Tatiana; Johnson, Britt; Fraser, Nisa; Chen, Wyman; Rascon, Rafael; Yin, Xue; Goli, Harish; Bodamer, Olaf A; Lai, Kent

    2014-01-01

    The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies. PMID:24549051

  8. Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model.

    PubMed

    Tang, Manshu; Siddiqi, Anwer; Witt, Benjamin; Yuzyuk, Tatiana; Johnson, Britt; Fraser, Nisa; Chen, Wyman; Rascon, Rafael; Yin, Xue; Goli, Harish; Bodamer, Olaf A; Lai, Kent

    2014-10-01

    The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies. PMID:24549051

  9. How strict is galactose restriction in adults with galactosaemia? International practice.

    PubMed

    Adam, S; Akroyd, R; Bernabei, S; Bollhalder, S; Boocock, S; Burlina, A; Coote, T; Corthouts, K; Dalmau, J; Dawson, S; Defourny, S; De Meyer, A; Desloovere, A; Devlin, Y; Diels, M; Dokoupil, K; Donald, S; Evans, S; Fasan, I; Ferguson, C; Ford, S; Forga, M; Gallo, G; Grünert, S C; Heddrich-Ellerbrok, M; Heidenborg, C; Jonkers, C; Lefebure, K; Luyten, K; MacDonald, A; Meyer, U; Micciche, A; Müller, E; Portnoi, P; Ripley, S; Robert, M; Robertson, L V; Rosenbaum-Fabian, S; Sahm, K; Schultz, S; Singleton, K; Sjöqvist, E; Stoelen, L; Terry, A; Thompson, S; Timmer, C; Vande Kerckhove, K; van der Ploeg, L; Van Driessche, M; van Rijn, M; van Teeffelen-Heithoff, A; Vitoria, I; Voillot, C; Wenz, J; Westbrook, M; Wildgoose, J; Zweers, H

    2015-05-01

    Dietary management of 418 adult patients with galactosaemia (from 39 centres/12 countries) was compared. All centres advised lactose restriction, 6 restricted galactose from galactosides ± fruits and vegetables and 12 offal. 38% (n=15) relaxed diet by: 1) allowing traces of lactose in manufactured foods (n=13) or 2) giving fruits, vegetables and galactosides (n=2). Only 15% (n=6) calculated dietary galactose. 32% of patients were lost to dietetic follow-up. In adult galactosaemia, there is limited diet relaxation. PMID:25873073

  10. Intracerebroventricular D-galactose administration impairs memory and alters activity and expression of acetylcholinesterase in the rat.

    PubMed

    Rodrigues, André Felipe; Biasibetti, Helena; Zanotto, Bruna Stela; Sanches, Eduardo Farias; Pierozan, Paula; Schmitz, Felipe; Parisi, Mariana Migliorini; Barbé-Tuana, Florencia; Netto, Carlos Alexandre; Wyse, Angela T S

    2016-05-01

    Tissue accumulation of galactose is a hallmark in classical galactosemia. Cognitive deficit is a symptom of this disease which is poorly understood. The aim of this study was to investigate the effects of intracerebroventricular administration of galactose on memory (inhibitory avoidance and novel object recognition tasks) of adult rats. We also investigated the effects of galactose on acetylcholinesterase (AChE) activity, immunocontent and gene expression in hippocampus and cerebral cortex. Wistar rats received a single injection of galactose (4mM) or saline (control). For behavioral parameters, galactose was injected 1h or 24h previously to the testing. For biochemical assessment, animals were decapitated 1h, 3h or 24h after galactose or saline injection; hippocampus and cerebral cortex were dissected. Results showed that galactose impairs the memory formation process in aversive memory (inhibitory avoidance task) and recognition memory (novel object recognition task) in rats. The activity of AChE was increased, whereas the gene expression of this enzyme was decreased in hippocampus, but not in cerebral cortex. These findings suggest that these changes in AChE may, at least in part, to lead to memory impairment caused by galactose. Taken together, our results can help understand the etiopathology of classical galactosemia. PMID:26948151

  11. Possible role of metal ionophore against zinc induced cognitive dysfunction in D-galactose senescent mice.

    PubMed

    Bharti, Kanchan; Majeed, Abu Bakar Abdul; Prakash, Atish

    2016-06-01

    Metal ionophores are considered as potential anti-dementia agents, and some are currently undergoing clinical trials. Many metals are known to accumulate and distribute abnormally in the aging brain. Alterations in zinc metal homeostasis in the glutaminergic synapse could contribute to ageing and the pathophysiology of Alzheimer's disease (AD). The present study was designed to investigate the effect of metal ionophores on long term administration of zinc in D-galactose induced senescent mice. The ageing model was established by combined administration of zinc and D-galactose to mice for 6 weeks. A novel metal ionophore, PBT-2 was given daily to zinc-induced d-galactose senescent mice. The cognitive behaviour of mice was monitored using the Morris Water Maze. The anti-oxidant status and amyloidogenic activity in the ageing mouse was measured by determining mito-oxidative parameters and deposition of amyloid β (Aβ) in the brain. Systemic administration of both zinc and D-galactose significantly produced memory deficits, mito-oxidative damage, heightened acetylcholinesterase enzymatic activity and deposition of amyloid-β. Treatment with PBT-2 significantly improved behavioural deficits, biochemical profiles, cellular damage, and curbed the deposition of APP in zinc-induced senescent mice. These findings suggest that PBT-2, acting as a metal protein attenuating compound, may be helpful in the prevention of AD or alleviation of ageing. PMID:26923568

  12. Delayed Anaphylaxis to Red Meat Associated With Specific IgE Antibodies to Galactose

    PubMed Central

    Wen, Liping; Zhou, Junxiong; Sun, Jin-lu; Sun, Yi; Wu, Kai; Katial, Rohit

    2015-01-01

    A novel delayed anaphylactic reaction to red meat, associated with tick bites and IgE antibodies against galactose-α-1, 3-galactose (α-gal), was reported in 2009 in the US, Australia and Europe. In this case, serum specific IgE to galactose-α-1, 3-galactose (>100 kU/L) and IgE to multiple non-primate mammalian proteins were positive. However, the pathogenesis of this disease remains unclear. We report the first case in Asia of delayed anaphylactic reaction to red meat, which was induced by bites from the hard tick, Hematophagous ixodidae. We confirmed the increased concentration of IgE reactive epitopes in non-primate mammalian organs, which may be rich in α-gal proteins in lymphatic and endothelial tissues. All confirmed ticks associated with this disorder in the literature and in our case belonged to the hard tick family. We hypothesize that hard tick saliva is enriched with blood-type substances, such as oligosaccharides, from the non-primate mammal victim's blood after days to weeks of blood sucking, which sensitizes humans through the injection route while blood sucking. PMID:25553269

  13. Moderate exercise prevents neurodegeneration in D-galactose-induced aging mice.

    PubMed

    Li, Li; Xu, Meng; Shen, Bo; Li, Man; Gao, Qian; Wei, Shou-Gang

    2016-05-01

    D-galactose has been widely used in aging research because of its efficacy in inducing senescence and accelerating aging in animal models. The present study investigated the benefits of exercise for preventing neurodegeneration, such as synaptic plasticity, spatial learning and memory abilities, in mouse models of aging. D-galactose-induced aging mice were administered daily subcutaneous injections of D-galactose at the base of the neck for 10 consecutive weeks. Then, the mice were subjected to exercise training by running on a treadmill for 6 days a week. Shortened escape latency in a Morris water maze test indicated that exercise improved learning and memory in aging mice. The ameliorative changes were likely induced by an upregulation of Bcl-2 and brain-derived neurotrophic factor, the repression of apoptosis factors such as Fas and Bax, and an increase in the activity of glucose transporters-1 and 4. The data suggest moderate exercise may retard or inhibit neurodegeneration in D-galactose-induced aging mice. PMID:27335566

  14. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES...

  15. Moderate exercise prevents neurodegeneration in D-galactose-induced aging mice

    PubMed Central

    Li, Li; Xu, Meng; Shen, Bo; Li, Man; Gao, Qian; Wei, Shou-gang

    2016-01-01

    D-galactose has been widely used in aging research because of its efficacy in inducing senescence and accelerating aging in animal models. The present study investigated the benefits of exercise for preventing neurodegeneration, such as synaptic plasticity, spatial learning and memory abilities, in mouse models of aging. D-galactose-induced aging mice were administered daily subcutaneous injections of D-galactose at the base of the neck for 10 consecutive weeks. Then, the mice were subjected to exercise training by running on a treadmill for 6 days a week. Shortened escape latency in a Morris water maze test indicated that exercise improved learning and memory in aging mice. The ameliorative changes were likely induced by an upregulation of Bcl-2 and brain-derived neurotrophic factor, the repression of apoptosis factors such as Fas and Bax, and an increase in the activity of glucose transporters-1 and 4. The data suggest moderate exercise may retard or inhibit neurodegeneration in D-galactose-induced aging mice. PMID:27335566

  16. Fap2 of Fusobacterium nucleatum Is a Galactose-Inhibitable Adhesin Involved in Coaggregation, Cell Adhesion, and Preterm Birth

    PubMed Central

    Coppenhagen-Glazer, S.; Sol, A.; Abed, J.; Naor, R.; Zhang, X.

    2015-01-01

    Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria. PMID:25561710

  17. A re-evaluation of life-long severe galactose restriction for the nutrition management of classic galactosemia.

    PubMed

    Van Calcar, Sandra C; Bernstein, Laurie E; Rohr, Frances J; Scaman, Christine H; Yannicelli, Steven; Berry, Gerard T

    2014-07-01

    The galactose-restricted diet is life-saving for infants with classic galactosemia. However, the benefit and extent of dietary galactose restriction required after infancy remain unclear and variation exists in practice. There is a need for evidence-based recommendations to better standardize treatment for this disorder. This paper reviews the association between diet treatment and outcomes in classic galactosemia and evaluates the contribution of food sources of free galactose in the diet. Recommendations include allowing all fruits, vegetables, legumes, soy products that are not fermented, various aged cheeses and foods containing caseinates. Further research directions are discussed. PMID:24857409

  18. Delayed Anaphylaxis to Red Meat in Patients with IgE Specific for Galactose alpha-1,3-Galactose (alpha-gal)

    PubMed Central

    Platts-Mills, Thomas A. E.

    2012-01-01

    Anaphylaxis is a severe allergic reaction that can be rapidly progressing and fatal. In instances where the triggering allergen is not known, establishing the etiology of anaphylaxis is pivotal to long-term risk management. Our recent work has identified a novel IgE antibody (Ab) response to a mammalian oligosaccharide epitope, galactose-alpha-1,3-galactose (alpha-gal), that has been associated with two distinct forms of anaphylaxis: (1) immediate onset anaphylaxis during first exposure to intravenous cetuximab, and (2) delayed onset anaphylaxis 3–6 h after ingestion of mammalian food products (e.g., beef and pork). The results of our studies strongly suggest that tick bites are a cause, if not the only significant cause, of IgE Ab responses to alpha-gal in the southern, eastern and central United States. Patients with IgE Ab to alpha-gal continue to emerge and, increasingly, these cases involve children. This IgE Ab response cross-reacts with cat and dog but does not appear to pose a risk for asthma; however, it may impair diagnostic testing in some situations. PMID:23054628

  19. Galactose-functionalized multi-responsive nanogels for hepatoma-targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Lou, Shaofeng; Gao, Shan; Wang, Weiwei; Zhang, Mingming; Zhang, Ju; Wang, Chun; Li, Chen; Kong, Deling; Zhao, Qiang

    2015-02-01

    We report here a hepatoma-targeting multi-responsive biodegradable crosslinked nanogel, poly(6-O-vinyladipoyl-d-galactose-ss-N-vinylcaprolactam-ss-methacrylic acid) P(ODGal-VCL-MAA), using a combination of enzymatic transesterification and emulsion copolymerization for intracellular drug delivery. The nanogel exhibited redox, pH and temperature-responsive properties, which can be adjusted by varying the monomer feeding ratio. Furthermore, the volume phase transition temperature (VPTT) of the nanogels was close to body temperature and can result in rapid thermal gelation at 37 °C. Scanning electron microscopy also revealed that the P(ODGal-VCL-MAA) nanogel showed uniform spherical monodispersion. With pyrene as a probe, the fluorescence excitation spectra demonstrated nanogel degradation in response to glutathione (GSH). X-ray diffraction (XRD) showed an amorphous property of DOX within the nanogel, which was used in this study as a model anti-cancer drug. Drug-releasing characteristics of the nanogel were examined in vitro. The results showed multi-responsiveness of DOX release by the variation of environmental pH values, temperature or the availability of GSH, a biological reductase. An in vitro cytotoxicity assay showed a higher anti-tumor activity of the galactose-functionalized DOX-loaded nanogels against human hepatoma HepG2 cells, which was, at least in part, due to specific binding between the galactose segments and the asialoglycoprotein receptors (ASGP-Rs) in hepatic cells. Confocal laser scanning microscopy (CLSM) and flow cytometric profiles further confirmed elevated cellular uptake of DOX by the galactose-functionalised nanogels. Thus, we report here a multi-responsive P(ODGal-VCL-MAA) nanogel with a hepatoma-specific targeting ability for anti-cancer drug delivery.We report here a hepatoma-targeting multi-responsive biodegradable crosslinked nanogel, poly(6-O-vinyladipoyl-d-galactose-ss-N-vinylcaprolactam-ss-methacrylic acid) P(ODGal-VCL-MAA), using

  20. Metabolic pathway of 3,6-anhydro-D-galactose in carrageenan-degrading microorganisms.

    PubMed

    Lee, Sun Bok; Kim, Jeong Ah; Lim, Hyun Seung

    2016-05-01

    Complete hydrolysis of κ-carrageenan produces two sugars, D-galactose and 3,6-anhydro-D-galactose (D-AnG). At present, however, we do not know how carrageenan-degrading microorganisms metabolize D-AnG. In this study, we investigated the metabolic pathway of D-AnG degradation by comparative genomic analysis of Cellulophaga lytica LIM-21, Pseudoalteromonas atlantica T6c, and Epulopiscium sp. N.t. morphotype B, which represent the classes Flavobacteria, Gammaproteobacteria, and Clostridia, respectively. In this bioinformatic analysis, we found candidate common genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the D-AnG cluster. In all three microorganisms, D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named as the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions in the following route: 3,6-anhydro-D-galactose → 3,6-anhydro-D-galactonate → 2-keto-3-deoxy-D-galactonate (D-KDGal) → 2-keto-3-deoxy-6-phospho-D-galactonate → pyruvate + D-glyceraldehyde-3-phosphate. The pathway of D-AnG degradation is composed of two parts: transformation of D-AnG to D-KDGal using two D-AnG specific enzymes and breakdown of D-KDGal to two glycolysis intermediates using two DeLey-Doudoroff pathway enzymes. To our knowledge, this is the first report on the metabolic pathway of D-AnG degradation. PMID:26875872

  1. A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein

    NASA Technical Reports Server (NTRS)

    Salins, L. L.; Ware, R. A.; Ensor, C. M.; Daunert, S.

    2001-01-01

    The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible. Copyright 2001 Academic Press.

  2. Galactose-functionalized multi-responsive nanogels for hepatoma-targeted drug delivery.

    PubMed

    Lou, Shaofeng; Gao, Shan; Wang, Weiwei; Zhang, Mingming; Zhang, Ju; Wang, Chun; Li, Chen; Kong, Deling; Zhao, Qiang

    2015-02-21

    We report here a hepatoma-targeting multi-responsive biodegradable crosslinked nanogel, poly(6-O-vinyladipoyl-D-galactose-ss-N-vinylcaprolactam-ss-methacrylic acid) P(ODGal-VCL-MAA), using a combination of enzymatic transesterification and emulsion copolymerization for intracellular drug delivery. The nanogel exhibited redox, pH and temperature-responsive properties, which can be adjusted by varying the monomer feeding ratio. Furthermore, the volume phase transition temperature (VPTT) of the nanogels was close to body temperature and can result in rapid thermal gelation at 37 °C. Scanning electron microscopy also revealed that the P(ODGal-VCL-MAA) nanogel showed uniform spherical monodispersion. With pyrene as a probe, the fluorescence excitation spectra demonstrated nanogel degradation in response to glutathione (GSH). X-ray diffraction (XRD) showed an amorphous property of DOX within the nanogel, which was used in this study as a model anti-cancer drug. Drug-releasing characteristics of the nanogel were examined in vitro. The results showed multi-responsiveness of DOX release by the variation of environmental pH values, temperature or the availability of GSH, a biological reductase. An in vitro cytotoxicity assay showed a higher anti-tumor activity of the galactose-functionalized DOX-loaded nanogels against human hepatoma HepG2 cells, which was, at least in part, due to specific binding between the galactose segments and the asialoglycoprotein receptors (ASGP-Rs) in hepatic cells. Confocal laser scanning microscopy (CLSM) and flow cytometric profiles further confirmed elevated cellular uptake of DOX by the galactose-functionalised nanogels. Thus, we report here a multi-responsive P(ODGal-VCL-MAA) nanogel with a hepatoma-specific targeting ability for anti-cancer drug delivery. PMID:25613320

  3. Galacturonic Acid Inhibits the Growth of Saccharomyces cerevisiae on Galactose, Xylose, and Arabinose

    PubMed Central

    Huisjes, Eline H.; de Hulster, Erik; van Dam, Jan C.; Pronk, Jack T.

    2012-01-01

    The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pKa value of galacturonic acid (3.51), the addition of 10 g · liter−1 galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter−1 galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks. PMID:22582063

  4. Separation of galactose, 5-hydroxymethylfurfural and levulinic acid in acid hydrolysate of agarose by nanofiltration and electrodialysis.

    PubMed

    Kim, Jae Hyung; Na, Jeong-Geol; Yang, Ji-Won; Chang, Yong Keun

    2013-07-01

    A two-stage membrane process for the separation of galactose, 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA) has been proposed. The first step of nanofiltration (NF) is to remove 5-HMF and LA from galactose solution obtained by the hydrolysis of agarose, the main component of red algal galactan for the reduction of its microbial toxicity. 5-HMF and LA are inhibitory to fermentation but at the same time useful compounds themselves with many applications. The second step of electrodialysis (ED) is to separate 5-HMF and LA in the permeate from NF. More than 91% of 5-HMF and up to 62% of LA could be removed from agarose hydrolysate, while galactose was almost completely retained by NF. Further removal of LA was expected to be possible with no loss of galactose by operating the NF process in a diafiltration mode. 5-HMF and LA could be effectively separated from each other by ED. PMID:23672940

  5. A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species 1

    PubMed Central

    Gross, Kenneth C.; Pharr, David M.

    1982-01-01

    Conversion of [14C]galactose (Gal) 1-P, UDP-[14C]Gal, or UDP-[14C]glucose to [14C]sucrose was observed when cell-free homogenates of cucumber (Cucumis sativus L.) fruit peduncles were incubated with individual 14C-labeled substrates, appropriate cofactors, and fructose. The sucrose product was labeled only in the glucose moiety. Conversion of [14C]Gal-1-P to [14C]sucrose was catalyzed by extracts of peduncles from all other stachyose transporting species tested, as well as green bean (a sucrose transporter) but was not catalyzed by peduncle extracts from three other sucrose transporting species. In cucumber, the ability of extracts to form [14C]sucrose from [14C]Gal-1-P was greater when peduncles were harvested from growing fruit than from unpollinated ovaries. [14C]Sucrose formation from [14C]Gal-1-P was inhibited by Mg · PPi, Mg · UDP, UMP, and sucrose. α-Galactosidase, galactokinase, UDP-gal pyrophosphorylase, UDP-Gal-4′-epimerase, UDP-glucose pyrophosphorylase, and sucrose synthase activities were detected in peduncle extracts. Neither sucrose phosphate synthetase nor hexose-1-P uridyltransferase were detected. Peduncle tissue contained a small pool of free galactose. These results suggest a potential pathway for the metabolism of galactose moieties hydrolyzed from stachyose, the major sugar transported by cucumber plants. PMID:16662141

  6. Structure-function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family.

    PubMed

    Yin, DeLu Tyler; Urresti, Saioa; Lafond, Mickael; Johnston, Esther M; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H; Davies, Gideon J; Brumer, Harry

    2015-01-01

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure-function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

  7. Crystal structure of rice alpha-galactosidase complexed with D-galactose.

    PubMed

    Fujimoto, Zui; Kaneko, Satoshi; Momma, Mitsuru; Kobayashi, Hideyuki; Mizuno, Hiroshi

    2003-05-30

    alpha-Galactosidases catalyze the hydrolysis of alpha-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice alpha-galactosidase has been determined at 1.5A resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of alpha-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (beta/alpha)8-barrel structure, and the C-terminal domain was made up of eight beta-strands containing a Greek key motif. The structure was solved as a complex with d-galactose, providing a mode of substrate binding in detail. The d-galactose molecule was found bound in the active site pocket on the C-terminal side of the central beta-barrel of the catalytic domain. The d-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice alpha-galactosidase with chicken alpha-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes. PMID:12657636

  8. [Neonatal diarrhea due to congenital glucose-galactose malabsorption: report of seven cases].

    PubMed

    Chedane-Girault, C; Dabadie, A; Maurage, C; Piloquet, H; Chailloux, E; Colin, E; Pelatan, C; Giniès, J-L

    2012-12-01

    Congenital glucose-galactose malabsorption (CGGM) is a rare autosomal recessive disorder, which presents as a protracted diarrhea in early neonatal life. We describe the clinical history, diagnostic evaluation, and management of 7 children with CGGM in western France. There were 4 girls and 3 boys from 5 families, born between 1984 and 2010. The principal complaint was a neonatal onset of watery and acidic severe diarrhea complicated by hypertonic dehydration. The diarrhea stopped with fasting. In 2 cases, the family history supported the diagnosis. In the other cases, elimination of glucose and galactose (lactose) from the diet resulted in the complete resolution of diarrhea symptoms. In 2 cases, the H2 breath tests were positive. In 2 cases, the HGPO or oral glucose tolerance test (OGTT) demonstrated an abnormal curve with glucose and a normal curve with fructose. DNA sequencing was not used. When glucose and galactose were eliminated from the diet, the infants had normal growth and development. In conclusion, CGGM is a rare etiology of neonatal diarrhea; however, the diagnosis is easy to make and the prognosis is excellent. PMID:23107089

  9. Yulangsan polysaccharide improves redox homeostasis and immune impairment in D-galactose-induced mimetic aging.

    PubMed

    Doan, Van Minh; Chen, Chunxia; Lin, Xing; Nguyen, Van Phuc; Nong, Zhihuan; Li, Weisi; Chen, Qingquan; Ming, Jianjun; Xie, Qiuqiao; Huang, Renbin

    2015-05-01

    Yulangsan polysaccharide (YLSP) is a traditional Chinese medicine used in long-term treatment as a modulator of brain dysfunction and immunity. In this study, we evaluated the protective effect of YLSP against D-galactose-induced impairment of oxidative stress and the immune system and evaluated its possible mechanism of action. D-galactose was subcutaneously injected into the dorsal neck of mice daily for 8 weeks to establish the aging model. YLSP was simultaneously administered once daily. The results indicate that YLSP significantly improves the general appearance of the aging mice. YLSP significantly increased the levels of antioxidant enzymes, such as super oxide dismutase, glutathione peroxidase, catalase and total anti-oxidation capability, while decreasing the content of malondialdehyde in different tissues, including the liver, brain, and serum. YLSP also increased the interleukin-2 level while decreasing the interleukin-6 level. Moreover, YLSP significantly inhibited advanced glycation end product formation. Furthermore, YLSP decreased p21 and p53 gene expressions in the liver and brain of D-galactose-treated mice. These results suggest that YLSP may have a protective effect suppressing the aging process by enhancing antioxidant activity and immunity, as well as modulating aging-related gene expression. PMID:25920068

  10. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

    PubMed Central

    Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

    2015-01-01

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

  11. Effects of rhein lysinate on D-galactose-induced aging mice

    PubMed Central

    ZHEN, YONG-ZHAN; LIN, YA-JUN; LI, KAI-JI; ZHANG, GUANG-LING; ZHAO, YU-FANG; WANG, MEI-MEI; WEI, JING-BO; WEI, JIE; HU, GANG

    2016-01-01

    The aim of the present study was to investigate the anti-aging effects of rhein lysinate (RHL), and to explore its mechanism of action in a D-galactose-induced aging mouse model. Aging was induced by D-galactose (100 mg/kg/day) that was subcutaneously injected to animals for 8 weeks. RHL was simultaneously administered once a day by intragastric gavage. The appetite, mental condition, body weight and organ index of the mice were monitored. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined, and the levels of malondialdehyde (MDA) in the liver, kidney and serum were measured by appropriate assay kits. Western blot analysis was used to detect proteins associated with age. The results indicated that RHL may improve the appetite, mental state and organ conditions of the model mice, improve the activities of SOD and GSH-Px, reduce MDA levels and modulate the expression of age-associated proteins (Sirtuin 1, p21 and p16) in D-galactose-induced mice. Therefore, RHL may be effective at suppressing the aging process through a combination of enhancing antioxidant activity, scavenging free radicals and modulating aging-associated gene expression. PMID:26889258

  12. IgG N-Glycosylation Galactose Incorporation Ratios for the Monitoring of Classical Galactosaemia.

    PubMed

    Stockmann, Henning; Coss, Karen P; Rubio-Gozalbo, M Estela; Knerr, Ina; Fitzgibbon, Maria; Maratha, Ashwini; Wilson, James; Rudd, Pauline; Treacy, Eileen P

    2016-01-01

    Classical galactosaemia (OMIM #230400) is a rare disorder of carbohydrate metabolism caused by deficiency of the galactose-1-phosphate uridyltransferase enzyme (EC 2.7.7.12). The cause of the long-term complications, including neurological, cognitive and fertility problems in females, remains poorly understood. The relatively small number of patients with galactosaemia and the lack of validated biomarkers pose a substantial challenge for determining prognosis and monitoring disease progression and responses to new therapies. We report an improved method of automated robotic hydrophilic interaction ultra-performance liquid chromatography N-glycan analysis for the measurement of IgG N-glycan galactose incorporation ratios applied to the monitoring of adult patients with classical galactosaemia. We analysed 40 affected adult patients and 81 matched healthy controls. Significant differences were noted between the G0/G1 and G0/G2 incorporation ratios between galactosaemia patients and controls (p < 0.001 and <0.01, respectively). Our data indicate that the use of IgG N-glycosylation galactose incorporation analysis may be now applicable for monitoring patient dietary compliance, determining prognosis and the evaluation of potential new therapies. PMID:26419375

  13. Galactose utilization sheds new light on sugar metabolism in the sequenced strain Dekkera bruxellensis CBS 2499.

    PubMed

    Moktaduzzaman, Md; Galafassi, Silvia; Capusoni, Claudia; Vigentini, Ileana; Ling, Zhihao; Piškur, Jure; Compagno, Concetta

    2015-03-01

    Dekkera bruxellensis and Saccharomyces cerevisiae are considered two phylogenetically distant relatives, but they share several industrial relevant traits such as the ability to produce ethanol under aerobic conditions (Crabtree effect), high tolerance towards ethanol and acids, and ability to grow without oxygen. Beside a huge adaptability, D. bruxellensis exhibits a broader spectrum in utilization of carbon and nitrogen sources in comparison to S. cerevisiae. With the aim to better characterize its carbon source metabolism and regulation, the usage of galactose and the role that glucose plays on sugar metabolism were investigated in D. bruxellensis CBS 2499. The results indicate that in this yeast galactose is a non-fermentable carbon source, in contrast to S. cerevisiae that can ferment it. In particular, its metabolism is affected by the nitrogen source. Interestingly, D. bruxellensis CBS 2499 exhibits the 'short-term Crabtree effect', and the expression of genes involved in galactose utilization and in respiratory metabolism is repressed by glucose, similarly to what occurs in S. cerevisiae. PMID:25673757

  14. Piperine and curcumin exhibit synergism in attenuating D-galactose induced senescence in rats.

    PubMed

    Banji, David; Banji, Otilia J F; Dasaroju, Swetha; Annamalai, A R

    2013-03-01

    Aging is associated with progressive decline in mental abilities and functional capacities. Postmitotic tissues are most vulnerable to alteration due to oxidative damage leading to behavioral and biochemical changes. We hypothesized that the anatomical and functional facets of the brain could be protected with powerful antioxidants such as piperine and curcumin by examining their effects individually and in combination in delaying senescence induced by d-galactose. Young adult male Wistar rats were treated with piperine (12 mg/kg) alone, and curcumin (40 mg/kg) alone; and in combination for a period of 49 days by the oral route with treatment being initiated a week prior to d-galactose (60 mg/kg, i.p.). A control group, d-galactose alone and naturally aged control were also evaluated. Behavioral tests, hippocampal volume, CA1 neuron number, oxidative parameters, formation of lipofuscin like autofluorescent substances, neurochemical estimation, and histopathological changes in CA1 region of hippocampus were established. Our results suggest that the combination exerted a superior response compared to monotherapy as evidenced by improved spatial memory, reduced oxidative burden, reduced accumulation of lipofuscin; improvement in signaling, increase in hippocampal volume and protection of hippocampal neurons. We speculate that the powerful antioxidant nature of both, augmented response of curcumin in the presence of piperine and enhanced serotoninergic signaling was responsible for improved cognition and prevention in senescence. PMID:23200897

  15. Identification of PblB mediating galactose-specific adhesion in a successful Streptococcus pneumoniae clone

    PubMed Central

    Hsieh, Yu-Chia; Lin, Tzu-Lung; Lin, Che-Ming; Wang, Jin-Town

    2015-01-01

    The pneumococcal genome is variable and there are minimal data on the influence of the accessory genome on phenotype. Pneumococcal serotype 14 sequence type (ST) 46 had been the most prevalent clone causing pneumonia in children in Taiwan. A microarray was constructed using the genomic DNA of a clinical strain (NTUH-P15) of serotype 14 ST46. Using DNA hybridization, genomic variations in NTUH-P15 were compared to those of 3 control strains. Microarray analysis identified 7 genomic regions that had significant increases in hybridization signals in the NTUH-P15 strain compared to control strains. One of these regions encoded PblB, a phage-encoded virulence factor implicated (in Streptococcus mitis) in infective endocarditis. The isogenic pblB mutant decreased adherence to A549 human lung epithelial cell compared to wild-type NTUH-P15 strain (P = 0.01). Complementation with pblB restored the adherence. PblB is predicted to contain a galactose-binding domain-like region. Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner. Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung. PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success. PMID:26193794

  16. Assessment of liver function in dogs using the 13C-galactose breath test.

    PubMed

    Silva, S; Wyse, C A; Goodfellow, M R; Yam, P S; Preston, T; Papasouliotis, K; Hall, E J

    2010-08-01

    The aim of this study was to evaluate the application of the 13C-galactose breath test (13C-GBT) in assessing canine liver function by applying it to a group of healthy dogs, and to a group with clinicopathological evidence of liver dysfunction. Breath samples were collected 30 min before ingestion of 13C-galactose, and then at regular intervals thereafter for 6 h. The proportion of 13CO2/12CO2 in the breath samples was measured by isotope-ratio mass spectrometry. There was no significant difference in recovery of 13CO2 in the diseased group, compared to the healthy controls, but there was considerable inter-subject variation in both groups, possibly due to differences in the rate of gastric emptying, which could preclude detection of alterations in hepatic metabolism of galactose. The results of this study do not support the application of the 13C-GBT for assessment of canine liver function. PMID:19546016

  17. Study on galactose-poly(ethylene glycol)-poly(L-lysine) as novel gene vector for targeting hepatocytes in vitro.

    PubMed

    Hu, Hai-mei; Zhang, Xuan; Zhong, Nv-qi; Pan, Shi-rong

    2012-01-01

    A non-viral gene-delivery system has been used to deliver plasmid DNA into specific cell types because of its safety and ease of manufacture. Receptor-mediated gene transfer is currently a promising gene-delivery technique. To specifically target genes to asialoglycoprotein receptor of hepatocytes, a galactose moiety was combined into the poly(ethylene glycol) (PEG)-terminal end by reductive coupling using lactose and sodium cyanoborohydride. A synthesis method of conjugating poly(L-lysine) (PLL) derivatives with terminally galactose-graft-PEG was developed using ring-opening polymerization of N(ε)-benzyloxycarbonyl-L-lysine-N(α)-carboxyan-hydride (Z-Lys-NCA) initiated onto galactose graft amine-terminated PEG (galactose-PEG-NH₂) as a macro-initiator. The synthesis was characterized with ¹H-, ¹³C-NMR, IR and UV spectroscopy, and all of them successfully verified the formation of the co-polymers. The gel-retardation assay of the complexes between galactose-PEG-PLL and plasmid DNA indicated that these polymeric gene carriers demonstrated the potent ability to condense plasmid DNA electrostatically as well as PLL. The particle size and zeta potential of polymer/DNA complexes were measured, and their cytotoxicity and transfection efficiency in different cells were evaluated. The results indicate that galactose-PEG-PLL can form a complex with plasmid DNA and serve as an effective gene-delivery carrier with lower cytotoxicity compared to that of PLL. Transfection experiments clearly showed that galactose-PEG-PLL effectively delivered DNA into hepatoma cells in vitro. Such data demonstrates that galactose and its complex with plasmid DNA may serve as a safe and effective gene-transfer system targeting hepatocytes. PMID:21375808

  18. Metadynamics simulations reveal a Na+ independent exiting path of galactose for the inward-facing conformation of vSGLT.

    PubMed

    Bisha, Ina; Rodriguez, Alex; Laio, Alessandro; Magistrato, Alessandra

    2014-12-01

    Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11-12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters. PMID:25522004

  19. Cyclic voltammetry at TCNQ and TTF-TCNQ modified platinum electrodes: A study of the glucose oxidase/glucose and galactose oxidase/galactose systems

    SciTech Connect

    Hale, P.D.; Skotheim, T.A.

    1988-01-01

    Recent work has shown that the synthetic metal TTF-TCNQ can be used as an electrode material for the oxidation of enzymes containing the prosthetic group flavin adenine dinucleotide (FAD). This direct electron transfer (direct in the sense that oxygen is not a mediator) between reduced enzyme and electrode, a process which does not occur to any measurable extent at a typical metal electrode, is not very well understood. In the present work, electron transfer between reduced glucose oxidase and TTF-TCNQ is investigated using cyclic voltammetry, and it is also shown that TCNQ itself can mediate this electron transfer between the enzyme and a platinum electrode. In addition to the glucose oxidase studies, cyclic voltammetric experiments have been performed on the galactose oxidase system, which contains a copper redox center rather than FAD. The results of these experiments demonstrate that the catalytic ability of TTF-TCNQ in enzyme-based electrochemical sensors is quite general. 15 refs., 4 figs.

  20. Delayed anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for galactose-α-1,3-galactose

    PubMed Central

    Commins, Scott P.; Satinover, Shama M.; Hosen, Jacob; Mozena, Jonathan; Borish, Larry; Lewis, Barrett D.; Woodfolk, Judith A.; Platts-Mills, Thomas A. E.

    2012-01-01

    Background Carbohydrate moieties are frequently encountered in food and can elicit IgE responses, the clinical significance of which has been unclear. Recent work, however, has shown that IgE antibodies to galactose-α-1,3-galactose (α-gal), a carbohydrate commonly expressed on nonprimate mammalian proteins, are capable of eliciting serious, even fatal, reactions. Objective We sought to determine whether IgE antibodies to α-gal are present in sera from patients who report anaphylaxis or urticaria after eating beef, pork, or lamb. Methods Detailed histories were taken from patients presenting to the University of Virginia Allergy Clinic. Skin prick tests (SPTs), intradermal skin tests, and serum IgE antibody analysis were performed for common indoor, outdoor, and food allergens. Results Twenty-four patients with IgE antibodies to α-gal were identified. These patients described a similar history of anaphylaxis or urticaria 3 to 6 hours after the ingestion of meat and reported fewer or no episodes when following an avoidance diet. SPTs to mammalian meat produced wheals of usually less than 4 mm, whereas intradermal or fresh-food SPTs provided larger and more consistent wheal responses. CAP-RAST testing revealed specific IgE antibodies to beef, pork, lamb, cow’s milk, cat, and dog but not turkey, chicken, or fish. Absorption experiments indicated that this pattern of sensitivity was explained by an IgE antibody specific for α-gal. Conclusion We report a novel and severe food allergy related to IgE antibodies to the carbohydrate epitope α-gal. These patients experience delayed symptoms of anaphylaxis, angioedema, or urticaria associated with eating beef, pork, or lamb. PMID:19070355

  1. The relevance of tick bites to the production of IgE antibodies to the mammalian oligosaccharide galactose-α-1,3-galactose

    PubMed Central

    Commins, Scott P.; James, Hayley R.; Kelly, Elizabeth A.; Pochan, Shawna L.; Workman, Lisa J.; Perzanowski, Matthew S.; Kocan, Katherine M.; Fahy, John V.; Nganga, Lucy W.; Ronmark, Eva; Cooper, Philip J.; Platts-Mills, Thomas A. E.

    2011-01-01

    Background In 2009, we reported a novel form of delayed anaphylaxis to red meat, which is related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Most of these patients had tolerated meat for many years previously. The implication is that some exposure in adult life had stimulated the production of these IgE antibodies. Objectives To investigate possible causes of this IgE antibody response, focusing on evidence related to tick bites, which are common in the region where these reactions occur. Methods Serum assays were carried out using biotinylated proteins and extracts bound to a streptavidin ImmunoCAP. Results Prospective studies on IgE antibodies in three subjects following tick bites showed an increase in IgE to alpha-gal of twenty-fold or greater. Other evidence included i) a strong correlation between histories of tick bites and IgE to alpha-gal (χ2=26.8, p<0.001), ii) evidence that these IgE antibodies are common in areas where the tick Amblyomma americanum is common, and iii) a significant correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from A. americanum (rs=0.75, p<0.001). Conclusion The results presented here provide evidence that tick bites are a cause, or possibly the only cause, of IgE specific for alpha-gal in this area of the United States. Both the number of subjects becoming sensitized and the titer of IgE antibodies to alpha-gal are striking. Here we report the first example of a response to an ectoparasite giving rise to an important form of food allergy. PMID:21453959

  2. Qing'E formula alleviates the aging process in D-galactose-induced aging mice

    PubMed Central

    ZHONG, LIN; HUANG, FEI; SHI, HAILIAN; WU, HUI; ZHANG, BEIBEI; WU, XIAOJUN; WEI, XIAOHUI; WANG, ZHENGTAO

    2016-01-01

    Qing'E formula (QEF) is a clinically used prescription with four ingredients, Eucommiae Cortex, Psoraleae Fructus, Juglandis Semen and Garlic Rhizoma, from the Song dynasty (10th century CE). The present study aimed to investigate the anti-aging effect and mechanisms of QEF on D-galactose-induced aging mice. A mouse subacute aging model was established by subcutaneous injection of D-galactose at the neck consecutively for 8 weeks. Motor activity and memory impairment of the mice were evaluated by the rotarod test and passive avoidance test, respectively. Serum and liver parameters were analyzed with biochemical kits. Hippocampal mRNA and protein expression levels were examined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. QEF administration significantly ameliorated the impaired motor and memory of aging mice. In the serum, QEF reduced blood urea nitrogen, creatinine, nitric oxide (NO) and malondialdehyde (MDA) levels, and inhibited alanine aminotransferase and aspartate aminotransferase activities. In the liver, QEF increased the glutathione level, enhanced total antioxidant capacity and catalase activity, deceased NO and MDA production, and reduced NO synthase activity. In the hippocampus, QEF elevated gene expression levels of Klotho, sirtuin 1 (SIRT1), forkhead box transcription factor O3, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), insulin-like growth factor-1 and peroxiredoxin-3. QEF increased protein expression levels of Klotho and SIRT1, and decreased that of PGC-1α in the hippocampus. In conclusion, QEF attenuated the aging process in D-galactose-treated mice, which may be mediated through enhancing the antioxidants in the body, protecting renal and hepatic health, and balancing hippocampal expression levels of the longevity-related genes. PMID:27347412

  3. Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans

    PubMed Central

    Ramya, T N C; Weerapana, Eranthie; Cravatt, Benjamin F; Paulson, James C

    2013-01-01

    In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of “robust enrichment” afforded by covalent-labeling techniques and “specificity for glycoproteins” typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status. PMID:23070960

  4. Genetics and Evolution of the Salmonella Galactose-Initiated Set of O Antigens

    PubMed Central

    Reeves, Peter R.; Cunneen, Monica M.; Liu, Bin; Wang, Lei

    2013-01-01

    This paper covers eight Salmonella serogroups, that are defined by O antigens with related structures and gene clusters. They include the serovars that are now most frequently isolated. Serogroups A, B1, B2, C2-C3, D1, D2, D3 and E have O antigens that are distinguished by having galactose as first sugar, and not N-acetyl glucosamine or N-acetyl galactosamine as in the other 38 serogroups, and indeed in most Enterobacteriaceae. The gene clusters for these galactose-initiated appear to have entered S. enterica since its divergence from E. coli, but sequence comparisons show that much of the diversification occurred long before this. We conclude that the gene clusters must have entered S. enterica in a series of parallel events. The individual gene clusters are discussed, followed by analysis of the divergence for those genes shared by two or more gene clusters, and a putative phylogenic tree for the gene clusters is presented. This set of O antigens provides a rare case where it is possible to examine in detail the relationships of a significant number of O antigens. In contrast the more common pattern of O-antigen diversity within a species is for there to be only a few cases of strains having related gene clusters, suggesting that diversity arose through gain of individual O-antigen gene clusters by lateral gene transfer, and under these circumstances the evolution of the diversity is not accessible. This paper on the galactose-initiated set of gene clusters gives new insights into the origins of O-antigen diversity generally. PMID:23874940

  5. Mechanism of the anticataract effect of liposomal magnesium taurate in galactose-fed rats

    PubMed Central

    Iezhitsa, Igor; Saad, Sarah Diyana Bt; Zakaria, Fatin Kamilah Bt; Agarwal, Puneet; Krasilnikova, Anna; Rahman, Thuhairah Hasrah Abdul; Rozali, Khairul Nizam Bin; Spasov, Alexander; Ozerov, Alexander; Alyautdin, Renad; Ismail, Nafeeza Mohd

    2016-01-01

    Purpose Increased lenticular oxidative stress and altered calcium/magnesium (Ca/Mg) homeostasis underlie cataractogenesis. We developed a liposomal formulation of magnesium taurate (MgT) and studied its effects on Ca/Mg homeostasis and lenticular oxidative and nitrosative stress in galactose-fed rats. Methods The galactose-fed rats were topically treated with liposomal MgT (LMgT), liposomal taurine (LTau), or corresponding vehicles twice daily for 28 days with weekly anterior segment imaging. At the end of the experimental period, the lenses were removed and subjected to analysis for oxidative and nitrosative stress, Ca and Mg levels, ATP content, Ca2+-ATPase, Na+,K+-ATPase, and calpain II activities. Results The LTau and LMgT groups showed significantly lower opacity index values at all time points compared to the corresponding vehicle groups (p<0.001). However, the opacity index in the LMgT group was lower than that in the LTau group (p<0.05). Significantly reduced oxidative and nitrosative stress was observed in the LTau and LMgT groups. The lens Ca/Mg ratio in LMgT group was decreased by 1.15 times compared to that in the LVh group. Calpain II activity in the LMgT group was decreased by 13% compared to the LVh group. The ATP level and Na+,K+-ATPase and Ca2+-ATPase activities were significantly increased in the LMgT group compared to the LVh group (p<0.05). Conclusions Topical liposomal MgT delays cataractogenesis in galactose-fed rats by maintaining the lens mineral homeostasis and reducing lenticular oxidative and nitrosative stress. PMID:27440992

  6. Hepatic uptake and metabolism of galactose can be quantified in vivo by 2-[18F]fluoro-2-deoxygalactose positron emission tomography.

    PubMed

    Sørensen, Michael; Munk, Ole Lajord; Mortensen, Frank Viborg; Olsen, Aage Kristian; Bender, Dirk; Bass, Ludvik; Keiding, Susanne

    2008-07-01

    Metabolism of galactose is a specialized liver function. The purpose of this PET study was to use the galactose analog 2-[(18)F]fluoro-2-deoxygalactose (FDGal) to investigate hepatic uptake and metabolism of galactose in vivo. FDGal kinetics was studied in 10 anesthetized pigs at blood concentrations of nonradioactive galactose yielding approximately first-order kinetics (tracer only; n = 4), intermediate kinetics (0.5-0.6 mmol galactose/l blood; n = 2), and near-saturation kinetics (>3 mmol galactose/l blood; n = 4). All animals underwent liver C15O PET (blood volume) and FDGal PET (galactose kinetics) with arterial and portal venous blood sampling. Flow rates in the hepatic artery and the portal vein were measured by ultrasound transit-time flowmeters. The hepatic uptake and net metabolic clearance of FDGal were quantified by nonlinear and linear regression analyses. The initial extraction fraction of FDGal from blood-to-hepatocyte was unity in all pigs. Hepatic net metabolic clearance of FDGal, K(FDGal), was 332-481 ml blood.min(-1).l(-1) tissue in experiments with approximately first-order kinetics and 15.2-21.8 ml blood.min(-1).l(-1) tissue in experiments with near-saturation kinetics. Maximal hepatic removal rates of galactose were on average 600 micromol.min(-1).l(-1) tissue (range 412-702), which was in agreement with other studies. There was no significant difference between K(FDGal) calculated with use of the dual tracer input (Kdual(FDGal)) or the single arterial input (Karterial(FDGal)). In conclusion, hepatic galactose kinetics can be quantified with the galactose analog FDGal. At near-saturated kinetics, the maximal hepatic removal rate of galactose can be calculated from the net metabolic clearance of FDGal and the blood concentration of galactose. PMID:18483186

  7. Effectiveness of topical caffeine in cataract prevention: Studies with galactose cataract

    PubMed Central

    Varma, Shambhu D.; Kovtun, Svitlana

    2010-01-01

    Purpose The primary objective of the study was to investigate the possible inhibition of cataract formation by topical administration of caffeine using the galactosemic rat model. It was hypothesized that caffeine will do so by acting as scavenger of reactive oxygen species known to be generated under hyperglycemic conditions. Methods Cataract was induced by feeding young rats a diet containing 24% galactose for a period of 25 days. A control group of such rats was treated with a placebo eye drop preparation containing hydroxy propyl methyl cellulose as a wetting agent. In the experimental group, the rats were treated with the above preparation mixed with 72 mM caffeine. Results Administration of caffeine eye drops was found to significantly inhibit the onset as well as the progress of cataract formation. By day 25 on the galactose diet, all the animals in the control group developed advanced white opacity spread over the entire area of the lens. In the caffeine group, the formation of such opacity remained strikingly inhibited. The lenses remained largely transparent. The transparency data paralleled the higher concentration of glutathione maintained by caffeine treatment. Its levels in the placebo group were 0.8, 0.5, and 0.4 µmoles/g lens wt. on days 5, 15, and 25 against a consistent basal control value of ~3 µmoles/g over the entire period. In the caffeine group, the corresponding values were nearly 3 µmoles/g till day 15, but decreasing to ~2 µmoles/g on day 25. The levels were hence significantly higher than in the caffeine untreated group, remaining relatively closer to the basal controls. In addition, the compound was found effective in inhibiting morphological changes induced by galactose. Conclusions Micromolar amounts of topical caffeine have been found to be significantly effective in inhibiting the formation of galactose cataract, strongly suggesting its possible usefulness against diabetic cataracts. The effects are attributed to its ability to

  8. Adsorption characteristics of metal ions on chitosan chemically modified by D-galactose

    SciTech Connect

    Kondo, Kazuo; Sumi, Hisaharu; Matsumoto, Michiaki

    1996-07-01

    The adsorption characteristics of metal ions on chitosan chemically modified by D-galactose were examined. The pH dependency on the distribution ratio was found to be affected by the valency of the metal ion, and the apparent adsorption equilibrium constants of the metal ions were determined. The order of adsorption of the metal ions is Ga > In > Nd > Eu for the trivalent metal ions and Cu > Ni > Co for the divalent metal ions. It is believed that amino and hydroxyl groups in the chitosan act as a chelating ligand.

  9. Synthesis of three different galactose-based methacrylate monomers for the production of sugar-based polymers.

    PubMed

    Desport, Jessica S; Mantione, Daniele; Moreno, Mónica; Sardón, Haritz; Barandiaran, María J; Mecerreyes, David

    2016-09-01

    Glycopolymers, synthetic sugar-containing macromolecules, are attracting ever-increasing interest from the chemistry community. Glycidyl methacrylate (GMA) is an important building block for the synthesis of sugar based methacrylate monomers and polymers. Normally, glycidyl methacrylate shows some advantages such as reactivity against nucleophiles or milder synthetic conditions such as other reactive methacrylate monomers. However, condensation reactions of glycidyl methacrylate with for instance protected galactose monomer leads to a mixture of two products due to a strong competition between the two possible pathways: epoxide ring opening or transesterification. In this paper, we propose two alternative routes to synthesize regiospecific galactose-based methacrylate monomers using the epoxy-ring opening reaction. In the first alternative route, the protected galactose is first oxidized to the acid in order to make it more reactive against the epoxide of GMA. In the second route, the protected sugar was first treated with epichlorohydrin followed by the epoxy ring opening reaction with methacrylic acid, to create an identical analogue of the ring-opening product of GMA. These two monomers were polymerized using conventional radical polymerization and were compared to the previously known galactose-methacrylate one. The new polymers show similar thermal stability but lower glass transition temperature (Tg) with respect to the known galactose methacrylate polymer. PMID:27394038

  10. Host Glycan Sugar-Specific Pathways in Streptococcus pneumonia: Galactose as a Key Sugar in Colonisation and Infection

    PubMed Central

    Paixão, Laura; Oliveira, Joana; Veríssimo, André; Vinga, Susana; Lourenço, Eva C.; Ventura, M. Rita; Kjos, Morten; Veening, Jan-Willem; Fernandes, Vitor E.; Andrew, Peter W.; Yesilkaya, Hasan; Neves, Ana Rute

    2015-01-01

    The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis. PMID:25826206

  11. [Urinary metabolomics study of the effects of Scutellaria baicalensis Georgi ethanol extract on D-galactose-induced rats].

    PubMed

    Chang, Yan-fen; Gong, Wen-xia; Zheng, Yan-hong; Li, Jian-wei; Zhou, Yu-zhi; Qin, Xue-mei; Du, Guan-hua

    2016-01-01

    The purpose of this study is to evaluate the anti-aging effects and reveal the underlying mechanism of Scutellaria baicalensis Georgi ethanol extract (SBG) in D-galactose-induced rats. Fifty rats were randomly divided into five groups: vehicle control group, D-galactose group, and D-galactose combined with 50, 100, 200 mg x kg(-1) SBG. A rat aging model was induced by injecting subcutaneously D-galactose (100 mg x kg(-1)) for ten weeks. At the tenth week, the locomotor activity (in open-field test) and the learning and memory abilities (in Morris water maze test) were examined respectively. The urine was collected using metabolic cages and analyzed by high-resolution 1H NMR spectroscopy combined with multivariate statistical analyses. The SBG at doses of 50, 100 and 200 mg x kg(-1) treatments groups could significantly ameliorate aging process in rats' cognitive performance. The 50, 100, 200 mg x kg(-1) SBG regulated citrate, pyruvate, lactate, trimethylamine (TMA), pantothenate, β-hydroxybutyrate in urine favorably toward the control group. These biochemical changes are related to the disturbance in energy metabolism, glycometabolism and microbiome metabolism, which is helpful to further understanding the D-galactose induced aging rats and the therapeutic mechanism of SBG. PMID:27405167

  12. Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei.

    PubMed

    Rodríguez-Díaz, Jesús; Yebra, María J

    2011-07-20

    UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides. PMID:21663774

  13. The Molecular Dynamics of Trypanosoma brucei UDP-Galactose 4′-Epimerase: A Drug Target for African Sleeping Sickness

    PubMed Central

    Friedman, Aaron J; Durrant, Jacob D; Pierce, Levi C T; McCorvie, Thomas J; Timson, David J; McCammon, J Andrew

    2012-01-01

    During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4′-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein. PMID:22487100

  14. Molecular cloning and characterization of L-galactose-1-phosphate phosphatase from tobacco (Nicotiana tabacum).

    PubMed

    Sakamoto, Shingo; Fujikawa, Yukichi; Tanaka, Nobukazu; Esaka, Muneharu

    2012-01-01

    L-Galactose-1-phosphate phosphatase (GPPase) is an enzyme involved in ascorbate biosynthesis in higher plants. We isolated a cDNA encoding GPPase from tobacco, and named it NtGPPase. The putative amino acid sequence of NtGPPase contained inositol monophosphatase motifs and metal binding sites. Recombinant NtGPPase hydrolyzed not only L-galactose-1-phosphate, but also myo-inositol-1-phosphate. The optimum pH for the GPPase activity of NtGPPase was 7.5. Its enzyme activity required Mg2+, and was inhibited by Li+ and Ca2+. Its fluorescence, fused with green fluorescence protein in onion cells and protoplasts of tobacco BY-2 cells, was observed in both the cytosol and nucleus. The expression of NtGPPase mRNA and protein was clearly correlated with L-ascorbic acid (AsA) contents of BY-2 cells during culture. The AsA contents of NtGPPase over expression lines were higher than those of empty lines at 13 d after subculture. This suggests that NtGPPase contributes slightly to AsA biosynthesis. PMID:22790939

  15. Inhalation with Fucose and Galactose for Treatment of Pseudomonas Aeruginosa in Cystic Fibrosis Patients

    PubMed Central

    Hauber, Hans-Peter; Schulz, Maria; Pforte, Almuth; Mack, Dietrich; Zabel, Peter; Schumacher, Udo

    2008-01-01

    Background: Colonisation of cystic fibrosis (CF) lungs with Pseudomonas aeruginosa is facilitated by two lectins, which bind to the sugar coat of the surface lining epithelia and stop the cilia beating. Objectives: We hypothesized that P. aeruginosa lung infection should be cleared by inhalation of fucose and galactose, which compete for the sugar binding site of the two lectins and thus inhibit the binding of P. aeruginosa. Methods: 11 adult CF patients with chronic infection with P. aeruginosa were treated twice daily with inhalation of a fucose/galactose solution for 21 days (4 patients only received inhalation, 7 patients received inhalation and intravenous antibiotics). Microbial counts of P. aeruginosa, lung function measurements, and inflammatory markers were determined before and after treatment. Results: The sugar inhalation was well tolerated and no adverse side effects were observed. Inhalation alone as well as combined therapy (inhalation and antibiotics) significantly decreased P. aeruginosa in sputum (P < 0.05). Both therapies also significantly reduced TNFα expression in sputum and peripheral blood cells (P < 0.05). No change in lung function measurements was observed. Conclusions: Inhalation of simple sugars is a safe and effective measure to reduce the P. aeruginosa counts in CF patients. This may provide an alternative therapeutical approach to treat infection with P. aeruginosa. PMID:19043609

  16. Elemental distribution in frozen-hydrated rat lenses with galactose cataract

    SciTech Connect

    Koyama-Ito, H. )

    1990-01-01

    The elemental distributions in frozen-hydrated rat lenses with galactose cataract were compared before and after the onset of the nuclear cataract to investigate the possible role of ion levels in the lens opacification due to the phase separation of the lens cytoplasm. The maps of the weight concentrations of the minor elements, S, Cl, K and Ca, on the basis of wet weight in the central plane of lens were obtained by X-ray analysis with the high energy ion microprobe at a resolution of 50 microns. Before the onset of the nuclear cataract, the distributions of Cl and K, were almost normal, except in the lens posterior periphery with high Cl and low K. In the lens with the nuclear opacity, sudden changes were observed. The Cl increased throughout the lens, and K decreased throughout the lens except at lens anterior thin layer. However, the totalized monovalent ion level changed only slightly. The Ca level increased throughout the lens after the onset of the nuclear cataract, suggesting a possible role of Ca in the nuclear opacification of galactose cataract of rats. The distributions of S were similar to the protein density distributions previously known both in the normal and in the cataractous lenses.

  17. Metallization of double-stranded DNA triggered by bound galactose-modified naphthalene diimide.

    PubMed

    Komizo, Kohei; Ikedo, Hideyuki; Sato, Shinobu; Takenaka, Shigeori

    2014-08-20

    Naphthalene diimide (NDI) derivatives bearing galactose moieties through different spacers, NDI-DS1 and NDI-DS2, were synthesized by the click reaction of the acetylene derivatives of NDI with galactose azide. They bound to double-stranded DNA with threading intercalation, as confirmed by the topoisomerase I assay and circular dichroism spectroscopy. The binding affinities of these ligands were on the order of 10(5) M(-1) with several-fold higher affinity for double-stranded DNA than for single-stranded DNA. The silver mirror reaction on the double-stranded DNA bound to these ligands afforded silver nanowires that were converted to gold nanowires. In the atomic force microscopy measurements, the increased height of DNA areas on a mica plate was observed in the case of double-stranded DNA after NDI-DS2 treatment and subsequently silver mirror reaction, whereas the increased height of DNA areas was not observed in the case of single-stranded DNA after the same treatment. PMID:25011665

  18. Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT

    PubMed Central

    Adelman, Joshua L.; Sheng, Ying; Choe, Seungho; Abramson, Jeff; Wright, Ernest M.; Rosenberg, John M.; Grabe, Michael

    2014-01-01

    Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1. PMID:24655503

  19. [Biological assay for galactose-1 phosphate measurement application in subjects with galactosemia].

    PubMed

    Braham, Imene; Charfeddine, Bassem; Ben Othmene, Leila; Neffati, Souhir; Mtar, Aida; Ben Abdallah, Jihene; Ali Smach, Med; Dridi, Hedi; Limem, Khalifa

    2012-01-01

    Congenital galactosemia is a hereditary, autosomal recessive and metabolic disease. It is linked to an enzyme deficiency, more commonly known by the deficiency of galactose-1- phosphate uridyltransferase (GALT), which is responsible for an accumulation of galactose-1- phosphate in the blood. Clinical symptoms appear early in infancy from the second week of life. They generally manifested by some disorders within liver, kidney, eye, gastrointestinal, neurological and also with cataracts. Currently, the clinical diagnosis remains difficult hence the importance of further investigations based on effective biological assessments to highlight the disease. The diagnosis of galactosemia is made by the laboratory test. The latter includes the determination of Gal-1-P which is done by a fluorometric method spot test. This study was conducted in order to assess the repeatability, reproducibility, accuracy, and effectiveness of the techniques used. We have found the CV for a repeatability (CV = 5 %), reproducibility (CV = 4 %) which confirms the accuracy of the method proceeded in this study. This method allows us to have a degree of inaccuracy less than 1%. According to the study of the effectiveness of "spot test", we found that our technique is specific (Sp = 93 %) and sensitive (Se = 83 %). PMID:22294140

  20. A De Novo Variant in Galactose-1-P Uridylyltransferase (GALT) Leading to Classic Galactosemia.

    PubMed

    Tran, Thanh-Thanh Claire V; Liu, Ying; Zwick, Michael E; Ramachandran, Dhanya; Cutler, David J; Huang, Xiaoping; Berry, Gerard T; Fridovich-Keil, Judith L

    2015-01-01

    Classic galactosemia (CG) is a potentially lethal genetic disease that results from profound impairment of galactose-1-P uridylyltransferase (GALT), the middle enzyme in the Leloir pathway of galactose metabolism. Patients with CG carry pathogenic loss-of-function mutations in both of their GALT alleles; the parents of patients are considered obligate carriers. We report here a first exception to that rule - a de novo GALT variant in a patient with classic galactosemia. The new variant, c.563A>C (p.Q188P), which introduces a missense substitution near the active site of the GALT enzyme, was found in the compound heterozygous state in a child with classic galactosemia, but not in either of her parents. Extensive genomic studies of DNA from the child and both parents confirmed the expected degrees of relationship in the trio as well as inheritance of a common c.563A>G (p.Q188R) GALT mutation from the mother. This result demonstrates that not all pathogenic GALT mutations are inherited and raises concern that GALT may have a higher new mutation rate than previously believed. PMID:25681079

  1. Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

    PubMed

    Machuka, J S; Okeola, O G; Van Damme Els, J M; Chrispeels, M J; Van Leuven, F; Peumans, W J

    1999-07-01

    A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1. PMID:10389271

  2. The cloning and sequencing of the UDP-galactose 4-epimerase gene (galE) from Avibacterium paragallinarum.

    PubMed

    Roodt, Yolande; Bragg, Robert; Albertyn, Jacobus

    2007-08-01

    The putative uridine diphosphate (UDP)-galactose 4-epimerase encoding gene, galE, was isolated from Avibacterium paragallinarum with the use of degenerate primers, colony hybridization and inverse PCR. The data revealed an open reading frame of 1017 bp encoding a protein of 338 amino acids with a molecular weight of 37 kDa and an isoelectric point of 5.5. High sequence homology was obtained with an 87, 91 and 89% sequence identity on protein level towards the galE genes from Actinobacillus pleuropneumoniae, Haemophilus influenza and Pasteurella multocida, respectively. To verify that the cloned galE gene encodes for a UDP-galactose 4-epimeras, this gene was cloned into the pYES-2 expression vector, followed by transformation in a Saccharomyces cerevisiae gal10 deletion strain. Complementation of the gal10 deletion mutant with the galE gene confirmed that this gene encodes a UDP-galactose 4-epimerase. PMID:17541831

  3. Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy Streptococcus thermophilus Strains Reveals Four Different Fermentation Profiles

    PubMed Central

    de Vin, Filip; Rådström, Peter; Herman, Lieve; De Vuyst, Luc

    2005-01-01

    Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related. PMID:16000774

  4. Ovotoxic Effects of Galactose Involve Attenuation of Follicle-Stimulating Hormone Bioactivity and Up-Regulation of Granulosa Cell p53 Expression

    PubMed Central

    Banerjee, Sayani; Chakraborty, Pratip; Saha, Piyali; Bandyopadhyay, Soma Aditya; Banerjee, Sutapa; Kabir, Syed N.

    2012-01-01

    Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented

  5. [Protective effect of Angelica sinensis polysaccharides on subacute renal damages induced by D-galactose in mice and its mechanism].

    PubMed

    Fan, Yan-ling; Xia, Jie-yu; Jia, Dao-yong; Zhang, Meng-si; Zhang, Yan-yan; Wang, Lu; Huang, Guo-ning; Wang, Ya-ping

    2015-11-01

    To explore the protective effect of Angelica sinensis polysaccharides(ASP) on subacute renal damages induced by D-galactose in mice and its mechanism. Male C57BL/6J mice were randomly divided into 3 groups, with 10 mice in each group. The D-galactose model group was subcutaneously injected with D-galactose (120 mg x kg(-1)), qd x 42; the ASP + D-galactose model group was intraperitoneally injected with ASP since the 8th day of the replication of the D-galactose model, qd x 35; and the normal control group was subcutaneously injected with saline at the same dose and time. On the 2nd day of after the injection, the peripheral blood was collected to measure the content of BUN, Crea, UA, Cys-C; paraffin sections were made to observe the renal histomorphology by HE staining; senescence-associated β-g-alactosidase (SA-β-Gal) stain was used to observe the relative optical density (ROD) in renal tissues; transmission electron microscopy was assayed to observe the renal ultrastructure; the renal tissue homogenate was prepared to measure the content of SOD, GSH-PX, MDA; the content of AGEs and 8-OH-dG were measured by ELISA. According to the result, compared with the D-galactose model group, the ASP + D-galactose model group showed obviously decreases in the content of BUN, Crea, UA, Cysc, AGES, 8-OH-dG, the number of hardening renal corpuscle, renal capsular space and renal tubular lumen, ROD of SA-β-Gal staining positive kidney cells, mesangial cells, basement membrane thickness, podocyte secondary processes fusion and MDA and increases in the number of normal renal corpuscle, ribosome and rough endoplasmic reticulum in podocytes, the activity of SOD and GSH-PX. In Conclusion, A. sinensis polysaccharides can antagonize kidney subacute damages induced by D-galactose in mice. Its protective mechanism may be correlated with the inhibition of the oxidative stress injury. PMID:27071262

  6. Multifunctional supramolecular vesicles based on the complex of ferrocenecarboxylic acid capped pillar[5]arene and a galactose derivative for targeted drug delivery.

    PubMed

    Chang, Yincheng; Hou, Chenxi; Ren, Jingli; Xin, Xiaoting; Pei, Yuxin; Lu, Yuchao; Cao, Shoupeng; Pei, Zhichao

    2016-07-21

    Supramolecular vesicles based on the host-guest complexation of ferrocenecarboxylic acid capped pillar[5]arene and a galactose derivative have been constructed, which showed dual-responsiveness and cancer cells targetability resulting from its ferrocenecarboxylic acid units and galactose units, respectively. This work provides a good example for the construction of multifunctional nanocarriers for targeted drug delivery. PMID:27387299

  7. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress

    PubMed Central

    Vu, Hieu Sy; Roth, Mary R.; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A.; Williams, Todd D.; Welti, Ruth

    2014-01-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

  8. Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

    PubMed Central

    Li, Yijun; Ptolemy, Adam S.; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T.

    2013-01-01

    Background The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based assay for GALT enzyme activity measurement. Method Our assay used stable isotope-labeled α-galactose-1-phosphate ([13C6]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard. Results The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) µmol · (gHgb)−1 · h−1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 µmol · (g Hgb)−1 · h−1 (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent Km of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. Conclusions This LC-MS/MS–based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities. PMID:20348403

  9. Arabidopsis VTC2 Encodes a GDP-l-Galactose Phosphorylase, the Last Unknown Enzyme in the Smirnoff-Wheeler Pathway to Ascorbic Acid in Plants*

    PubMed Central

    Linster, Carole L.; Gomez, Tara A.; Christensen, Kathryn C.; Adler, Lital N.; Young, Brian D.; Brenner, Charles; Clarke, Steven G.

    2008-01-01

    The first committed step in the biosynthesis of l-ascorbate from d-glucose in plants requires conversion of GDP-l-galactose to l-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-l-galactose to l-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from Arabidopsis thaliana as a specific GDP-l-galactose/GDP-d-glucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature. PMID:17462988

  10. Role of Snf1p in regulation of intracellular sorting of the lactose and galactose transporter Lac12p in Kluyveromyces lactis.

    PubMed

    Wiedemuth, Christian; Breunig, Karin D

    2005-04-01

    The protein kinase Snf1/AMPK plays a central role in carbon and energy homeostasis in yeasts and higher eukaryotes. To work out which aspects of the Snf1-controlled regulatory network are conserved in evolution, the Snf1 requirement in galactose metabolism was analyzed in the yeast Kluyveromyces lactis. Whereas galactose induction was only delayed, K. lactis snf1 mutants failed to accumulate the lactose/galactose H+ symporter Lac12p in the plasma membran,e as indicated by Lac12-green fluorescent protein fusions. In contrast to wild-type cells, the fusion protein was mostly intracellular in the mutant. Growth on galactose and galactose uptake could be restored by the KHT3 gene, which encodes a new transporter of the HXT subfamily of major facilitators These findings indicate a new role of Snf1p in regulation of sugar transport in K. lactis. PMID:15821131

  11. The organization and transcription of the galactose gene cluster of Kluyveromyces lactis.

    PubMed Central

    Webster, T D; Dickson, R C

    1988-01-01

    The yeast Kluyveromyces lactis grows on galactose by inducing the Leloir pathway enzymes-kinase, epimerase, and transferase. To investigate the molecular mechanism for regulating expression of this metabolic pathway we isolated GAL1, GAL7, GAL10, which code for kinase, transferase, and epimerase, respectively, and characterized their size, organization, and transcriptional regulation. Our results indicate that induction of the Leloir pathway in K. lactis occurs at the level of transcription and that the organization and regulation of the GAL gene cluster in K. lactis is closely related to the homologous gene cluster in Saccharomyces cerevisiae. Likewise, the Upstream Activator Sequences that regulate induction of the GAL genes are similar in base sequence, number and relative location in the two yeasts. Images PMID:3047676

  12. Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose.

    PubMed Central

    Bourne, Yves; Astoul, Corinne Houlès; Zamboni, Véronique; Peumans, Willy J; Menu-Bouaouiche, Laurence; Van Damme, Els J M; Barre, Annick; Rougé, Pierre

    2002-01-01

    Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galbeta1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin-alpha-methyl-mannose complex at 2 A resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the beta-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities to a single lectin. PMID:11988090

  13. Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose.

    PubMed

    Bourne, Yves; Astoul, Corinne Houlès; Zamboni, Véronique; Peumans, Willy J; Menu-Bouaouiche, Laurence; Van Damme, Els J M; Barre, Annick; Rougé, Pierre

    2002-05-15

    Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galbeta1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin-alpha-methyl-mannose complex at 2 A resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the beta-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities to a single lectin. PMID:11988090

  14. Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry.

    PubMed

    Sakashita, Mizuha; Mochizuki, Shinichi; Sakurai, Kazuo

    2014-10-01

    Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed 'lipoplex', we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis. PMID:25155912

  15. Characterization of pyranose dehydrogenase from Agaricus meleagris and its application in the C-2 specific conversion of D-galactose.

    PubMed

    Sygmund, Christoph; Kittl, Roman; Volc, Jindrich; Halada, Petr; Kubátová, Elena; Haltrich, Dietmar; Peterbauer, Clemens K

    2008-02-01

    Pyranose dehydrogenase (PDH) of the mushroom Agaricus meleagris was purified from mycelial culture media to substantial homogeneity using ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric polypeptide with a molecular mass of 66,547Da as determined by matrix-assisted laser desorption/ionisation mass spectrometry containing approximately 7% carbohydrate and covalently bound flavin adenine dinucleotide. The enzyme exhibited a broad sugar substrate tolerance, oxidizing different aldopyranoses to the corresponding C-2 or C-2,3 (di)dehydro sugars. Preferred electron donors with the highest k(cat)/K(m) values were major sugar constituents of cellulose and hemicellulose, namely d-glucose, D-galactose, l-arabinose, D-xylose and cellobiose. This indicates a possible physiological role of the enzyme in lignocellulose breakdown. PDH showed no detectable activity with oxygen, and its reactivity towards electron acceptors was limited to various substituted benzoquinones and complexed metal ions, with the ferricenium ion and the benzoquinone imine 2,6-dichloroindophenole displaying the highest k(cat)/K(m). The enzyme catalyzed in up to 95% yields the regiospecific conversion of D-galactose to 2-dehydro-D-galactose, an intermediate in a possible biotechnologically interesting process for redox isomerization of D-galactose to the prebiotic sugar D-tagatose. PMID:18083263

  16. Molecular cloning and characterization of the mouse CGT gene encoding UDP-galactose ceramide-galactosyltransferase (cerebroside synthetase)

    SciTech Connect

    Bosio, A.; Binczek, E.; Stoffel, W.

    1996-07-01

    UDP-galactose ceramide galactosyltransferase, CGT, EC 2.4.1.45, is the key enzyme in the biosynthesis of cerebrosides and sulfatides, which are the most abundant glycosphingolipids in the myelin of the central nervous system and the peripheral nervous system.

  17. The Rapid and Sensitive Quantitative Determination of Galactose by Combined Enzymatic and Colorimetric Method: Application in Neonatal Screening.

    PubMed

    Kianmehr, Anvarsadat; Mahrooz, Abdolkarim; Ansari, Javad; Oladnabi, Morteza; Shahbazmohammadi, Hamid

    2016-05-01

    The quantitative measurement of galactose in blood is essential for the early diagnosis, treatment, and dietary monitoring of galactosemia patients. In this communication, we aimed to develop a rapid, sensitive, and cost-effective combined method for galactose determination in dry blood spots. This procedure was based on the combination of enzymatic reactions of galactose dehydrogenase (GalDH), dihydrolipoyl dehydrogenase (DLD), and alkaline phosphates with a colorimetric system. The incubation time and the concentration of enzymes used in new method were also optimized. The analytical performance was studied by the precision, recovery, linearity, and sensitivity parameters. Statistical analysis was applied to method comparison experiment. The regression equation and correlation coefficient (R (2)) were Y = 0.0085x + 0.032 and R (2) = 0.998, respectively. This assay exhibited a recovery in the range of 91.7-114.3 % and had the limit detection of 0.5 mg/dl for galactose. The between-run coefficient of variation (CV) was between 2.6 and 11.1 %. The within-run CV was between 4.9 and 9.2 %. Our results indicated that the new and reference methods were in agreement because no significant biases exist between them. Briefly, a quick and reliable combined enzymatic and colorimetric assay was presented for application in newborn mass screening and monitoring of galactosemia patients. PMID:26821257

  18. Arginine Functionally Improves Clinically Relevant Human Galactose-1-Phosphate Uridylyltransferase (GALT) Variants Expressed in a Prokaryotic Model.

    PubMed

    Coelho, Ana I; Trabuco, Matilde; Silva, Maria João; de Almeida, Isabel Tavares; Leandro, Paula; Rivera, Isabel; Vicente, João B

    2015-01-01

    Classic galactosemia is a rare genetic disease of the galactose metabolism, resulting from deficient activity of galactose-1-phosphate uridylyltransferase (GALT). The current standard of care is lifelong dietary restriction of galactose, which however fails to prevent the development of long-term complications. Structural-functional studies demonstrated that the most prevalent GALT mutations give rise to proteins with increased propensity to aggregate in solution. Arginine is a known stabilizer of aggregation-prone proteins, having already shown a beneficial effect in other inherited metabolic disorders.Herein we developed a prokaryotic model of galactose sensitivity that allows evaluating in a cellular context the mutations' impact on GALT function, as well as the potential effect of arginine in functionally rescuing clinically relevant variants.This study revealed that some hGALT variants, previously described to exhibit no detectable activity in vitro, actually present residual activity when determined in vivo. Furthermore, it revealed that arginine presents a mutation-specific beneficial effect, particularly on the prevalent p.Q188R and p.K285N variants, which led us to hypothesize that it might constitute a promising therapeutic agent in classic galactosemia. PMID:25814382

  19. Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

    PubMed

    Ramos, Kristine Rose M; Valdehuesa, Kris Niño G; Liu, Huaiwei; Nisola, Grace M; Lee, Won-Keun; Chung, Wook-Jin

    2014-12-01

    An engineered Escherichia coli strain was developed for enhanced isoprene production using D-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley-Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from D-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct D-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze D-galactose conversion to D-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD-MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD-MEP pathway. PMID:24928200

  20. Bitterness of glucose/galactose: novel mutations in the SLC5A1 gene.

    PubMed

    Pode-Shakked, Ben; Reish, Orit; Aktuglu-Zeybek, Cigdem; Kesselman, Dafna; Dekel, Benjamin; Bujanover, Yoram; Anikster, Yair

    2014-01-01

    Glucose galactose malabsorption (GGM) is a rare autosomal recessive disorder characterized by life-threatening osmotic diarrhea at infancy. When the intake of the offending sugars (namely, glucose, galactose and lactose) is ceased, the diarrhea promptly stops. Mutations in the SLC5A1 gene, encoding the sodium-glucose co-transporter located in the brush border of enterocytes, have been shown to cause the disease. More than 300 subjects of diverse origin have been reported worldwide, most of whom are a result of a consanguineous union. We examined 6 patients from 4 families presenting with complaints consistent with GGM and responsive to the appropriate fructose-based diet. Genomic DNA of the patients was polymerase chain reaction amplified for each of the 15 exons of the SLC5A1 gene and analyzed by nucleotide sequencing. The analysis lead to the identification of 2 novel mutations: a 1915 del C mutation, a frameshift mutation leading to a premature stop at codon 645; and a substitution missense mutation of T to C on nucleotide 947 (exon 9) causing a L316P substitution. In addition, G426R and C255W mutations previously described were identified; in both cases, the patients were shown to be homozygous and their parents heterozygous for the mutation. Of note, additional patients who underwent a similar evaluation at our center for suspected GGM did not show mutations in the SLC5A1 gene. Because the latter did not previously undergo a diagnostic algorithm in full, for instance, one that may consist of a glucose breath hydrogen test and an empiric attempt of a dietary switch to galactomin, we suggest that molecular genotyping of such patients should only follow such appropriate clinical evaluation. PMID:24048166

  1. Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli

    PubMed Central

    Paukner, Regina; Staudigl, Petra; Choosri, Withu; Haltrich, Dietmar; Leitner, Christian

    2015-01-01

    A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4 mg enzyme per L of growth culture with a specific activity of 159 U mg−1. By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 68.5 kDa. The pH optimum for the oxidation of galactose was in the range of pH 6–7.5. Optimum temperature for the enzyme activity was 35 °C, with a half-life of 11.2 min, 5.3 min, and 2.7 min for incubation at 40 °C, 50 °C, and 60 °C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226 U mg−1) and the highest catalytic efficiency (kcat/Km) for melibiose (2700 mM−1 s−1). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN. PMID:25543085

  2. Lactose metabolism in Streptococcus lactis: phosphorylation of galactose and glucose moieties in vivo.

    PubMed Central

    Thompson, J

    1979-01-01

    Starved cells of Streptococcus lactis ML3 grown previously on lactose, galactose, or maltose were devoid of adenosine 5'-triphosphate contained only three glycolytic intermediates: 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (PEP). The three metabolites (total concentration, ca 40 mM) served as the intracellular PEP potential for sugar transport via PEP-dependent phosphotransferase systems. When accumulation of [14C]lactose by iodoacetate-inhibited starved cells was abolished within 1 s of commencement of transport, a phosphorylated disaccharide was identified by autoradiography. The compound was isolated by ion-exchange (borate) chromatography, and enzymatic analysis showed that the derivative was 6-phosphoryl-O-beta-D-galactopyranosyl (1 leads to 4')-alpha-D-glucopyranose (lactose 6-phosphate). After maximum lactose uptake (ca. 15 mM in 15 s) the cells were collected by membrane filtration and extracted with trichloroacetic acid. Neither free nor phosphorylated lactose was detected in cell extracts, but enzymatic analysis revealed high levels of galactose 6-phosphate and glucose 6-phosphate. The starved organisms rapidly accumulated glucose, 2-deoxy-D-glucose, methyl-beta-D-thiogalactopyranoside, and o-nitrophenyl-beta-D-galactopyranoside in phosphorylated form to intracellular concentrations of 32, 32, 42, and 38.5 mM, respectively. In contrast, maximum accumulation of lactose (ca. 15 mM) was only 40 to 50% that of the monosaccharides. From the stoichiometry of PEP-dependent lactose transport and the results of enzymatic analysis, it was concluded that (i) ca. 60% of the PEP potential was utilized via the lactose phosphotransferase system for phosphorylation of the galactosyl moiety of the disaccharide, and (ii) the residual potential (ca. 40%) was consumed during phosphorylation of the glucose moiety. Images PMID:118155

  3. Galactose-Depleted Xyloglucan Is Dysfunctional and Leads to Dwarfism in Arabidopsis1

    PubMed Central

    Kong, Yingzhen; Peña, Maria J.; Renna, Luciana; Avci, Utku; Pattathil, Sivakumar; Tuomivaara, Sami T.; Li, Xuemei; Reiter, Wolf-Dieter; Brandizzi, Federica; Hahn, Michael G.; Darvill, Alan G.; York, William S.; O’Neill, Malcolm A.

    2015-01-01

    Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes. PMID:25673778

  4. Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose.

    PubMed

    Hu, Dan; Tateno, Hiroaki; Kuno, Atsushi; Yabe, Rikio; Hirabayashi, Jun

    2012-06-01

    6-sulfo-galactose (6S-Gal) is a prevalent motif observed in highly sulfated keratan sulfate, which is closely associated with the glioblastoma malignancy while acting as a critical determinant for endogenous lectins. However, facile detection of this unique glycoepitope is greatly hampered because of a lack of appropriate probes. We have previously reported tailoring an α2-6-linked sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein, EW29Ch, by a reinforced ribosome display system following an error-prone PCR. In this study, we challenged the creation of novel lectins to recognize 6S-Gal-terminated glycans by incorporating a high-throughput screening system with a glycoconjugate microarray. After two rounds of selection procedures, 20 mutants were obtained and 12 were then successfully expressed in Escherichia coli, 8 of which showed a significant affinity for 6'-Sulfo-LN (6-O-sulfo-Galβ1-4GlcNAc), which the parental EW29Ch lacked. Analysis of two representative mutants by frontal affinity chromatography revealed a substantial affinity (K(d) ∼3 μm) for a 6S-Gal-terminated glycan. On the basis of the observation that all eight mutants have a common mutation at Glu-20 to Lys, site-directed mutagenesis experiments were performed focusing on this aspect. The results clearly indicated that the E20K mutation is necessary and sufficient to acquire the specificity for 6S-Gal. We also confirmed a difference in binding between E20K and EW29Ch to CHO cells, in which enzymes to catalyze the synthesis of 6S-Gal were overexpressed. The results clearly demonstrate that these mutants have potential to distinguish between cells containing different amounts of 6S-Gal-terminated glycans. This new technology will be used to provide novel tools essential for sulfoglycomics. PMID:22493425

  5. Enzymatic generation of galactose-rich oligosaccharides/oligomers from potato rhamnogalacturonan I pectic polysaccharides.

    PubMed

    Khodaei, Nastaran; Karboune, Salwa

    2016-04-15

    Potato pulp by-product rich in galactan-rich rhamnogalacturonan I (RG I) was investigated as a new source of oligosaccharides with potential prebiotic properties. The efficiency of selected monocomponent enzymes and multi-enzymatic preparations to generate oligosaccharides/oligomers from potato RG I was evaluated. These overall results of yield were dependent on the activity profile of the multi-enzymatic preparations. Highest oligo-RG I yield of 93.9% was achieved using multi-enzymatic preparation (Depol 670L) with higher hydrolytic activity toward side chains of RG I as compared to its backbone. Main oligo-RG I products were oligosaccharides with DP of 2-12 (79.8-100%), while the oligomers with DP of 13-70 comprised smaller proportion (0.0-20.2%). Galactose (58.9-91.2%, w/w) was the main monosaccharide of oligo-RG I, while arabinose represented 0.0-12.1%. An understanding of the relationship between the activity profile of multi-enzymatic preparations and the yield/DP of oligo-RG I was achieved. This is expected to provide the capability to generate galacto- and galacto(arabino) oligosaccharides and their corresponding oligomers from an abundant by-product. PMID:26616968

  6. Blueberry treatment decreased D-galactose-induced oxidative stress and brain damage in rats.

    PubMed

    Çoban, Jale; Doğan-Ekici, Işın; Aydın, A Fatih; Betül-Kalaz, Esra; Doğru-Abbasoğlu, Semra; Uysal, Müjdat

    2015-06-01

    D-galactose (GAL) causes aging-related changes and oxidative stress in the organism. We investigated the effect of whole fresh blueberry (BB) (Vaccinium corymbosum L.) treatment on oxidative stress in age-related brain damage model. Rats received GAL (300 mg/kg; s.c.; 5 days per week) alone or together with 5 % (BB1) and 10 % (BB2) BB containing chow for two months. Malondialdehyde (MDA),protein carbonyl (PC) and glutathione (GSH) levels, and Cu Zn-superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities as well as acetylcholinesterase (AChE) activities were determined. Expressions of B cell lymphoma-2 (Bcl-2), Bax and caspase-3 were also evaluated in the brain by immunohistochemistry. MDA and PC levels and AChE activity increased, but GSH levels, SOD and GSH-Px activities decreased together with histopathological structural damage in the brain of GAL-treated rats. BB treatments, especially BB2 reduced MDA and PC levels and AChE activity and elevated GSH levels and GSH-Px activity. BB1 and BB2 treatments diminished apoptosis and ameliorated histopathological findings in the brain of GAL-treated rats. These results indicate that BB partially prevented the shift towards an imbalanced prooxidative status and apoptosis together with histopathological amelioration by acting as an antioxidant (radical scavenger) itself in GAL-treated rats. PMID:25511550

  7. Evaluation of Galactose Adapted Yeasts for Bioethanol Fermentation from Kappaphycus alvarezii Hydrolyzates.

    PubMed

    Nguyen, Trung Hau; Ra, Chae Hun; Sunwoo, In Yung; Jeong, Gwi-Taek; Kim, Sung-Koo

    2016-07-28

    Bioethanol was produced from Kappaphycus alvarezii seaweed biomass using separate hydrolysis and fermentation (SHF). Pretreatment was evaluated for 60 min at 121°C using 12% (w/v) biomass slurry with 364 mM H2SO4. Enzymatic saccharification was then carried out at 45°C for 48 h using Celluclast 1.5 L. Ethanol fermentation with 12% (w/v) K. alvarezii hydrolyzate was performed using the yeasts Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 with or without prior adaptation to high concentrations of galactose. When non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 11.5 g/l, 6.7 g/l, and 6.0 g/l of ethanol were produced, respectively. When adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 15.8 g/l, 11.6 g/l, and 13.4 g/l of ethanol were obtained, respectively. The highest ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and YT% = 84.3%, which was obtained using adapted S. cerevisiae. PMID:27056472

  8. Antiaging Effect of Inula britannica on Aging Mouse Model Induced by D-Galactose.

    PubMed

    Chen, Hui; Long, Yuanyuan; Guo, Lei

    2016-01-01

    The antiaging effect of Inula britannica flower total flavonoids (IBFTF) on aging mice induced by D-galactose and its mechanism was examined in this study. From the results, the biochemical indexes and histological analysis of skin tissues showed that IBFTF could effectively improve the antioxidant enzyme activity of the aging mice, enhance the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) of skin tissue, and decrease the malondialdehyde (MDA) content. Besides, IBFTF could maintain the skin collagen, hydroxyproline (Hyp), dermal thickness, and moisture content. Meanwhile, IBFTF could significantly reduce the number of cells arrested in G0/G1 phase, and from the point of view of protein and mRNA expression level in skin tissue, IBFTF could significantly increase the expression of Sirt1 and CyclinD1 but decrease the expression of p16 and p21, and its effect was not less than that of the well-known vitamin E (VE). Overall, these results seem to be implying that IBFTF is a potential natural anti-skin aging agent with great antioxidant ability. PMID:27066100

  9. Antiaging Effect of Inula britannica on Aging Mouse Model Induced by D-Galactose

    PubMed Central

    Chen, Hui; Long, Yuanyuan; Guo, Lei

    2016-01-01

    The antiaging effect of Inula britannica flower total flavonoids (IBFTF) on aging mice induced by D-galactose and its mechanism was examined in this study. From the results, the biochemical indexes and histological analysis of skin tissues showed that IBFTF could effectively improve the antioxidant enzyme activity of the aging mice, enhance the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) of skin tissue, and decrease the malondialdehyde (MDA) content. Besides, IBFTF could maintain the skin collagen, hydroxyproline (Hyp), dermal thickness, and moisture content. Meanwhile, IBFTF could significantly reduce the number of cells arrested in G0/G1 phase, and from the point of view of protein and mRNA expression level in skin tissue, IBFTF could significantly increase the expression of Sirt1 and CyclinD1 but decrease the expression of p16 and p21, and its effect was not less than that of the well-known vitamin E (VE). Overall, these results seem to be implying that IBFTF is a potential natural anti-skin aging agent with great antioxidant ability. PMID:27066100

  10. Protective effect of tetrahydropalmatine against d-galactose induced memory impairment in rat.

    PubMed

    Qu, Zhuo; Zhang, Jingze; Yang, Honggai; Huo, Liqin; Gao, Jing; Chen, Hong; Gao, Wenyuan

    2016-02-01

    Aging is associated with Alzheimer's disease (AD), cardiovascular disease and cancer. Oxidative stress is considered as a major factor that accelerates the aging process. d-galactose (d-gal), a reducing sugar, induces oxidative stress resulting in alteration in mitochondrial dynamics and apoptosis of neurons. To understand the ability of tetrahydropalmatine (THP) to ameliorate memory impairment caused by aging, we investigated the effect of THP on d-gal induced memory impairment in rats. Subcutaneous injection of d-gal (100mg/kg/d) for 8weeks caused memory loss as detected by the Morris water maze and morphologic abnormalities of neurons in the hippocampus regions and cortex of rat brain. THP treatment ameliorated d-gal induced memory impairment associated with the decrease of malondialdehyde (MDA) and nitric oxide (NO) contents, as well as the increase of glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. THP treatment was also found to reverse the abnormality of acetylcholine (ACh) levels and acetylcholinesterase (AChE) activities. In addition, treatment with THP could decrease the expression of nuclear factor κ (NF-κB) and glial fibrillary acidic protein (GFAP) which prevented the neuroinflammation and memory impairment in the d-gal treated rats. Taken together, these results clearly demonstrated that subcutaneous injection of d-gal produced memory deficits, meanwhile THP could protect neuron from d-gal insults and improve cognition. This study provided an experimental basis for clinical application of THP in AD therapy. PMID:26592138

  11. Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon

    PubMed Central

    Møller, Thorleif; Franch, Thomas; Udesen, Christina; Gerdes, Kenn; Valentin-Hansen, Poul

    2002-01-01

    The physiological role of Escherichia coli Spot 42 RNA has remained obscure, even though the 109-nucleotide RNA was discovered almost three decades ago. Structural features of Spot 42 RNA and previous work suggested to us that the RNA might be a regulator of discoordinate gene expression of the galactose operon, a control that is only understood at the phenomenological level. The effects of controlled expression of Spot 42 RNA or deleting the gene (spf) encoding the RNA supported this hypothesis. Down-regulation of galK expression, the third gene in the gal operon, was only observed in the presence of Spot 42 RNA and required growth conditions that caused derepression of the spf gene. Subsequent biochemical studies showed that Spot 42 RNA specifically bound at the galK Shine-Dalgarno region of the galETKM mRNA, thereby blocking ribosome binding. We conclude that Spot 42 RNA is an antisense RNA that acts to differentially regulate genes that are expressed from the same transcription unit. Our results reveal an interesting mechanism by which the expression of a promoter distal gene in an operon can be modulated and underline the importance of antisense control in bacterial gene regulation. PMID:12101127

  12. Effect of galactose on acid induced molten globule state of Soybean Agglutinin: Biophysical approach

    NASA Astrophysics Data System (ADS)

    Alam, Parvez; Naseem, Farha; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

    2015-11-01

    In the present study the formation of molten globule-like unfolding intermediate Soybean Agglutinin (SBA) in acidic pH range has been established with the help of acrylamide quenching, intrinsic fluorescence, ANS fluorescence measurement, far UV CD and dynamic light scattering measurement. A marked increase in ANS fluorescence was observed at pH 2.2. Ksv of acrylamide quenching was found to be higher at pH 2.2 than that of native SBA at pH 7. Far UV CD spectra of pH induced state suggest that SBA shows significant retention of secondary structure closure to native. Hydrodynamic radius of SBA at pH 2.2 was found be more as compared to native state and also in other pH induced states. Further we checked the effect of galactose on the molten globule state of SBA. This study suggests that SBA exist as molten globule at pH 2.2 and this study will help in acid induced molten globule state of other proteins.

  13. Carbohydrate affinity for the glucose-galactose binding protein is regulated by allosteric domain motions.

    PubMed

    Ortega, Gabriel; Castaño, David; Diercks, Tammo; Millet, Oscar

    2012-12-01

    Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding. PMID:23148479

  14. Structures and energies of D-galactose and galabiose conformers as calculated by ab initio and semiempirical methods.

    PubMed

    Rahal-Sekkal, Majda; Sekkal, Nezha; Kleb, Dirk C; Bleckmann, Paul

    2003-05-01

    Optimized geometries and total energies of some conformers of alpha- and beta-D-galactose have been calculated using the RHF/6-31G* ab initio method. Vibrational frequencies were computed at the 6-31G* level for the conformers that favor internal hydrogen bonding, in order to evaluate their enthalpies, entropies, Gibbs free energies, and then their structural stabilities. The semiempirical AM1, PM3, MNDO methods have also been performed on the conformers GG, GT, and TG of alpha- and beta-D-galactose. In order to test the reliability of each semiempirical method, the obtained structures and energies from the AM1, PM3, and MNDO methods have been compared to those achieved using the RHF/6-31G* ab initio method. The MNDO method has not been investigated further, because of the large deviation in the structural parameters compared with those obtained by the ab initio method for the galactose. The semiempirical method that has yielded the best results is AM1, and it has been chosen to perform structural and energy calculations on the galabiose molecule (the disaccharides constituted by two galactose units alpha 1,4 linked). The goal of such calculations is to draw the energy surface maps for this disaccharide. To realize each map, 144 different possible conformations resulting from the rotations of the two torsional angles psi and phi of the glycosidic linkage are considered. In each calculation, at each increment of psi and phi, using a step of 30 degrees from 0 to 330 degrees, the energy optimization is employed. In this article, we report also calculations concerning the galabiose molecule using different ab initio levels such as RHF/6-31G*, RHF/6-31G**, and B3Lyp/6-31G*. PMID:12692790

  15. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    PubMed

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production. PMID:26922727

  16. Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.

    PubMed

    Azar, Shadi Rokhsartalab; Naiebi, Raika; Homami, Ameneh; Akbari, Zahra; Kianmehr, Anvarsadat; Mahdizadehdehosta, Rahman; Najafzadeh, Faezeh

    2015-02-01

    The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of β-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD'-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery. PMID:26040113

  17. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats

    PubMed Central

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-01-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficial effects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacification was observed, and swelling and membrane rupture of the lens fiber cells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficial cortical fibers during cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significantly inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers, via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  18. HRT dependent performance and bacterial community population of granular hydrogen-producing mixed cultures fed with galactose.

    PubMed

    Kumar, Gopalakrishnan; Sivagurunathan, Periyasamy; Park, Jeong-Hoon; Park, Jong-Hun; Park, Hee-Deung; Yoon, Jeong-Jun; Kim, Sang-Hyoun

    2016-04-01

    The effects of hydraulic retention times (HRTs-6, 3 and 2 h) on H2 production, operational stability and bacterial population response in a continuously stirred tank reactor (CSTR) were evaluated using galactose. A peak hydrogen production rate (HPR) of 25.9 L H2/L-d was obtained at a 3 h HRT with an organic loading rate (OLR) of 120 g/L-d, while the maximum hydrogen yield (HY) of 2.21 mol H2/mol galactose was obtained at a 6 h HRT (60 g galactose/L-d). Butyrate was dominant and the lactate concentration increased as HRT decreased, which significantly affected the HY. Biomass concentration (VSS) decreased from 16 to 3g/L at a 2 h HRT, leading to failure. A 3 h HRT supported the favorable growth of Clostridium species, as indicated by an increase in their populations from 25.4% to 27%, while significantly reducing Bacilli populations from 61.6% to 54.2%, indicating that this was the optimal condition. PMID:26859326

  19. A galactose-binding lectin isolated from Aplysia kurodai (sea hare) eggs inhibits streptolysin-induced hemolysis.

    PubMed

    Hasan, Imtiaj; Watanabe, Miharu; Ishizaki, Naoto; Sugita-Konishi, Yoshiko; Kawakami, Yasushi; Suzuki, Jun; Dogasaki, Chikaku; Rajia, Sultana; Kawsar, Sarkar M A; Koide, Yasuhiro; Kanaly, Robert A; Sugawara, Shigeki; Hosono, Masahiro; Ogawa, Yukiko; Fujii, Yuki; Iriko, Hideyuki; Hamako, Jiharu; Matsui, Taei; Ozeki, Yasuhiro

    2014-01-01

    A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes α-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of d-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth. PMID:25197935

  20. Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages

    SciTech Connect

    Schlepper-Schaefer, J.; Huelsmann, D.; Djovkar, A.; Meyer, H.E.; Herbertz, L.; Kolb, H.; Kolb-Bachofen, V.

    1986-01-01

    The intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues is rat liver was followed. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au/sub 5/), 17 nm (Au/sub 17/), 50 nm (Au/sub 50/) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au/sub 5/ in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc.

  1. Osmanthus fragrans Flower Extract and Acteoside Protect Against d-Galactose-Induced Aging in an ICR Mouse Model.

    PubMed

    Xiong, Lina; Mao, Shuqin; Lu, Baiyi; Yang, Jiajia; Zhou, Fei; Hu, Yinzhou; Jiang, Yirong; Shen, Canxi; Zhao, Yajing

    2016-01-01

    Osmanthus fragrans flower extract (OFE) is an organic extract from O. fragrans flower, which exhibits neuroprotective, free radical scavenging, and antioxidant effects. Therefore, the protective effect of OFE and acteoside against aging was studied. An aging ICR mouse model was established by chronically administering d-galactose (250 mg/kg) for 8 weeks. d-galactose induced spatial learning and memory impairments that were successfully inhibited by OFE and acteoside, which could shorten escape latency, improve platform crossing times, and increase zone time. The antioxidant potential of OFE and acteoside in vivo was evaluated by estimating the following: activities of antioxidant enzymes, such as glutathione peroxidase and aging-related enzyme, particularly monoamine oxidase; contents of lipid peroxidation methane dicarboxylic aldehyde, advanced glycation end products, and 8-hydroxy-2'-deoxyguanosine (a DNA damage product); and levels of nuclear factor-erythroid 2-related factor 2. OFE and acteoside also inhibited d-galactose-induced neurological aging by suppressing the increase in glial fibrillary acidic protein and neurotrophin-3. Considering the dose-dependent protective effects of OFE and acteoside, we concluded that OFE, rich in acteoside, was a good source of natural antiaging compounds. PMID:26181905

  2. The D-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates.

    PubMed

    Jimbo, M; Yanohara, T; Koike, K; Koike, K; Sakai, R; Muramoto, K; Kamiya, H

    2000-02-01

    A D-galactose binding lectin (SLL-2) was isolated from Sinularia lochmodes, an octocoral, by a combination of affinity chromatography on acid-treated agarose and FPLC on Superdex 200. SLL-2 agglutinated rabbit and horse erythrocytes while SLL-1, a minor component, reacted only with rabbit erythrocytes. SLL-2 is a glycoprotein with a molecular mass of 122 kDa and is composed of eight identical subunits (15 kDa). The sequence of the amino terminal region of SLL-2 did not show any apparent homology to the sequences of other animal and plant lectins. D-Galactose, N-acetyl-D-galactosamine, lactose, and melibiose were moderate inhibitors to the agglutination of rabbit erythrocytes. In contrast, horse erythrocytes were much more susceptible to agglutination by SLL-2, which was inhibited by sugars and glycoproteins such as D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and porcine stomach mucin. SLL-2 showed considerable tolerance to heating and kept its activity after heating at 80 degrees C for 60 min. In immuno-histochemical studies using an anti-SLL-2 antiserum and protein A gold conjugate, SLL-2 was found to be present in high amounts in the nematocysts. SLL-2 was also detected on the surface of symbiotic dinoflagellate, Symbiodinium sp. cells irrespective whether they were surrounded with or without host cells. These observations suggest the presence of lectin-mediated interaction between symbiotic dinoflagellates and S. lochmodes. PMID:10817910

  3. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats.

    PubMed

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-03-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficialeffects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacificationwas observed, and swelling and membrane rupture of the lens fibercells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficialcortical fibersduring cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significanty inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers,via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  4. Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif.

    PubMed

    Hatakeyama, Tomomitsu; Kamiya, Takuro; Kusunoki, Masami; Nakamura-Tsuruta, Sachiko; Hirabayashi, Jun; Goda, Shuichiro; Unno, Hideaki

    2011-03-25

    CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity. PMID:21247895

  5. UDP-galactose transporter gene hUGT1 expression in tobacco plants leads to hyper-galactosylated cell wall components.

    PubMed

    Abedi, Tayebeh; Khalil, Mohamed Farouk Mohamed; Asai, Toshihiko; Ishihara, Nami; Kitamura, Kenji; Ishida, Nobuhiro; Tanaka, Nobukazu

    2016-05-01

    We reported previously that tobacco plants transformed with the human UDP-galactose transporter 1 gene (hUGT1-transgenic plants) displayed morphological, architectural, and physiological alterations, such as enhanced growth, increased accumulation of chlorophyll and lignin, and a gibberellin-responsive phenotype. In the present study, we demonstrated that hUGT1 expression altered the monosaccharide composition of cell wall matrix polysaccharides, such as pectic and hemicellulosic polysaccharides, which are biosynthesized in the Golgi lumen. An analysis of the monosaccharide composition of the cell wall matrix polysaccharides revealed that the ratio of galactose to total monosaccharides was significantly elevated in the hemicellulose II and pectin fractions of hUGT1-transgenic plants compared with that of control plants. A hyper-galactosylated xyloglucan structure was detected in hemicellulose II using oligosaccharide mass profiling. These results indicated that, because of the enhanced UDP-galactose transport from the cytosol to the Golgi apparatus by hUGT1, galactose incorporation in the cell wall matrix polysaccharides increased. This increased galactose incorporation may have contributed to increased galactose tolerance in hUGT1-transgenic plants. PMID:26507776

  6. Genes Involved in Control of Galactose Uptake in Lactobacillus brevis and Reconstitution of the Regulatory System in Bacillus subtilis

    PubMed Central

    Djordjevic, Gordana M.; Tchieu, Jason H.; Saier, Milton H.

    2001-01-01

    The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-β-galactoside (TMG) by a permease-catalyzed sugar:H+ symport mechanism. Addition of glucose to L. brevis cells loaded with [14C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H+ symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H+ symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H+ symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response

  7. Oral administration of d-galactose induces cognitive impairments and oxidative damage in rats.

    PubMed

    Budni, Josiane; Pacheco, Robson; da Silva, Sabrina; Garcez, Michelle Lima; Mina, Francielle; Bellettini-Santos, Tatiani; de Medeiros, Jesiel; Voss, Bruna Constantino; Steckert, Amanda Valnier; Valvassori, Samira da Silva; Quevedo, João

    2016-04-01

    d-Galactose (d-gal) is a reducing sugar that can be used to mimic the characteristics of aging in rodents; however, the effects of d-gal administration by oral route are not clear. Therefore, the aim of this study was to elucidate if the oral administration of d-gal induces cognitive impairments, neuronal loss, and oxidative damage, mimicking an animal model of aging. Male adult Wistar rats (4 months old) received d-gal (100mg/kg) via the oral route for a period of 1, 2, 4, 6 or 8 weeks. The results showed cognitive impairments in the open-field test in the 4th and 6th weeks after d-gal administration, as well as an impairment in spatial memory in the radial maze test after the 6th week of d-gal administration. The results indicated increase of levels of thiobarbituric acid reactive species-TBARS-and carbonyl group content in the prefrontal cortex from the 4th week, and in all weeks of d-gal administration, respectively. An increase in the levels of TBARS and carbonyl group content was observed in the hippocampus over the entire period of d-gal treatment. In the 8th week of d-gal administration, we also observed reductions in synaptophysin and TAU protein levels in the prefrontal cortex. Thus, d-gal given by oral route caused cognitive impairments which were accompanied by oxidative damage. Therefore, these results indicate that orally administered d-gal can induce the behavioral and neurochemical alterations that are observed in the natural aging process. However, oral d-gal effect in rats deserve further studies to be better described. PMID:26748256

  8. Galactose-1-phosphate Uridyltransferase Dried Blood Spot Quality Control Materials for Newborn Screening Tests

    PubMed Central

    Adam, Barbara W.; Flores, Sharon R.; Hou, Yu; Allen, Todd W.; De Jesus, Victor R.

    2015-01-01

    Objectives We aimed to prepare dried-blood-spot (DBS) quality control (QC) materials for galactose-1-phosphate uridyltransferase (GALT), to evaluate their stability during storage and use, and to evaluate their performance in five DBS GALT test methods. Design and Methods We prepared and characterized GALT-normal and GALT-deficient DBS materials and compared GALT activities in DBSs after predetermined storage intervals at controlled temperatures and humidities. External evaluators documented the suitability of the DBS QC materials for use in five GALT test methods. Results GALT activity losses from DBSs stored in low (<30%) humidity for 14 days at 45°C, 35 days at 37°C, 91 days at room temperature, 182 days at 4°C, and 367 days at −20°C were 54%, 53%, 52% 23%, and 7% respectively. In paired DBSs stored in high humidity (>50%) for identical intervals, losses were: 45°C—68%; 37°C—79%; room temperature—72%, and 4°C—63%. GALT activities in DBSs stored at 4°C were stable throughout 19 excursions to room temperature. Twenty-five of 26 external evaluators, using five different GALT test methods, classified the GALT-deficient DBSs as “outside normal limits”. All evaluators classified the GALT-normal DBSs as “within normal limits”. Conclusions Most of the GALT activity loss from DBSs stored at elevated or room temperature was attributable to the effects of storage temperature. Most of the loss from DBSs stored at 4°C was attributable to the effects of elevated humidity. Loss from DBSs stored at −20°C was insignificant. The DBS materials were suitable for monitoring performance of all five GALT test methods. PMID:25528144

  9. Isorhynchophylline improves learning and memory impairments induced by D-galactose in mice.

    PubMed

    Xian, Yan-Fang; Su, Zi-Ren; Chen, Jian-Nan; Lai, Xiao-Ping; Mao, Qing-Qiu; Cheng, Christopher H K; Ip, Siu-Po; Lin, Zhi-Xiu

    2014-10-01

    Isorhynchophylline (IRN), an alkaloid isolated from Uncaria rhynchophylla, has been reported to improve cognitive impairment induced by beta-amyloid in rats. However, whether IRN could also ameliorate the D-galactose (D-gal)-induced mouse memory deficits is still not clear. In the present study, we aimed to investigate whether IRN had potential protective effect against the D-gal-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (100mg/kg) and orally administered IRN (20 or 40mg/kg) daily for 8weeks, followed by assessing spatial learning and memory function by the Morris water maze test. The results showed that IRN significantly improved spatial learning and memory function in the D-gal-treated mice. In the mechanistic studies, IRN significantly increased the level of glutathione (GSH) and the activities of superoxide dismutase (SOD) and catalase (CAT), while decreased the level of malondialdehyde (MDA) in the brain tissues of the D-gal-treated mice. Moreover, IRN (20 or 40mg/kg) significantly inhibited the production of prostaglandin E 2 (PGE2) and nitric oxide (NO), and the mRNA expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the activation of nuclear factor kappa B (NF-κB) in the brain tissues of D-gal-treated mice. Our results amply demonstrated that IRN was able to ameliorate cognitive deficits induced by D-gal in mice, and the observed cognition-improving action may be mediated, at least in part, through enhancing the antioxidant status and anti-inflammatory effect of brain tissues via NFκB signaling. PMID:24984171

  10. Carnosine and taurine treatments diminished brain oxidative stress and apoptosis in D-galactose aging model.

    PubMed

    Aydın, A Fatih; Çoban, Jale; Doğan-Ekici, Işın; Betül-Kalaz, Esra; Doğru-Abbasoğlu, Semra; Uysal, Müjdat

    2016-04-01

    D-galactose (GAL) has been used as an animal model for brain aging and antiaging studies. GAL stimulates oxidative stress in several tissues including brain. Carnosine (CAR; β-alanil-L-histidine) and taurine (TAU; 2-aminoethanesulfonic acid) exhibit antioxidant properties. CAR and TAU have anti-aging and neuroprotective effects. We investigated the effect of CAR and TAU supplementations on oxidative stress and brain damage in GAL-treated rats. Rats received GAL (300 mg/kg; s.c.; 5 days per week) alone or together with CAR (250 mg/kg/daily; i.p.; 5 days per week) or TAU (2.5% w/w; in rat chow) for 2 months. Brain malondialdehyde (MDA), protein carbonyl (PC) and glutathione (GSH) levels and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST) and acetylcholinesterase (AChE) activities were determined. Expressions of B cell lymphoma-2 (Bcl-2), Bax and caspase-3 were also evaluated in the brains by immunohistochemistry. GAL treatment increased brain MDA and PC levels and AChE activities. It decreased significantly brain GSH levels, SOD and GSH-Px but not GST activities. GAL treatment caused histopathological changes and increased apoptosis. CAR and TAU significantly reduced brain AChE activities, MDA and PC levels and elevated GSH levels in GAL-treated rats. CAR, but not TAU, significantly increased low activities of SOD and GSH-Px. Both CAR and TAU diminished apoptosis and ameliorated histopathological findings in the brain of GAL-treated rats. Our results indicate that CAR and TAU may be effective to prevent the development of oxidative stress, apoptosis and histopathological deterioration in the brain of GAL-treated rats. PMID:26518192

  11. Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction.

    PubMed

    Low, Diana Hooi Ping; Frecer, Vladimir; Le Saux, Agnès; Srinivasan, Ganesh Anand; Ho, Bow; Chen, Jianzhu; Ding, Jeak Ling

    2010-03-26

    Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human. PMID:20118243

  12. Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase

    PubMed Central

    Carney, Amanda E.; Sanders, Rebecca D.; Garza, Kerry R.; McGaha, Lee Anne; Bean, Lora J. H.; Coffee, Bradford W.; Thomas, James W.; Cutler, David J.; Kurtkaya, Natalie L.; Fridovich-Keil, Judith L.

    2009-01-01

    Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5′ proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5′ deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT–PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5′ deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms. PMID:19224951

  13. Adipose stem cells' antagonism in glycosylation of D-galactose-induced skin aging of nude mice and its skin recovery function.

    PubMed

    Wang, Haiying; Wei, Shuyue; Xue, Xinxin; You, Yuntian; Ma, Qiang

    2016-09-01

    This study aims to discuss adipose stem cells' (ASCs) antagonism in glycosylation of D-galactose-induced skin aging of nude mice and its skin recovery function; the study also aims to explore a new mechanism of anti-aging to provide clinical anti-aging therapy with new thoughts and methods. We selected 40 healthy specific pathogen-free (SPF) nude mice and divided them randomly into four groups which were: blank control group; D-galactose + phosphate buffer saline (PBS) group; D-galactose + ASCs treatment group; and D-galactose + aminoguanidine (AG) group. Results showed that the superoxide dismutase (SOD) level of mice in the D-galactose-induced model group (87.15 ± 4.95 U/g) decreased significantly compared with that of control group (146.21 ± 4.76 U/g), while malonaldehyde (MDA) level of mice in D-galactose induced model group (11.12 ± 2.08 nmol/mg) increased significantly compared with that of control group (5.46 ± 2.05 nmol/mg) (P <0.05); thus D-galactose induced sub-acutely aging mice models were duplicated successfully. Results also indicated that transplantation of ASCs could reverse expression of aging-related biomarkers such as MDA, SOD, and advanced glycosylation end products (AGEs); hematoxylin and eosin (HE) staining showed that thickness of the dermis layer as well as the collagen content of mice in the D-galactose-induced model group increased significantly after ASC transplantation compared with that of control group. In addition, immunohistochemical assay showed that expression quantity of CD31 and vascular endothelial growth factor (VEGF) of mice in the D-galactose-induced model group increased significantly after ASC transplantation compared with that of control group. In conclusion, ASCs can trace cell distribution successfully through bioluminescence, and they survive for a short time in the skin after transplantation, which provides a basis for the application of ASC transplantation in clinical practices. Moreover, ASCs can control

  14. Identification of a Fusobacterium nucleatum PK1594 galactose-binding adhesin which mediates coaggregation with periopathogenic bacteria and hemagglutination.

    PubMed Central

    Shaniztki, B; Hurwitz, D; Smorodinsky, N; Ganeshkumar, N; Weiss, E I

    1997-01-01

    Attachment of Fusobacterium nucleatum to various oral surfaces is mediated by several adhesins anchored on its outer surface. Monoclonal antibodies (MAbs) were prepared and used to identify the putative galactose-binding adhesin of F. nucleatum PK1594. Four unique MAbs, 8G7, 26B9, 28G11, and 29D4, were isolated on the basis of their ability to inhibit coaggregation of F. nucleatum PK1594 with Porphyromonas gingivalis PK1924. All four MAbs were also capable of inhibiting galactose-inhibitable interactions of F. nucleatum PK1594 with other oral gram-negative bacteria and with erythrocytes. Preincubation of F. nucleatum PK1594 with MAb 26B9 or its Fab fragments at concentrations lower than 1 microg/ml resulted in complete inhibition of coaggregation with P. gingivalis PK1924 or hemagglutination. F. nucleatum PK1594 surface components prepared by mild sonication or by extracting whole cells with detergents were subjected to Western blot analysis. None of the MAbs were able to recognize any polypeptide in these experiments. Therefore, detergent extracts of F. nucleatum PK1594 surface components were subjected to three experimental procedures: (i) separation by ion-exchange chromatography and testing of fractions for reaction with MAb 26B9 in an enzyme-linked immunosorbent assay (ELISA), (ii) lactose-Sepharose affinity chromatography and testing of the lactose eluate in ELISA with MAb 26B9, and (iii) immunoseparation with either MAb 26B9 or 8G7. Collectively, the results suggest that the putative adhesin is a 30-kDa outer membrane polypeptide which mediates the coaggregation with P. gingivalis PK1924 as well as other galactose-sensitive interactions of F. nucleatum PK1594. PMID:9393820

  15. Ultra Fast and Sensitive Liquid Chromatography Tandem Mass Spectrometry Based Assay for Galactose-1-Phosphate Uridylyltransferase and Galactokinase Deficiencies

    PubMed Central

    Li, Yijun; Ptolemy, Adam S.; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T.

    2013-01-01

    The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([13C6]-uridine diphosphate galactose in GALT assay and [13C6]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4 ± 4.2 and GALK activity of 1.8 ± 0.47 (mean ± SD) µmol·(g Hgb) −1·hr−1. Erythrocyte GALT activities in a cohort of 16 patients with classic galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analzyed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test. PMID:20863731

  16. Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies.

    PubMed

    Li, Yijun; Ptolemy, Adam S; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T

    2011-01-01

    The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) μmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test. PMID:20863731

  17. Influence of a family 29 carbohydrate binding module on the recombinant production of galactose oxidase in Pichia pastoris

    PubMed Central

    Mollerup, Filip; Master, Emma

    2015-01-01

    Herein, we report the extracellular expression of carbohydrate active fusion enzymes in Pichia pastoris. Particularly, CBM29-1-2 from Piromyces equi was separately fused to the N- and C-terminus of galactose 6-oxidase (GaO, D-galactose: oxygen 6-oxidoreductase, EC 1.1.13.9, CAZy family AA5) from Fusarium graminearum, generating CBM29-GaO and GaO-CBM29, respectively. P. pastoris was transformed with expression vectors encoding GaO, CBM29-GaO and GaO-CBM29, and the fusion proteins were expressed in both shake-flask and 2L bioreactor systems. Volumetric production yields and specific GaO activity increased when expression was performed in a bioreactor system compared to shake-flask cultivation. This was observed for both CBM29-GaO and GaO-CBM29, and is consistent with previous reports of GaO expression in P. pastoris (Spadiut et al., 2010; Anasontzis et al., 2014) [1], [2]. Fusion of CBM29 to the C-terminal of GaO (GaO-CBM29) resulted in a stable uniform protein at the expected calculated size (107 kDa) when analyzed with SDS-PAGE. By comparison, the expression of the N-terminal fusion protein (CBM29-GaO) was low, and two truncated versions of CBM29-GaO were coexpressed with the full-sized protein. Despite differences in protein yield, the specific GaO activity on galactose was not affected by CBM29 fusion to either the N- or C-terminus of the enzyme. A detailed description of the catalytic and physiochemical properties of CBM29-GaO and GaO-CBM29 is available in the parent publication (Mollerup et al., 2015) [3]. PMID:26858983

  18. Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

    PubMed Central

    Pickup, John C.; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J. S.

    2013-01-01

    We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.Fluorescence technique is one of the major solutions for achieving the continuous and noninvasive glucose sensor for diabetes. In this article, a highly sensitive nanostructured sensor is developed to detect extremely small amounts of aqueous glucose by applying fluorescence energy transfer (FRET). A one-pot method is applied to produce the dextran-fluorescein isothiocyanate (FITC)-conjugating mesoporous silica nanoparticles (MSNs), which afterward interact with the tetramethylrhodamine isothiocyanate (TRITC)-labeled concanavalin A (Con A) to form the FRET nanoparticles (FITC-dextran-Con A-TRITC@MSNs). The nanostructured glucose sensor is then formed via the self-assembly of the FRET nanoparticles on a transparent, flexible, and biocompatible substrate, e.g., poly(dimethylsiloxane). Our results indicate the diameter of the MSNs is 60 ± 5 nm. The difference in the images before and after adding 20 μl of glucose (0.10 mmol/liter) on the FRET sensor can be detected in less than 2 min by the laser confocal laser scanning microscope. The correlation between the ratio of fluorescence intensity, I(donor)/I(acceptor), of the FRET sensor and the concentration of aqueous glucose in the range of 0.04–4 mmol/liter has been investigated; a linear relationship is found. Furthermore, the durability of the nanostructured FRET sensor is evaluated for 5 days. In addition, the recorded images can be converted to digital images by

  19. Optimizing a Male Reproductive Aging Mouse Model by d-Galactose Injection

    PubMed Central

    Liao, Chun-Hou; Chen, Bing-Huei; Chiang, Han-Sun; Chen, Chiu-Wei; Chen, Mei-Feng; Ke, Chih-Chun; Wang, Ya-Yun; Lin, Wei-Ning; Wang, Chi-Chung; Lin, Ying-Hung

    2016-01-01

    The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies

  20. Optimizing a Male Reproductive Aging Mouse Model by D-Galactose Injection.

    PubMed

    Liao, Chun-Hou; Chen, Bing-Huei; Chiang, Han-Sun; Chen, Chiu-Wei; Chen, Mei-Feng; Ke, Chih-Chun; Wang, Ya-Yun; Lin, Wei-Ning; Wang, Chi-Chung; Lin, Ying-Hung

    2016-01-01

    The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8-10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or

  1. Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins.

    PubMed

    Gabrielsen, Mads; Abdul-Rahman, Puteri Shafinaz; Othman, Shatrah; Hashim, Onn H; Cogdell, Richard J

    2014-06-01

    Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding. PMID:24915077

  2. Production of Normal Mammalian Organ Culture Using a Medium Containing Mem-Alpha, Leibovitz L 15, Glucose Galactose Fructose

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor)

    1999-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under micro- gravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel. The medium used for culturing the cells, especially a mixture of epithelial and mesenchymal cells contains a mixture of Mem-alpha and Leibovits L15 supplemented with glucose, galactose and fructose.

  3. Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins

    PubMed Central

    Gabrielsen, Mads; Abdul-Rahman, Puteri Shafinaz; Othman, Shatrah; Hashim, Onn H.; Cogdell, Richard J.

    2014-01-01

    Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding. PMID:24915077

  4. Saponins from Aralia taibaiensis attenuate D-galactose-induced aging in rats by activating FOXO3a and Nrf2 pathways.

    PubMed

    Li, Ying-Na; Guo, Yu; Xi, Miao-Miao; Yang, Pei; Zhou, Xue-Ying; Yin, Shuang; Hai, Chun-Xu; Li, Jin-Gang; Qin, Xu-Jun

    2014-01-01

    Reactive oxygen species (ROS) are closely related to the aging process. In our previous studies, we found that the saponins from Aralia taibaiensis have potent antioxidant activity, suggesting the potential protective activity on the aging. However, the protective effect of the saponins and the possible underlying molecular mechanism remain unknown. In the present study, we employed a D-galactose-induced aging rat model to investigate the protective effect of the saponins. We found that D-galactose treatment induced obvious aging-related changes such as the decreased thymus and spleen coefficients, the increased advanced glycation end products (AGEs) level, senescence-associated β-galactosidase (SAβ-gal) activity, and malondialdehyde (MDA) level. Further results showed that Forkhead box O3a (FOXO3a), nuclear factor-erythroid 2-related factor 2 (Nrf2), and their targeted antioxidants such as superoxide dismutase 2 (SOD2), catalase (CAT), glutathione reductase (GR), glutathione (GSH), glutamate-cysteine ligase (GCL), and heme oxygenase 1 (HO-1) were all inhibited in the aging rats induced by D-galactose treatment. Saponins supplementation showed effective protection on these changes. These results demonstrate that saponins from Aralia taibaiensis attenuate the D-galactose-induced rat aging. By activating FOXO3a and Nrf2 pathways, saponins increase their downstream multiple antioxidants expression and function, at least in part contributing to the protection on the D-galactose-induced aging in rats. PMID:24669284

  5. Galactose-PEG dual conjugation of beta-(1-->3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake.

    PubMed

    Karinaga, Ryouji; Anada, Takahisa; Minari, Jusaku; Mizu, Masami; Koumoto, Kazuya; Fukuda, Junji; Nakazawa, Kohji; Hasegawa, Teruaki; Numata, Munenori; Shinkai, Seiji; Sakurai, Kazuo

    2006-03-01

    Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery. PMID:16174528

  6. Inhibitory Effect of Trigonella Foenum-Graecum on Galactose Induced Cataracts in a Rat Model; in vitro and in vivo Studies

    PubMed Central

    Kumar Gupta, Suresh; Kalaiselvan, Vivekananthan; Srivastava, Sushma; Saxena, Rohit; Sunder Agrawal, Shyam

    2009-01-01

    Purpose To evaluate the in vitro and in vivo anti-cataract potential of Trigonella foenumgraecum (TF) on galactose induced cataracts in an animal model. Methods In the in vitro group,enucleated rat lenses were maintained in organ culture containing Dulbecco’s Modified Eagles Medium alone (normal group),or with the addition of 30 mM galactose (control group). The medium in the test group was supplemented with both galactose and TF. All lenses were incubated at 37°C for 24 hours and then processed for determination of levels of reduced glutathione and malondialdehyde. In the in vivo group, cataracts were induced in rats by a 30% galactose diet alone (control) or with the addition of TF (treated group). Results Reduction (26%) in glutathione level and elevation (31%) in malondialdehyde content were observed in controls as compared to normal lenses. TF significantly (P<0.01) restored glutathione and reduced malondialdehyde levels as compared to controls. A significant delay in the onset and progression of cataract was observed with 2.5% TF diet; after 30 days none of the treated eyes developed mature cataracts as compared to 100% of control eyes. Conclusion TF can delay the onset and progression of cataracts in an experimental rat model of galactose induced cataracts both in vitro and in vivo. PMID:23198077

  7. Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

    PubMed Central

    Anasontzis, George E; Salazar Penã, Margarita; Spadiut, Oliver; Brumer, Harry; Olsson, Lisbeth

    2014-01-01

    Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014 PMID:24493559

  8. Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

    PubMed Central

    Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk R.; Lüder, Carsten G. K.; Ferguson, Michael A. J.; Routier, Françoise H.

    2015-01-01

    Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential. PMID:26529232

  9. Structural and functional mimic of galactose oxidase by a copper complex of a sterically demanding [N2O2] ligand.

    PubMed

    John, Alex; Shaikh, Mobin M; Ghosh, Prasenjit

    2008-06-01

    A structural and functional mimic of the galactose oxidase (GOase) enzyme active-site by a copper complex supported over a sterically demanding ligand having [N2O2] donor sites is reported. Specifically, the binding of the histidine (496 and 581) and tyrosine (272 and 495) residues to the copper center in a square-pyramidal fashion in the active-site of galactose oxidase (GOase) enzyme has been modeled in a copper complex, ([(3-tert-butyl-5-methyl-2-hydoxybenzyl)(3'-tert-butyl-5'-methyl-2'-oxobenzyl)(2-pyridylmethyl)]amine)Cu(OAc)) (1b), stabilized over a sterically demanding ligand in which the two phenolate-O atoms mimicked the tyrosine binding while an amine-N and pyridyl-N atoms emulated the histidine binding to the metal center, similar to that in the enzyme active-site. Furthermore, the copper complex 1b is found to be an effective functional model of the enzyme as it efficiently catalyzed the chemoselective oxidation of primary alcohols to aldehydes in high turnover numbers under ambient conditions. An insight into the nature of the active-species was obtained by EPR and CV studies, which in conjunction with the DFT studies, revealed that the active-species is an anti-ferromagnetically coupled diamagnetic radical cation, (1)1b+, obtained by one electron oxidation at the equatorial phenolate-O atom of the ligand in the 1b complex. PMID:18478142

  10. Effects of Dimethylaminoethanol and Compound Amino Acid on D-Galactose Induced Skin Aging Model of Rat

    PubMed Central

    Liu, Su; Chen, Zhenyu; Cai, Xia; Sun, Ying; Zhao, Cailing

    2014-01-01

    A lasting dream of human beings is to reverse or postpone aging. In this study, dimethylaminoethanol (DMAE) and compound amino acid (AA) in Mesotherapy were investigated for their potential antiaging effects on D-galactose induced aging skin. At 18 days after D-gal induction, each rat was treated with intradermal microinjection of saline, AA, 0.1% DMAE, 0.2% DMAE, 0.1% DMAE + AA, or 0.2% DMAE + AA, respectively. At 42 days after treatment, the skin wound was harvested and assayed. Measurement of epidermal and dermal thickness in 0.1% DMAE + AA and 0.2% DMAE + AA groups appeared significantly thicker than aging control rats. No differences were found in tissue water content among groups. Hydroxyproline in 0.1% DMAE + AA, 0.2% DMAE + AA, and sham control groups was much higher than all other groups. Collagen type I, type III, and MMP-1 expression was highly upregulated in both 0.1% DMAE + AA and 0.2% DMAE + AA groups compared with aging control. In contrast, TIMP-1 expression levels of various aging groups were significantly reduced when compared to sham control. Coinjection of DMAE and AA into target tissue has marked antiaging effects on D-galactose induced skin aging model of rat. PMID:25133239

  11. Effects of dimethylaminoethanol and compound amino acid on D-galactose induced skin aging model of rat.

    PubMed

    Liu, Su; Chen, Zhenyu; Cai, Xia; Sun, Ying; Zhao, Cailing; Liu, Fangjun; Liu, Dalie

    2014-01-01

    A lasting dream of human beings is to reverse or postpone aging. In this study, dimethylaminoethanol (DMAE) and compound amino acid (AA) in Mesotherapy were investigated for their potential antiaging effects on D-galactose induced aging skin. At 18 days after D-gal induction, each rat was treated with intradermal microinjection of saline, AA, 0.1% DMAE, 0.2% DMAE, 0.1% DMAE + AA, or 0.2% DMAE + AA, respectively. At 42 days after treatment, the skin wound was harvested and assayed. Measurement of epidermal and dermal thickness in 0.1% DMAE + AA and 0.2% DMAE + AA groups appeared significantly thicker than aging control rats. No differences were found in tissue water content among groups. Hydroxyproline in 0.1% DMAE + AA, 0.2% DMAE + AA, and sham control groups was much higher than all other groups. Collagen type I, type III, and MMP-1 expression was highly upregulated in both 0.1% DMAE + AA and 0.2% DMAE + AA groups compared with aging control. In contrast, TIMP-1 expression levels of various aging groups were significantly reduced when compared to sham control. Coinjection of DMAE and AA into target tissue has marked antiaging effects on D-galactose induced skin aging model of rat. PMID:25133239

  12. Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major

    PubMed Central

    Lamerz, Anne-Christin; Damerow, Sebastian; Kleczka, Barbara; Wiese, Martin; van Zandbergen, Ger; Lamerz, Jens; Wenzel, Alexander; Hsu, Fong-Fu; Turk, John; Beverley, Stephen M.; Routier, Françoise H.

    2010-01-01

    The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:α-d-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis. PMID:20335578

  13. Galactose-Functionalized PolyHIPE Scaffolds for Use in Routine Three Dimensional Culture of Mammalian Hepatocytes

    PubMed Central

    2013-01-01

    Three-dimensional (3D) cell culture is regarded as a more physiologically relevant method of growing cells in the laboratory compared to traditional monolayer cultures. Recently, the application of polystyrene-based scaffolds produced using polyHIPE technology (porous polymers derived from high internal phase emulsions) for routine 3D cell culture applications has generated very promising results in terms of improved replication of native cellular function in the laboratory. These materials, which are now available as commercial scaffolds, are superior to many other 3D cell substrates due to their high porosity, controllable morphology, and suitable mechanical strength. However, until now there have been no reports describing the surface-modification of these materials for enhanced cell adhesion and function. This study, therefore, describes the surface functionalization of these materials with galactose, a carbohydrate known to specifically bind to hepatocytes via the asialoglycoprotein receptor (ASGPR), to further improve hepatocyte adhesion and function when growing on the scaffold. We first modify a typical polystyrene-based polyHIPE to produce a cell culture scaffold carrying pendent activated-ester functionality. This was achieved via the incorporation of pentafluorophenyl acrylate (PFPA) into the initial styrene (STY) emulsion, which upon polymerization formed a polyHIPE with a porosity of 92% and an average void diameter of 33 μm. Histological analysis showed that this polyHIPE was a suitable 3D scaffold for hepatocyte cell culture. Galactose-functionalized scaffolds were then prepared by attaching 2′-aminoethyl-β-d-galactopyranoside to this PFPA functionalized polyHIPE via displacement of the labile pentafluorophenyl group, to yield scaffolds with approximately ca. 7–9% surface carbohydrate. Experiments with primary rat hepatocytes showed that cellular albumin synthesis was greatly enhanced during the initial adhesion/settlement period of cells on

  14. Galactose-functionalized polyHIPE scaffolds for use in routine three dimensional culture of mammalian hepatocytes.

    PubMed

    Hayward, Adam S; Eissa, Ahmed M; Maltman, Daniel J; Sano, Naoko; Przyborski, Stefan A; Cameron, Neil R

    2013-12-01

    Three-dimensional (3D) cell culture is regarded as a more physiologically relevant method of growing cells in the laboratory compared to traditional monolayer cultures. Recently, the application of polystyrene-based scaffolds produced using polyHIPE technology (porous polymers derived from high internal phase emulsions) for routine 3D cell culture applications has generated very promising results in terms of improved replication of native cellular function in the laboratory. These materials, which are now available as commercial scaffolds, are superior to many other 3D cell substrates due to their high porosity, controllable morphology, and suitable mechanical strength. However, until now there have been no reports describing the surface-modification of these materials for enhanced cell adhesion and function. This study, therefore, describes the surface functionalization of these materials with galactose, a carbohydrate known to specifically bind to hepatocytes via the asialoglycoprotein receptor (ASGPR), to further improve hepatocyte adhesion and function when growing on the scaffold. We first modify a typical polystyrene-based polyHIPE to produce a cell culture scaffold carrying pendent activated-ester functionality. This was achieved via the incorporation of pentafluorophenyl acrylate (PFPA) into the initial styrene (STY) emulsion, which upon polymerization formed a polyHIPE with a porosity of 92% and an average void diameter of 33 μm. Histological analysis showed that this polyHIPE was a suitable 3D scaffold for hepatocyte cell culture. Galactose-functionalized scaffolds were then prepared by attaching 2'-aminoethyl-β-D-galactopyranoside to this PFPA functionalized polyHIPE via displacement of the labile pentafluorophenyl group, to yield scaffolds with approximately ca. 7-9% surface carbohydrate. Experiments with primary rat hepatocytes showed that cellular albumin synthesis was greatly enhanced during the initial adhesion/settlement period of cells on the

  15. Modest phenotypic improvements in ASA-deficient mice with only one UDP-galactose:ceramide-galactosyltransferase gene

    PubMed Central

    Franken, S; Wittke, D; Mansson, JE; D'Hooge, R; De Deyn, PP; Lüllmann-Rauch, R; Matzner, U; Gieselmann, V

    2006-01-01

    Background Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice. Results ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice. Conclusion Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology. PMID:16893448

  16. A systems approach to designing next generation vaccines: combining α-galactose modified antigens with nanoparticle platforms

    NASA Astrophysics Data System (ADS)

    Phanse, Yashdeep; Carrillo-Conde, Brenda R.; Ramer-Tait, Amanda E.; Broderick, Scott; Kong, Chang Sun; Rajan, Krishna; Flick, Ramon; Mandell, Robert B.; Narasimhan, Balaji; Wannemuehler, Michael J.

    2014-01-01

    Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4+ T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.

  17. A systems approach to designing next generation vaccines: combining α-galactose modified antigens with nanoparticle platforms

    PubMed Central

    Phanse, Yashdeep; Carrillo-Conde, Brenda R.; Ramer-Tait, Amanda E.; Broderick, Scott; Kong, Chang Sun; Rajan, Krishna; Flick, Ramon; Mandell, Robert B.; Narasimhan, Balaji; Wannemuehler, Michael J.

    2014-01-01

    Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4+ T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens. PMID:24441019

  18. Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey.

    PubMed

    Barrett, Eoin; Stanton, Catherine; Zelder, Oskar; Fitzgerald, Gerald; Ross, R Paul

    2004-05-01

    The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and beta-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited beta-galactosidase activity in excess of 1000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and beta-galactosidase genes exhibited beta-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability. PMID:15128544

  19. Selenoprotein R Protects Human Lens Epithelial Cells against d-Galactose-Induced Apoptosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress

    PubMed Central

    Dai, Jie; Liu, Hongmei; Zhou, Jun; Huang, Kaixun

    2016-01-01

    Selenium is an essential micronutrient for humans. Much of selenium’s beneficial influence on health is attributed to its presence within 25 selenoproteins. Selenoprotein R (SelR), known as methionine sulfoxide reductase B1 (MsrB1), is a selenium-dependent enzyme that, like other Msrs, is required for lens cell viability. In order to investigate the roles of SelR in protecting human lens epithelial (hLE) cells against damage, the influences of SelR gene knockdown on d-galactose-induced apoptosis in hLE cells were studied. The results showed that both d-galactose and SelR gene knockdown by siRNA independently induced oxidative stress. When SelR-gene-silenced hLE cells were exposed to d-galactose, glucose-regulated protein 78 (GRP78) protein level was further increased, mitochondrial membrane potential was significantly decreased and accompanied by a release of mitochondrial cytochrome c. At the same time, the apoptosis cells percentage and the caspase-3 activity were visibly elevated in hLE cells. These results suggested that SelR might protect hLE cell mitochondria and mitigating apoptosis in hLE cells against oxidative stress and endoplasmic reticulum (ER) stress induced by d-galactose, implying that selenium as a micronutrient may play important roles in hLE cells. PMID:26875981

  20. Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose.

    PubMed

    Kirkeby, S

    2016-01-01

    FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms. PMID:26472148

  1. Structural basis of UDP-galactose binding by alpha-1,3-galactosyltransferase (alpha3GT): role of negative charge on aspartic acid 316 in structure and activity.

    PubMed

    Tumbale, Percy; Jamaluddin, Haryati; Thiyagarajan, Nethaji; Brew, Keith; Acharya, K Ravi

    2008-08-19

    alpha-1,3-Galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-galactose to form an alpha 1-3 link with beta-linked galactosides; it is part of a family of homologous retaining glycosyltransferases that includes the histo-blood group A and B glycosyltransferases, Forssman glycolipid synthase, iGb3 synthase, and some uncharacterized prokaryotic glycosyltransferases. In mammals, the presence or absence of active forms of these enzymes results in antigenic differences between individuals and species that modulate the interplay between the immune system and pathogens. The catalytic mechanism of alpha3GT is controversial, but the structure of an enzyme complex with the donor substrate could illuminate both this and the basis of donor substrate specificity. We report here the structure of the complex of a low-activity mutant alpha3GT with UDP-galactose (UDP-gal) exhibiting a bent configuration stabilized by interactions of the galactose with multiple residues in the enzyme including those in a highly conserved region (His315 to Ser318). Analysis of the properties of mutants containing substitutions for these residues shows that catalytic activity is strongly affected by His315 and Asp316. The negative charge of Asp316 is crucial for catalytic activity, and structural studies of two mutants show that its interaction with Arg202 is needed for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation. PMID:18651752

  2. Galactose targeted pH-responsive copolymer conjugated with near infrared fluorescence probe for imaging of intelligent drug delivery.

    PubMed

    Fu, Liyi; Sun, Chunyang; Yan, Lifeng

    2015-01-28

    Theranostic polymeric nanomaterials are of special important in cancer treatment. Here, novel galactose targeted pH-responsive amphiphilic multiblock copolymer conjugated with both drug and near-infrared fluorescence (NIR) probe has been designed and prepared by a four-steps process: (1) ring-opening polymerization (ROP) of N-carboxy anhydride (NCA) monomers using propargylamine as initiator; (2) reversible addition-fragmentation chain transfer (RAFT) polymerization of oligo(ethylene glycol) methacrylate (OEGMA) and gal monomer by an azido modified RAFT agent; (3) combing the obtained two polymeric segments by click reaction; (4) NIR copolymer prodrug was synthesized by chemical linkage of both cyanine dye and anticancer drug doxorubicin to the block copolymer via amide bond and hydrazone, respectively. The obtained NIRF copolymers were characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), and its was measured by means of micelles dynamic light scattering (DLS), field emission transmission electron microscopy (FETEM), and UV-vis and fluorescence spectrophotometry. The prodrug has strong fluorescence in the near-infrared region, and a pH sensitive drug release was confirmed at pH of 5.4 via an in vitro drug release experiment. Confocal laser scanning microscopy (CLSM) and flow cytometry experiments of the prodrug on both HepG2 and NIH3T3 cells reveal that the galactose targeted polymeric prodrug shows a fast and enhanced endocytosis due to the specific interaction for HepG2 cells, indicating the as-prepared polymer is a candidate for theranosis of liver cancer. PMID:25569169

  3. Chlorogenic acid protects d-galactose-induced liver and kidney injury via antioxidation and anti-inflammation effects in mice.

    PubMed

    Feng, Yan; Yu, Ying-Hua; Wang, Shu-Ting; Ren, Jing; Camer, Danielle; Hua, Yu-Zhou; Zhang, Qian; Huang, Jie; Xue, Dan-Lu; Zhang, Xiao-Fei; Huang, Xu-Feng; Liu, Yi

    2016-06-01

    Context Oxidative stress and inflammation are implicated in the aging process and its related hepatic and renal function decline. Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in the human diet. Recently, CGA has shown in vivo and in vitro antioxidant properties. Objective The current study investigates the effects of protective effects of chlorogenic acid (CGA) on d-galactose-induced liver and kidney injury. Materials and methods Hepatic and renal injuries were induced in a mouse model by subcutaneously injection of d-galactose (d-gal; 100 mg/kg) once a day for 8 consecutive weeks and orally administered simultaneously with CGA included in the food (200 mg/kg of diet). The liver and renal functions were examined. Histological analyses of liver and kidney were done by haematoxylin and eosin staining. The oxidative stress markers and pro-inflammatory cytokines in the liver and the kidney were measured. Results CGA significantly reduced the serum aminotransferase, serum creatinine (SCr) and blood urea nitrogen (BUN) levels in d-gal mice (p <0.05). CGA also restored superoxide dismutase, catalase, and malondialdehyde levels and decreased glutathione content in the liver and kidney in d-gal mice (p <0.05). Improvements in liver and kidney were also noted in histopathological studies. CGA reduced tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) protein levels in the liver and kidney in d-gal mice (p <0.05). Discussion and conclusion These findings suggest that CGA attenuates d-gal-induced chronic liver and kidney injury and that this protection may be due to its antioxidative and anti-inflammatory activities. PMID:26810301

  4. Raffinose Family Oligosaccharides Act As Galactose Stores in Seeds and Are Required for Rapid Germination of Arabidopsis in the Dark.

    PubMed

    Gangl, Roman; Tenhaken, Raimund

    2016-01-01

    Raffinose synthase 5 (AtRS5, At5g40390) was characterized from Arabidopsis as a recombinant enzyme. It has a far higher affinity for the substrates galactinol and sucrose than any other raffinose synthase previously reported. In addition raffinose synthase 5 is also working as a galactosylhydrolase, degrading galactinol, and raffinose under certain conditions. Together with raffinose synthase 4, which is predominantly a stachyose synthase, both enzymes contribute to the raffinose family oligosaccharide (RFO) accumulation in seeds. A double knockout in raffinose synthase 4 and raffinose synthase 5 (ΔAtRS4,5) was generated, which is devoid of RFOs in seeds. Unstressed leaves of 4 week old ΔAtRS4,5 plants showed drastically 23.8-fold increased concentrations of galactinol. Unexpectedly, raffinose appeared again in drought stressed ΔAtRS4,5 plants, but not under other abiotic stress conditions. Drought stress leads to novel transcripts of raffinose synthase 6 suggesting that this isoform is a further stress inducible raffinose synthase in Arabidopsis. ΔAtRS4,5 seeds showed a 5 days delayed germination phenotype in darkness and an elevated expression of the transcription factor phytochrome interacting factor 1 (AtPIF1) target gene AtPIF6, being a repressor of germination. This prolonged dormancy is not seen during germination in the light. Exogenous galactose partially promotes germination of ΔAtRS4,5 seeds in the dark suggesting that RFOs act as a galactose store and repress AtPIF6 transcripts. PMID:27507985

  5. Galactose-α-1,3-Galactose–Specific IgE Is Associated with Anaphylaxis but Not Asthma

    PubMed Central

    Commins, Scott P.; Kelly, Libby A.; Rönmark, Eva; James, Hayley R.; Pochan, Shawna L.; Peters, Edward J.; Lundbäck, Bo; Nganga, Lucy W.; Cooper, Philip J.; Hoskins, Janelle M.; Eapen, Saju S.; Matos, Luis A.; McBride, Dane C.; Heymann, Peter W.; Woodfolk, Judith A.; Perzanowski, Matthew S.

    2012-01-01

    Rationale: IgE antibodies to the mammalian oligosaccharide galactose-α-1,3-galactose (α-gal) are common in the southeastern United States. These antibodies, which are induced by ectoparasitic ticks, can give rise to positive skin tests or serum assays with cat extract. Objectives: To evaluate the relationship between IgE antibodies to α-gal and asthma, and compare this with the relationship between asthma and IgE antibodies to Fel d 1 and other protein allergens. Methods: Patients being investigated for recurrent anaphylaxis, angioedema, or acute urticaria underwent spirometry, exhaled nitric oxide, questionnaires, and serum IgE antibody assays. The results were compared with control subjects and cohorts from the emergency department in Virginia (n = 130), northern Sweden (n = 963), and rural Kenya (n = 131). Measurements and Main Results: Patients in Virginia with high-titer IgE antibodies to α-gal had normal lung function, low levels of exhaled nitric oxide, and low prevalence of asthma symptoms. Among patients in the emergency department and children in Kenya, there was no association between IgE antibodies to α-gal and asthma (odds ratios, 1.04 and 0.75, respectively). In Sweden, IgE antibodies to cat were closely correlated with IgE antibodies to Fel d 1 (r = 0.83) and to asthma (P < 0.001). Conclusions: These results provide a model of an ectoparasite-induced specific IgE response that can increase total serum IgE without creating a risk for asthma, and further evidence that the main allergens that are causally related to asthma are those that are inhaled. PMID:22281828

  6. Raffinose Family Oligosaccharides Act As Galactose Stores in Seeds and Are Required for Rapid Germination of Arabidopsis in the Dark

    PubMed Central

    Gangl, Roman; Tenhaken, Raimund

    2016-01-01

    Raffinose synthase 5 (AtRS5, At5g40390) was characterized from Arabidopsis as a recombinant enzyme. It has a far higher affinity for the substrates galactinol and sucrose than any other raffinose synthase previously reported. In addition raffinose synthase 5 is also working as a galactosylhydrolase, degrading galactinol, and raffinose under certain conditions. Together with raffinose synthase 4, which is predominantly a stachyose synthase, both enzymes contribute to the raffinose family oligosaccharide (RFO) accumulation in seeds. A double knockout in raffinose synthase 4 and raffinose synthase 5 (ΔAtRS4,5) was generated, which is devoid of RFOs in seeds. Unstressed leaves of 4 week old ΔAtRS4,5 plants showed drastically 23.8-fold increased concentrations of galactinol. Unexpectedly, raffinose appeared again in drought stressed ΔAtRS4,5 plants, but not under other abiotic stress conditions. Drought stress leads to novel transcripts of raffinose synthase 6 suggesting that this isoform is a further stress inducible raffinose synthase in Arabidopsis. ΔAtRS4,5 seeds showed a 5 days delayed germination phenotype in darkness and an elevated expression of the transcription factor phytochrome interacting factor 1 (AtPIF1) target gene AtPIF6, being a repressor of germination. This prolonged dormancy is not seen during germination in the light. Exogenous galactose partially promotes germination of ΔAtRS4,5 seeds in the dark suggesting that RFOs act as a galactose store and repress AtPIF6 transcripts. PMID:27507985

  7. Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine

    SciTech Connect

    Khan, Faaizah; Pickup, John C.

    2013-08-30

    Highlights: •We showed that the NIR fluorophore, 651-Blue Oxazine, is solvatochromic (polarity sensitive). •Blue Oxazine was covalently attached to mutants of glucose/galactose-binding protein (GBP). •Fluorescence intensity of GBP-Blue Oxazine increased with addition of glucose. •Fluorescence from bead-immobilised GBP-Blue Oxazine was detectable through skin in vitro. •This shows proof-of-concept for non-invasive glucose sensing using GBP-Blue Oxazine. -- Abstract: Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25 mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.

  8. Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

    PubMed Central

    Vázquez, Alicia; de Dios Ruiz-Rosado, Juan; Terrazas, Luis I.; Juárez, Imelda; Gomez-Garcia, Lorena; Calleja, Elsa; Camacho, Griselda; Chávez, Ana; Romero, Miriam; Rodriguez, Tonathiu; Espinoza, Bertha; Rodriguez-Sosa, Miriam

    2014-01-01

    The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 104 T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection. PMID:25170304

  9. Characterization of a mutation and an alternative splicing of UDP-galactose transporter in MDCK-RCAr cell line.

    PubMed

    Olczak, Mariusz; Guillen, Eduardo

    2006-01-01

    The UDP-galactose (UDP-Gal) transporter present in the Golgi apparatus is a member of a transporter family comprising hydrophobic proteins with multiple transmembrane domains. Co-immunoprecipitation experiments showed that the full-length UDP-Gal transporter protein forms oligomeric structures in the MDCK cell. A ricin-resistant mutant of the MDCK cell line (MDCK-RCA(r)) is deficient in galactose linked to macromolecules because of a lower UDP-Gal transport rate into the Golgi apparatus. We cloned this mutated protein and found that it contains a stop codon close to the 5' terminus of its open reading frame. We also detected a shorter splicing variant of the UDP-Gal transporter which contains a 183-nt in-frame deletion in both the wild-type and the mutant mRNA. We showed that the protein, when overexpressed, is localized in the Golgi apparatus and could partially correct the phenotype of the MDCK-RCA(r) and CHO-Lec8 mutant cell lines. The level of mRNA of the UDP-Gal transporter is much lower (25-30 copies per cell) than those of the CMP-sialic acid transporter (100 copies per cell), UDP-N-acetylglucosamine transporter (80 copies per cell), and GDP-fucose transporter (65 copies per cell). The transcript level of the shorter splicing variant of the UDP-Gal transporter is extremely rare in wild-type MDCK cells (a few copies per cell), but it is significantly increased in the mutant, RCA-resistant cells. PMID:16434112

  10. Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway.

    PubMed

    Badejo, Adebanjo Ayobamidele; Wada, Keiko; Gao, Yongshun; Maruta, Takanori; Sawa, Yoshihiro; Shigeoka, Shigeru; Ishikawa, Takahiro

    2012-01-01

    The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-(14)C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits. PMID:21984649

  11. Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway

    PubMed Central

    Badejo, Adebanjo Ayobamidele; Wada, Keiko; Gao, Yongshun; Maruta, Takanori; Sawa, Yoshihiro; Shigeoka, Shigeru; Ishikawa, Takahiro

    2012-01-01

    The D-mannose/L-galactose pathway for the biosynthesis of vitamin C (L-ascorbic acid; AsA) has greatly improved the understanding of this indispensable compound in plants, where it plays multifunctional roles. However, it is yet to be proven whether the same pathway holds for all the different organs of plants, especially the fruit-bearing plants, at different stages of development. Micro-Tom was used here to elucidate the mechanisms of AsA accumulation and regulation in tomato fruits. The mRNA expression of the genes in the D-mannose/L-galactose pathway were inversely correlated with increasing AsA content of Micro-Tom fruits during ripening. Feeding L-[6-14C]AsA to Micro-Tom plants revealed that the bulk of the label from AsA accumulated in the source leaf was transported to the immature green fruits, and the rate of translocation decreased as ripening progressed. L-Galactose feeding, but neither D-galacturonate nor L-gulono-1,4-lactone, enhanced the content of AsA in immature green fruit. On the other hand, L-galactose and D-galacturonate, but not L-gulono-1,4-lactone, resulted in an increase in the AsA content of red ripened fruits. Crude extract prepared from insoluble fractions of green and red fruits showed D-galacturonate reductase- and aldonolactonase-specific activities, the antepenultimate and penultimate enzymes, respectively, in the D-galacturonate pathway, in both fruits. Taken together, the present findings demonstrated that tomato fruits could switch between different sources for AsA supply depending on their ripening stages. The translocation from source leaves and biosynthesis via the D-mannose/L-galactose pathway are dominant sources in immature fruits, while the alternative D-galacturonate pathway contributes to AsA accumulation in ripened Micro-Tom fruits. PMID:21984649

  12. A reinvestigation of the secondary structure of functionally active vSGLT, the vibrio sodium/galactose cotransporter.

    PubMed

    Turk, Eric; Gasymov, Oktay K; Lanza, Seren; Horwitz, Joseph; Wright, Ernest M

    2006-02-01

    The bacterial Na(+)/galactose cotransporter vSGLT of Vibrio parahaemolyticus is a member of the sodium:solute symporter family (SSS). Previous studies using electron microscopy have shown that vSGLT is a monomeric protein. Computational and experimental topological analyses have consistently indicated that this protein possesses 14 transmembrane alpha-helices. Our previous study using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to quantitate secondary structure content had indicated, in contrast, an alpha-helical content of only 35%, too little to be consistent with the 14-span model [le Coutre, J., et al. (2002) Biochemistry 41, 8082-6]. ATR-FTIR had also indicated that upon binding of Na(+) and d-galactose, the alpha-helical content increased to 53%. Here we revisit the vSGLT secondary structural distribution using an alternative approach, ultraviolet circular dichroism spectropolarimetry (CD), which is highly accurate in determining the alpha-helical content of a protein in solution. CD spectra were obtained from actively functional, soluble vSGLT and, as an internal check, from a fusion protein of vSGLT and the beta-barrel green fluorescent protein (GFP). Far-UV CD of vSGLT indicates a predominating 85% alpha-helical content, and an absence of beta-strands. Far-UV CD of the vSGLT-GFP fusion corroborates this profile, indicating an equivalent alpha-helical content, and a beta-strand content consistent with the GFP contribution. No detectable substrate-induced macroscopic changes in secondary structure are apparent in the far UV. In the near UV, increases in positive CD intensity occur in a stepwise manner with added substrates, implying changing environments of aromatic amino acid residues. CD thus confirms the current 14-transmembrane span model of vSGLT and reveals distinct substrate-induced conformational changes. The high percentage of alpha-helical structure found requires, when considered in the context of membrane

  13. Purification, some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin.

    PubMed

    Konozy, Emadeldin H E; Mulay, Ranjana; Faca, Vitor; Ward, Richard John; Greene, Lewis Joel; Roque-Barriera, Maria Cristina; Sabharwal, Sushma; Bhide, Shobhana V

    2002-10-01

    Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS. PMID:12504284

  14. Computer-Aided Identification of Trypanosoma brucei Uridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness

    PubMed Central

    2010-01-01

    Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4′-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 μM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization. PMID:20527952

  15. An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence.

    PubMed

    de Jesus, José Batista; de Sá Pinheiro, Ana Acacia; Lopes, Angela H C S; Meyer-Fernandes, José Roberto

    2002-01-01

    This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity. PMID:12440729

  16. A beta-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity.

    PubMed

    Wang, Jian-Hua; Kong, Jing; Li, Wei; Molchanova, Valentina; Chikalovets, Irina; Belogortseva, Natalia; Luk'yanov, Pavel; Zheng, Yong-Tang

    2006-01-01

    A 30 kDa beta-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC(50) values were 0.0043 and 0.057 microM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC(50) of 0.073 microM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 microM and 0.07 microM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells. PMID:16316787

  17. The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

    PubMed Central

    Ng, Wy Ching; Liong, Stella; Tate, Michelle D.; Irimura, Tatsuro; Denda-Nagai, Kaori; Brooks, Andrew G.; Londrigan, Sarah L.

    2014-01-01

    Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca2+-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca2+-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV. PMID:24257596

  18. A Laterally Acquired Galactose Oxidase-Like Gene Is Required for Aerial Development during Osmotic Stress in Streptomyces coelicolor

    PubMed Central

    Liman, Recep; Facey, Paul D.; van Keulen, Geertje; Dyson, Paul J.; Del Sol, Ricardo

    2013-01-01

    Phylogenetic reconstruction revealed that most Actinobacterial orthologs of S. coelicolor SCO2837, encoding a metal-dependent galactose oxidase-like protein, are found within Streptomyces and were probably acquired by horizontal gene transfer from fungi. Disruption of SCO2837 (glxA) caused a conditional bld phenotype that could not be reversed by extracellular complementation. Studies aimed at characterising the regulation of expression of glxA showed that it is not a target for other bld genes. We provide evidence that glxA is required for osmotic adaptation, although independently from the known osmotic stress response element SigB. glxA has been predicted to be part of an operon with the transcription unit comprising the upstream cslA gene and glxA. However, both phenotypic and expression studies indicate that it is also expressed from an independent promoter region internal to cslA. GlxA displays an in situ localisation pattern similar to that one observed for CslA at hyphal tips, but localisation of the former is independent of the latter. The functional role of GlxA in relation to CslA is discussed. PMID:23326581

  19. New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1.

    PubMed

    Rodríguez, Cristina; Sanz, Pascual; Gancedo, Carlos

    2003-03-01

    We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation. PMID:12702249

  20. Lignans from Schisandra chinensis ameliorate cognition deficits and attenuate brain oxidative damage induced by D-galactose in rats.

    PubMed

    Yan, Tingxu; Shang, Lei; Wang, Mengshi; Zhang, Chenning; Zhao, Xu; Bi, Kaishun; Jia, Ying

    2016-06-01

    The aim of this study was to explore the neuroprotective effects of active compounds from Schisandra chinensis (Trucz.) Baill. (Magnoliaceae) against the D-galactose (D-gal)-induced neurotoxicity in rat. The Wistar rats were subcutaneously injected with D-gal (150 mg/(kg day)) for six weeks and orally administered with water extract or 95 % ethanol extract (partitioned with petroleum ether (PE), chloroform (CF), ethyl acetate (EA) and n-Butanol (NB), respectively) of the fruits of Schisandra chinensis simultaneously. The alteration of cognitive functions was assessed by using Morris water maze and Step-down type passive avoidance test. The results demonstrated that PE fraction was the most effective fraction to ameliorate cognitive deficits. Further biochemical examination indicated that PE could attenuate the activities decreasing of superoxide dismutase (SOD), catalase (CAT), the total antioxidant (T-AOC) induced by D-gal, and maintain the normal levels of glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO) in the serum, prefrontal cortex, striatum and hippocampus of the brain of related rat, selectively. Meanwhile, the compounds of PE fraction were also identified as mainly lignans, thus, these results suggest that lignans from the PE fraction of Schisandra chinensis represented a potential source of medicine for the treatment of the aging-associated neurodegenerative diseases. PMID:26847610

  1. Cerebralcare Granule(®), a Chinese Herb Compound Preparation, Attenuates D-Galactose Induced Memory Impairment in Mice.

    PubMed

    Qu, Zhuo; Yang, Honggai; Zhang, Jingze; Huo, Liqin; Chen, Hong; Li, Yuming; Liu, Changxiao; Gao, Wenyuan

    2016-09-01

    Cerebralcare granule(®) (CG) is a preparation of Traditional Chinese Medicine that widely used in China. It was approved by the China State Food and Drug Administration for treatment of headache and dizziness associated with cerebrovascular diseases. In the present study, we aimed to investigate whether CG had protective effect against D-galactose (gal)-induced memory impairment and to explore the mechanism of its action. D-gal was administered (100 mg/kg, subcutaneously) once daily for 8 weeks to induced memory deficit and neurotoxicity in the brain of aging mouse and CG (7.5, 15, and 30 g/kg) were simultaneously administered orally. The present study demonstrates that CG can alleviate aging in the mouse brain induced by D-gal through improving behavioral performance and reducing brain cell damage in the hippocampus. CG prevents aging mainly via suppression of oxidative stress response, such as decreasing NO and MDA levels, renewing activities of SOD, CAT, and GPx, as well as decreasing AChE activity in the brain of D-gal-treated mice. In addition, CG prevents aging through inhibiting NF-κB-mediated inflammatory response and caspase-3-medicated neurodegeneration in the brain of D-gal treated mice. Taken together, these data clearly demonstrates that subcutaneous injection of D-gal produced memory deficit, meanwhile CG can protect neuron from D-gal insults and improve memory ability. PMID:27161371

  2. Unraveling the Conformational Landscape of Ligand Binding to Glucose/Galactose-Binding Protein by Paramagnetic NMR and MD Simulations.

    PubMed

    Unione, Luca; Ortega, Gabriel; Mallagaray, Alvaro; Corzana, Francisco; Pérez-Castells, Javier; Canales, Angeles; Jiménez-Barbero, Jesús; Millet, Oscar

    2016-08-19

    Protein dynamics related to function can nowadays be structurally well characterized (i.e., instances obtained by high resolution structures), but they are still ill-defined energetically, and the energy landscapes are only accessible computationally. This is the case for glucose-galactose binding protein (GGBP), where the crystal structures of the apo and holo states provide structural information for the domain rearrangement upon ligand binding, while the time scale and the energetic determinants for such concerted dynamics have been so far elusive. Here, we use GGBP as a paradigm to define a functional conformational landscape, both structurally and energetically, by using an innovative combination of paramagnetic NMR experiments and MD simulations. Anisotropic NMR parameters induced by self-alignment of paramagnetic metal ions was used to characterize the ensemble of conformations adopted by the protein in solution while the rate of interconversion between conformations was elucidated by long molecular dynamics simulation on two states of GGBP, the closed-liganded (holo_cl) and open-unloaded (apo_op) states. Our results demonstrate that, in its apo state, the protein coexists between open-like (68%) and closed-like (32%) conformations, with an exchange rate around 25 ns. Despite such conformational heterogeneity, the presence of the ligand is the ultimate driving force to unbalance the equilibrium toward the holo_cl form, in a mechanism largely governed by a conformational selection mechanism. PMID:27219646

  3. Antioxidant effects of the orientin and vitexin in Trollius chinensis Bunge in D-galactose-aged mice★

    PubMed Central

    An, Fang; Yang, Guodong; Tian, Jiaming; Wang, Shuhua

    2012-01-01

    Total flavonoids are the main pharmaceutical components of Trollius chinensis Bunge, and orientin and vitexin are the monomer components of total flavonoids in Trollius chinensis Bunge. In this study, an aged mouse model was established through intraperitoneal injection of D-galactose for 8 weeks, followed by treatment with 40, 20, or 10 mg/kg orientin, vitexin, or a positive control (vitamin E) via intragastric administration for an additional 8 weeks. Orientin, vitexin, and vitamin E improved the general medical status of the aging mice and significantly increased their brain weights. They also produced an obvious rise in total antioxidant capacity, superoxide dismutase, catalase, and glutathione peroxidase levels in the serum, and the levels of superoxide dismutase, catalase and glutathione peroxidase, Na+-K+-ATP enzyme, and Ca2+-Mg2+-ATP enzyme in the liver, brain and kidneys. In addition, they significantly reduced malondialdehyde levels in the liver, brain and kidney and lipofuscin levels in the brain. They also significantly improved the neuronal ultrastructure. The 40 mg/kg dose of orientin and vitexin had the same antioxidant capacity as vitamin E. These experimental findings indicate that orientin and vitexin engender anti-aging effects through their antioxidant capacities. PMID:25368632

  4. sup 19 F NMR studies of the D-galactose chemosensory receptor. (1) Sugar binding yields a global structural change

    SciTech Connect

    Luck, L.A.; Falke, J.J. )

    1991-04-30

    The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enable docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, the authors have used {sup 19}F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting {sup 19}F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylaline. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via {sup 19}F NMR frequency shifts at 10 of the 12 labeled sites. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.

  5. Antioxidant effects of the orientin and vitexin in Trollius chinensis Bunge in D-galactose-aged mice.

    PubMed

    An, Fang; Yang, Guodong; Tian, Jiaming; Wang, Shuhua

    2012-11-25

    Total flavonoids are the main pharmaceutical components of Trollius chinensis Bunge, and orientin and vitexin are the monomer components of total flavonoids in Trollius chinensis Bunge. In this study, an aged mouse model was established through intraperitoneal injection of D-galactose for 8 weeks, followed by treatment with 40, 20, or 10 mg/kg orientin, vitexin, or a positive control (vitamin E) via intragastric administration for an additional 8 weeks. Orientin, vitexin, and vitamin E improved the general medical status of the aging mice and significantly increased their brain weights. They also produced an obvious rise in total antioxidant capacity, superoxide dismutase, catalase, and glutathione peroxidase levels in the serum, and the levels of superoxide dismutase, catalase and glutathione peroxidase, Na(+)-K(+)-ATP enzyme, and Ca(2+)-Mg(2+)-ATP enzyme in the liver, brain and kidneys. In addition, they significantly reduced malondialdehyde levels in the liver, brain and kidney and lipofuscin levels in the brain. They also significantly improved the neuronal ultrastructure. The 40 mg/kg dose of orientin and vitexin had the same antioxidant capacity as vitamin E. These experimental findings indicate that orientin and vitexin engender anti-aging effects through their antioxidant capacities. PMID:25368632

  6. Identification of concanavalin A receptors and galactose-binding proteins in purified plasma membranes of Dictyostelium discoideum.

    PubMed

    West, C M; McMahon, D

    1977-07-01

    Two techniques have been modified to provide simple means for the identification of molecules which bind concanavalin A (Con A). Crossed immunoelectrophoresis was altered by replacing antibody with Con A, and receptors were identified by the precipitin arcs which they produced. Con A, tagged with fluorescein isothiocyanate, was also diffused into prefixed sodium dodecyl sulfate (SDS)-polyacrylamide gels, and additional receptors identified by fluorescence. More than 35 molecules in the plasma membranes of the cellular slime mold Dictyostelium discoideum which bind Con A were identified with these techniques. At least 12 of these diminish and 12 increase in importance as receptors during differentiation of the cells from the vegetative to the preculmination stage of development. In the course of these experiments, it was possible to confirm the presence of the galactose-binding protein discoidin, in the plasma membrane, by electrophoresing membrane proteins into an agarose gel. This lectin regains its sugar-binding activity after denaturation and electrophoresis in SDS. PMID:559679

  7. Genome Sequence of Vibrio sp. Strain EJY3, an Agarolytic Marine Bacterium Metabolizing 3,6-Anhydro-l-Galactose as a Sole Carbon Source

    PubMed Central

    Roh, Hanseong; Yun, Eun Ju; Lee, Saeyoung; Ko, Hyeok-Jin; Kim, Sujin; Kim, Byung-Yong; Song, Heesang; Lim, Kwang-il

    2012-01-01

    The metabolic fate of 3,6-anhydro-l-galactose (l-AHG) is unknown in the global marine carbon cycle. Vibrio sp. strain EJY3 is an agarolytic marine bacterium that can utilize l-AHG as a sole carbon source. To elucidate the metabolic pathways of l-AHG, we have sequenced the complete genome of Vibrio sp. strain EJY3. PMID:22535948

  8. Allergy to Red Meat: A Diagnosis Made by the Patient and Confirmed by an Assay for IgE Antibodies Specific for Alpha-1,3-Galactose.

    PubMed

    Kaloga, Mamadou; Kourouma, Sarah; Kouassi, Yao Isidore; Ecra, Elidje Joseph; Gbery, Ildevert Patrice; Allou, Ange S; Diabate, Almamy; Djeha, Djokouehi; Sangaré, Abdoulaye; Yoboue, Yao Pauline

    2016-01-01

    We report the first case of allergy to red meat observed in Ivory Coast. A 49-year-old male presented with pruritus. The diagnosis of allergy to red meat was confirmed by an assay for IgE antibodies specific for alpha-1,3 galactose. Interestingly, the disease was considered a spell to the patient who was suspected of being a sorcerer by the community. PMID:26933408

  9. L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae.

    PubMed

    Aro-Kärkkäinen, Niina; Toivari, Mervi; Maaheimo, Hannu; Ylilauri, Mikko; Pentikäinen, Olli T; Andberg, Martina; Oja, Merja; Penttilä, Merja; Wiebe, Marilyn G; Ruohonen, Laura; Koivula, Anu

    2014-12-01

    Four potential dehydrogenases identified through literature and bioinformatic searches were tested for L-arabonate production from L-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a D-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a L-arabinose/D-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP(+) but uses also NAD(+) as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards L-arabinose, D-galactose and D-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of L-arabinose, and the stable oxidation product detected is L-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear L-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for L-arabinose uptake, resulted in production of 18 g of L-arabonate per litre, at a rate of 248 mg of L-arabonate per litre per hour, with 86 % of the provided L-arabinose converted to L-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for L-arabonate production in yeast. PMID:25236800

  10. Protective Effects of Selenium, Vitamin E, and Purple Carrot Anthocyanins on D-Galactose-Induced Oxidative Damage in Blood, Liver, Heart and Kidney Rats.

    PubMed

    Li, Xia; Zhang, Yunlong; Yuan, Yuan; Sun, Yong; Qin, Yan; Deng, Zeyuan; Li, Hongyan

    2016-10-01

    The present study was performed to investigate the protective effects of selenium (Se), vitamin E (Vit E) and anthocyanins from purple carrots and their combination against the oxidative stress induced by D-galactose in rats. A total of 80 male rats were equally divided into 11 groups, one of which acted as control (I) just receiving intraperitoneal injections of physiological saline. The remaining ten groups (II-XI) were intraperitoneally injected with D-galactose at a dose of 400 mg kg(-1) body weight (BW) per day for 42 consecutive days. Rats in groups III-XI were treated with antioxidants via gavage per day as follows: group III: Se-methylselenocysteine (SeMSC), IV: Se as sodium selenite (Na2SeO3), V: Se-enriched yeast (SeY), VI: Vit E as α-tocopherol acetate, VII: anthocyanin from purple carrots (APC), VIII: APC + Vit E, IX: SeMSC + APC+ Vit E, X: Na2SeO3 + APC + Vit E, XI: SeY + Ant + Vit E. The results showed that the rats treated with antioxidants (III-XI) showed significant decreases in the levels of malondialdehyde (MDA) and carbonyl protein (PCO) compared with the D-galactose-treated group (II) in the heart, liver, kidneys, and blood. Moreover, there were significant increases in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), glutathione (GSH) concentration, and total antioxidant capacity (T-AOC) in the heart, liver, kidneys, and blood of antioxidant-treated animals (III-XI) than those in control group (I). In addition, the combined treatments of two or three antioxidants showed greater antioxidant activities than those of individual treatments, suggesting the synergistic antioxidant effects of Se, Vit E, and APC. In conclusion, all the antioxidants exhibited protective effects against D-galactose-induced oxidative damage in rats, and these antioxidants showed a synergistic effect. PMID:27025718

  11. Histochemical localization of galactose-containing glycoconjugates in sensory neurons and their processes in the central and peripheral nervous system of the rat.

    PubMed

    Streit, W J; Schulte, B A; Balentine, D J; Spicer, S S

    1985-10-01

    We studied the distribution of sugar residues in the oligosaccharide chains of complex carbohydrates in tissue sections of rat spinal cord, brainstem, and sensory ganglia using twelve lectin-horseradish peroxidase conjugates. Glycoconjugates containing terminal galactose residues were localized apparently in the Golgi apparatus in a population of predominantly small B-type neurons in spinal and trigeminal ganglia. Large A-type neurons rarely showed reactivity with galactose-binding lectins. A cells stained for glycoconjugates with N-glycosidically linked oligosaccharides and glycogen. The central and peripheral processes of the small neurons, mostly unmyelinated C fibers in sensory roots and spinal nerves, contained an abundance of glycoconjugates with terminal alpha-galactose residues. The central projections and terminals of small to medium-sized primary sensory neurons in the spinal and trigeminal ganglia were visualized in Lissauer's tract and the substantia gelatinosa in the spinal cord, and in the spinal trigeminal tract and the nucleus trigeminus in the lower medulla with lectins specific for terminal alpha-galactose residues. In addition, fibers of the solitary system and the area postrema were reactive with these lectins. The peripheral and central nervous system elements with affinity for galactopyranosyl-specific lectins correspond in distribution with neuroanatomical regions thought to be involved in the transmission and relay of somatic and visceral afferent inputs such as pain and temperature. Such specific localization of a glycosubstance to a distinct subpopulation of neurons and their peripheral and central processes suggests that the particular glycoconjugate may be of physiological significance. PMID:4045182

  12. Ginkgo Biloba Extract EGB761 Protects against Aging-Associated Diastolic Dysfunction in Cardiomyocytes of D-Galactose-Induced Aging Rat

    PubMed Central

    Liu, Jing; Wang, Junhong; Chen, Xiangjian; Guo, Changqing; Guo, Yan; Wang, Hui

    2012-01-01

    The aim of the present study was to make use of the artificially induced aging model cardiomyocytes to further investigate potential anti-aging-associated cellular diastolic dysfunction effects of EGB761 and explore underlying molecular mechanisms. Cultured rat primary cardiomyocytes were treated with either D-galactose or D-galactose combined with EGB761 for 48 h. After treatment, the percentage of cells positive for SA-β-gal, AGEs production, cardiac sarcoplasmic reticulum calcium pump (SERCA) activity, the myocardial sarcoplasmic reticulum calcium uptake, and relative protein levels were measured. Our results demonstrated that in vitro stimulation with D-galactose induced AGEs production. The addition of EGB761 significantly decreased the number of cells positive for SA-β-gal. Furthermore, decreased diastolic [Ca2+]i, curtailment of the time from the maximum concentration of Ca2+ to the baseline level and increased reuptake of Ca2+ stores in the SR were also observed. In addition, the level of p-Ser16-PLN protein as well as SERCA was markedly increased. The study indicated that EGb761 alleviates formation of AGEs products on SERCA2a in order to mitigate myocardial stiffness on one hand; on other hand, improve SERCA2a function through increase the amount of Ser16 sites PLN phosphorylation, which two hands finally led to ameliorate diastolic dysfunction of aging cardiomyocytes. PMID:22693651

  13. Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by Streptococcus thermophilus LY03

    PubMed Central

    Degeest, Bart; De Vuyst, Luc

    2000-01-01

    The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of α-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03. PMID:10919816

  14. Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

    PubMed

    Liu, Jintao; Wang, Jie; Fan, Li; Chen, Xinning; Hu, Dongdong; Deng, Xiancun; Poon, H Fai; Wang, Haibin; Liu, Xuping; Tan, Wen-Song

    2015-07-01

    Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system. PMID:25931375

  15. A galactose-functionalized dendritic siRNA-nanovector to potentiate hepatitis C inhibition in liver cells

    NASA Astrophysics Data System (ADS)

    Lakshminarayanan, Abirami; Reddy, B. Uma; Raghav, Nallani; Ravi, Vijay Kumar; Kumar, Anuj; Maiti, Prabal K.; Sood, A. K.; Jayaraman, N.; Das, Saumitra

    2015-10-01

    A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated region (UTR) of HCV RNA using a liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand (DG). Physico-chemical characterization revealed finer details of complexation of DG with siRNA, whereas molecular dynamic simulations demonstrated sugar moieties projecting ``out'' in the complex. Preferential delivery of siRNA to the liver was achieved through a highly specific ligand-receptor interaction between dendritic galactose and the asialoglycoprotein receptor. The siRNA-DG complex exhibited perinuclear localization in liver cells and co-localization with viral proteins. The histopathological studies showed the systemic tolerance and biocompatibility of DG. Further, whole body imaging and immunohistochemistry studies confirmed the preferential delivery of the nucleic acid to mice liver. Significant decrease in HCV RNA levels (up to 75%) was achieved in HCV subgenomic replicon and full length HCV-JFH1 infectious cell culture systems. The multidisciplinary approach provides the `proof of concept' for restricted delivery of therapeutic siRNAs using a target oriented dendritic nano-vector.A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated

  16. Silybum marianum oil attenuates oxidative stress and ameliorates mitochondrial dysfunction in mice treated with D-galactose

    PubMed Central

    Zhu, Shu Yun; Dong, Ying; Tu, Jie; Zhou, Yue; Zhou, Xing Hua; Xu, Bin

    2014-01-01

    Background: Silybum marianum has been used as herbal medicine for the treatment of liver disease, liver cirrhosis, and to prevent liver cancer in Europe and Asia since ancient times. Silybum marianum oil (SMO), a by-product of silymarin production, is rich in essential fatty acids, phospholipids, sterols, and vitamin E. However, it has not been very good development and use. Objective: In the present study, we used olive oil as a control to investigate the antioxidant and anti-aging effect of SMO in D-galactose (D-gal)-induced aging mice. Materials and Methods: D-gal was injected intraperitoneally (500 mg/kg body weight daily) for 7 weeks while SMO was simultaneously administered orally. The triglycerides (TRIG) and cholesterol (CHOL) levels were estimated in the serum. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), monoamine oxidase (MAO), malondialdehyde (MDA), caspase-3, and Bcl-2 were determined in the liver and brain. The activities of Na+-K+-adenosine triphosphatase (ATPase), Ca2+-Mg2+-ATPase, membrane potential (ΔΨm), and membrane fluidity of the liver mitochondrial were estimated. Results: SMO decreased levels of TRIG and CHOL in aging mice. SMO administration elevated the activities of SOD, GSH-Px, and T-AOC, which are suppressed by aging. The levels of MAO and MDA in the liver and brain were reduced by SMO administration in aging mice. Enzyme linked immunosorbent assay showed that SMO significantly decreased the concentration of caspase-3 and improved the activity of Bcl-2 in the liver and brain of aging mice. Furthermore, SMO significantly attenuated the D-gal induced liver mitochondrial dysfunction by improving the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase, membrane potential (ΔΨm), and membrane fluidity. Conclusion: These results indicate that SMO effectively attenuated oxidative damage and improved apoptosis related factors as well as liver mitochondrial dysfunction in aging mice. PMID:24914315

  17. Serological activity against galactosyl-alpha(1-3)galactose in sera from patients with several kinetoplastida infections.

    PubMed Central

    Avila, J L; Rojas, M; Towbin, H

    1988-01-01

    Using rabbit erythrocyte-derived neutral glycosphingolipids enriched for a defined ceramide pentasaccharide as antigens, we have detected elevated anti-galactosyl-alpha(1-3)galactose (anti-G alpha G) antibody values in patients with American cutaneous leishmaniasis (ACL), chronic Chagas' disease, and Trypanosoma rangeli infections compared with normal subjects or with patients suffering from any of 15 other infectious diseases. The specificity of the G alpha G antibodies was determined by inhibition enzyme-linked immunosorbent assays, which revealed that several alpha-galactosyl- but not beta-galactosyl-bearing sugars blocked absorption of G alpha G antibodies to the specific antigen used. G alpha G antibodies were mainly distributed between immunoglobulin classes G and M in three Kinetoplastida infections studied, with a lower increase in reactivity detected in immunoglobulin A. Absorption of highly reactive G alpha G antibodies with purified murine laminin and nidogen, two basement membrane proteins, almost abolished G alpha G reactivity, suggesting the identity of anti-G alpha G with laminin and nidogen antibodies previously reported as elevated in Kinetoplastida infections. In ACL, G alpha G antibodies were detected in 71% of patients having skin lesions with a clinical evolution time of 0.5 month. This percentage increased with the time of evolution of skin lesions, reaching 93% in lesions older than 3 months, and tended to decrease inversely to the induration diameter in the skin leishmanin test. It is proposed that similar epitopes may exist on kinetoplast protozoa and that the determination of G alpha G antibodies may be a highly sensitive assay for the detection of humoral responses to Kinetoplastida infections. PMID:2449451

  18. Macrophage Galactose-Type Lectin-1 Deficiency Is Associated with Increased Neutrophilia and Hyperinflammation in Gram-Negative Pneumonia.

    PubMed

    Jondle, Christopher N; Sharma, Atul; Simonson, Tanner J; Larson, Benjamin; Mishra, Bibhuti B; Sharma, Jyotika

    2016-04-01

    C-type lectin receptors (CLRs), the carbohydrate-recognizing molecules, orchestrate host immune response in homeostasis and in inflammation. In the present study we examined the function of macrophage galactose-type lectin-1 (MGL1), a mammalian CLR, in pneumonic sepsis, a deadly immune disorder frequently associated with a nonresolving hyperinflammation. In a murine model of pneumonic sepsis using pulmonary infection with Klebsiella pneumoniae, the expression of MGL1 was upregulated in the lungs of K. pneumoniae-infected mice, and the deficiency of this CLR in MGL1(-/-) mice resulted in significantly increased mortality to infection than in the MGL1-sufficient wild-type mice, despite a similar bacterial burden. The phagocytic cells from MGL1(-/-) mice did not exhibit any defects in bacterial uptake and intracellular killing and were fully competent in neutrophil extracellular trap formation, a recently identified extracellular killing modality of neutrophils. Instead, the increased susceptibility of MGL1(-/-) mice seemed to correlate with severe lung pathology, indicating that MGL1 is required for resolution of pulmonary inflammation. Indeed, the MGL1(-/-) mice exhibited a hyperinflammatory response, massive pulmonary neutrophilia, and an increase in neutrophil-associated immune mediators. Concomitantly, MGL1-deficient neutrophils exhibited an increased influx in pneumonic lungs of K. pneumoniae-infected mice. Taken together, these results show a previously undetermined role of MGL1 in controlling neutrophilia during pneumonic infection, thus playing an important role in resolution of inflammation. To our knowledge, this is the first study depicting a protective function of MGL1 in an acute pneumonic bacterial infection. PMID:26912318

  19. KSGal6ST Is Essential for the 6-Sulfation of Galactose within Keratan Sulfate in Early Postnatal Brain

    PubMed Central

    Hoshino, Hitomi; Foyez, Tahmina; Ohtake-Niimi, Shiori; Takeda-Uchimura, Yoshiko; Michikawa, Makoto; Kadomatsu, Kenji

    2014-01-01

    Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains. PMID:24152993

  20. Transplanted Adipose-Derived Stem Cells Ameliorate Testicular Dysfunction In A D-Galactose-Induced Aging Rat Model.

    PubMed

    Yang, Chun; Du, Yi-Kuan; Wang, Jun; Luan, Ping; Yang, Qin-Lao; Huang, Wen-Hua; Yuan, Lin

    2015-10-01

    Glycation product accumulation during aging of slowly renewing tissues may be an important mechanism underlying aging of the testis. Adipose-derived stem cells (ADSCs) have shown promise in a novel tissue regenerative technique and may have utility in treating sexual dysfunction. ADSCs have also been found to be effective in antiaging therapy, although the mechanism underlying their effects remains unknown. This study was designed to investigate the anti-aging effect of ADSCs in a D-galactose (D-gal)-induced aging animal model and to clarify the underlying mechanism. Randomly selected 6-week-old male Sprague-Dawley rats were subcutaneously injected with D-gal daily for 8 weeks. Two weeks after completion of treatment, D-gal-induced aging rats were randomized to receive caudal vein injections of 3 × 10(6) 5-bromo 2'deoxy-uridine-labeled ADSCs or an equal volume of phosphate-buffered saline. Serum testosterone level, steroidogenic enzymes (3-β-hydroxysteroid dehydrogenase), and superoxide dismutase (SOD) activity decreased significantly in aging rats compared with the control group; serum lipid peroxidation, spermatogenic cell apoptosis, and methane dicarboxylic aldehyde (MDA) expression increased significantly. ADSCs increased the SOD level and reduced the MDA level in the aging animal model and restored levels of serum testosterone, steroidogenic enzymes, and spermatogenic cell apoptosis. These results demonstrate that ADSCs can contribute to testicular regeneration during aging. ADSCs also provide functional benefits through glycation suppression and antioxidant effects in a rat model of aging. Although some ADSCs differentiated into Leydig cells, the paracrine pathway seems to play a main role in this process, resulting in the reduction of apoptosis. PMID:25728126

  1. Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans*

    PubMed Central

    Yan, Shi; Brecker, Lothar; Jin, Chunsheng; Titz, Alexander; Dragosits, Martin; Karlsson, Niclas G.; Jantsch, Verena; Wilson, Iain B. H.; Paschinger, Katharina

    2015-01-01

    The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans. PMID:26002521

  2. Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance.

    PubMed

    Zhang, Gui-Yun; Liu, Ru-Ru; Zhang, Chang-Quan; Tang, Ke-Xuan; Sun, Ming-Fa; Yan, Guo-Hong; Liu, Qiao-Quan

    2015-01-01

    Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress. PMID:25938231

  3. Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening

    PubMed Central

    Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A.; Waldron, Keith W.; Quesada, Miguel A.; Muñoz-Blanco, Juan; Mercado, José A.

    2016-01-01

    Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. PMID:26585222

  4. Galactose 6-O-sulfotransferases are not required for the generation of Siglec-F ligands in leukocytes or lung tissue.

    PubMed

    Patnode, Michael L; Cheng, Chu-Wen; Chou, Chi-Chi; Singer, Mark S; Elin, Matilda S; Uchimura, Kenji; Crocker, Paul R; Khoo, Kay-Hooi; Rosen, Steven D

    2013-09-13

    Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor. PMID:23880769

  5. Anti-Apoptotic and Pro-Survival Effect of Alpinate Oxyphyllae Fructus (AOF) in a d-Galactose-Induced Aging Heart.

    PubMed

    Chang, Yung-Ming; Chang, Hen-Hong; Kuo, Wei-Wen; Lin, Hung-Jen; Yeh, Yu-Lan; Padma Viswanadha, Vijaya; Tsai, Chin-Chuan; Chen, Ray-Jade; Chang, Hsin-Nung; Huang, Chih-Yang

    2016-01-01

    Aging, a natural biological/physiological phenomenon, is accelerated by reactive oxygen species (ROS) accumulation and identified by a progressive decrease in physiological function. Several studies have shown a positive relationship between aging and chronic heart failure (HF). Cardiac apoptosis was found in age-related diseases. We used a traditional Chinese medicine, Alpinate Oxyphyllae Fructus (AOF), to evaluate its effect on cardiac anti-apoptosis and pro-survival. Male eight-week-old Sprague-Dawley (SD) rats were segregated into five groups: normal control group (NC), d-Galactose-Induced aging group (Aging), and AOF of 50 (AL (AOF low)), 100 (AM (AOF medium)), 150 (AH (AOF high)) mg/kg/day. After eight weeks, hearts were measured by an Hematoxylin-Eosin (H&E) stain, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assays and Western blotting. The experimental results show that the cardiomyocyte apoptotic pathway protein expression increased in the d-Galactose-Induced aging groups, with dose-dependent inhibition in the AOF treatment group (AL, AM, and AH). Moreover, the expression of the pro-survival p-Akt (protein kinase B (Akt)), Bcl-2 (B-cell lymphoma 2), anti-apoptotic protein (Bcl-xL) protein decreased significantly in the d-Galactose-induced aging group, with increased performance in the AOF treatment group with levels of p-IGFIR and p-PI3K (Phosphatidylinositol-3' kinase (PI3K)) to increase by dosage and compensatory performance. On the other hand, the protein of the Sirtuin 1 (SIRT1) pathway expression decreased in the aging groups and showed improvement in the AOF treatment group. Our results suggest that AOF strongly works against ROS-induced aging heart problems. PMID:27043531

  6. Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening.

    PubMed

    Paniagua, Candelas; Blanco-Portales, Rosario; Barceló-Muñoz, Marta; García-Gago, Juan A; Waldron, Keith W; Quesada, Miguel A; Muñoz-Blanco, Juan; Mercado, José A

    2016-02-01

    Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness. PMID:26585222

  7. Sugar composition of the pectic polysaccharides of charophytes, the closest algal relatives of land-plants: presence of 3-O-methyl-d-galactose residues

    PubMed Central

    O’Rourke, Christina; Gregson, Timothy; Murray, Lorna; Sadler, Ian H.; Fry, Stephen C.

    2015-01-01

    Background and Aims During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed, during the momentous step whereby land-plants arose from charophytic algal ancestors. Methods Polysaccharides were extracted from four charophyte orders [Chlorokybales (Chlorokybus atmophyticus), Klebsormidiales (Klebsormidium fluitans, K. subtile), Charales (Chara vulgaris, Nitella flexilis), Coleochaetales (Coleochaete scutata)] and an early-diverging land-plant (Anthoceros agrestis). ‘Pectins’ and ‘hemicelluloses’, operationally defined as extractable in oxalate (100 °C) and 6 m NaOH (37 °C), respectively, were acid- or Driselase-hydrolysed, and the monosaccharides analysed chromatographically. One unusual monosaccharide, ‘U’, was characterized by 1H/13C-nuclear magnetic resonance spectroscopy and also enzymically. Key Results ‘U’ was identified as 3-O-methyl-d-galactose (3-MeGal). All pectins, except in Klebsormidium, contained acid- and Driselase-releasable galacturonate, suggesting homogalacturonan. All pectins, without exception, released rhamnose and galactose on acid hydrolysis; however, only in ‘higher’ charophytes (Charales, Coleochaetales) and Anthoceros were these sugars also efficiently released by Driselase, suggesting rhamnogalacturonan-I. Pectins of ‘higher’ charophytes, especially Chara, contained little arabinose, instead possessing 3-MeGal. Anthoceros hemicelluloses were rich in glucose, xylose, galactose and arabinose (suggesting xyloglucan and arabinoxylan), none of which was consistently present in charophyte hemicelluloses. Conclusions Homogalacturonan is an ancient streptophyte feature, albeit secondarily lost in Klebsormidium. When conquering the land, the first embryophytes already possessed rhamnogalacturonan-I. In contrast, charophyte and land-plant hemicelluloses differ

  8. Anti-Apoptotic and Pro-Survival Effect of Alpinate Oxyphyllae Fructus (AOF) in a d-Galactose-Induced Aging Heart

    PubMed Central

    Chang, Yung-Ming; Chang, Hen-Hong; Kuo, Wei-Wen; Lin, Hung-Jen; Yeh, Yu-Lan; Padma Viswanadha, Vijaya; Tsai, Chin-Chuan; Chen, Ray-Jade; Chang, Hsin-Nung; Huang, Chih-Yang

    2016-01-01

    Aging, a natural biological/physiological phenomenon, is accelerated by reactive oxygen species (ROS) accumulation and identified by a progressive decrease in physiological function. Several studies have shown a positive relationship between aging and chronic heart failure (HF). Cardiac apoptosis was found in age-related diseases. We used a traditional Chinese medicine, Alpinate Oxyphyllae Fructus (AOF), to evaluate its effect on cardiac anti-apoptosis and pro-survival. Male eight-week-old Sprague–Dawley (SD) rats were segregated into five groups: normal control group (NC), d-Galactose-Induced aging group (Aging), and AOF of 50 (AL (AOF low)), 100 (AM (AOF medium)), 150 (AH (AOF high)) mg/kg/day. After eight weeks, hearts were measured by an Hematoxylin–Eosin (H&E) stain, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assays and Western blotting. The experimental results show that the cardiomyocyte apoptotic pathway protein expression increased in the d-Galactose-Induced aging groups, with dose-dependent inhibition in the AOF treatment group (AL, AM, and AH). Moreover, the expression of the pro-survival p-Akt (protein kinase B (Akt)), Bcl-2 (B-cell lymphoma 2), anti-apoptotic protein (Bcl-xL) protein decreased significantly in the d-Galactose-induced aging group, with increased performance in the AOF treatment group with levels of p-IGFIR and p-PI3K (Phosphatidylinositol-3′ kinase (PI3K)) to increase by dosage and compensatory performance. On the other hand, the protein of the Sirtuin 1 (SIRT1) pathway expression decreased in the aging groups and showed improvement in the AOF treatment group. Our results suggest that AOF strongly works against ROS-induced aging heart problems. PMID:27043531

  9. Transcriptional Regulation and Evolution of Lactose Genes in the Galactose-Lactose Operon of Lactococcus lactis NCDO2054

    PubMed Central

    Vaughan, Elaine E.; Pridmore, R. David; Mollet, Beat

    1998-01-01

    The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes. Initially the β-galactosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment. Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order is galK-galT-lacA-lacZ-galE; the gal genes encode enzymes of the Leloir pathway for galactose metabolism, and lacA encodes a galactoside acetyltransferase. The galT and galE genes of L. lactis LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting L. lactis C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening lacA-lacZ strain NCDO2054. Analysis of mRNA for the gal and lac genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the lac genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless β-glucuronidase gene (gusA) from E. coli, which identified three lactose-inducible intergenic promoters in the gal-lac operon. The greater similarity of the lacA and lacZ genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the gal genes, suggests that the genes within the gal operon of L. lactis NCDO2054 have been recently acquired. Thus, the lacA-lacZ genes appear to have engaged the promoters of the gal operon in order to direct and control their expression. PMID:9733693

  10. A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

    PubMed Central

    Eggink, Laura L.; Spyroulias, Georgios A.; Jones, Norman G.; Hanson, Carl V.; Hoober, J. Kenneth

    2015-01-01

    We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has

  11. Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy

    PubMed Central

    Yasutake, Junichi; Suzuki, Yusuke; Suzuki, Hitoshi; Hiura, Naoko; Yanagawa, Hiroyuki; Makita, Yuko; Kaneko, Etsuji; Tomino, Yasuhiko

    2015-01-01

    Background Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Methods Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. Results Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. Conclusion This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease. PMID:26109484

  12. Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa.

    PubMed

    Duan, Weike; Ren, Jun; Li, Yan; Liu, Tongkun; Song, Xiaoming; Chen, Zhongwen; Huang, Zhinan; Hou, Xilin; Li, Ying

    2016-01-01

    Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-Mannose/L-Galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), B. oleracea (C genome) and B. napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. (i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. (ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. (iii) Under NaCl, Cu(2+), MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments. PMID:27313597

  13. Conservation and Expression Patterns Divergence of Ascorbic Acid d-mannose/l-galactose Pathway Genes in Brassica rapa

    PubMed Central

    Duan, Weike; Ren, Jun; Li, Yan; Liu, Tongkun; Song, Xiaoming; Chen, Zhongwen; Huang, Zhinan; Hou, Xilin; Li, Ying

    2016-01-01

    Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-Mannose/L-Galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), B. oleracea (C genome) and B. napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. (i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. (ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. (iii) Under NaCl, Cu2+, MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments. PMID:27313597

  14. Effects of aluminum on the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in the dentate gyrus of D-galactose-treated mice via increasing oxidative stress.

    PubMed

    Nam, Sung Min; Kim, Jong Whi; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Choi, Jung Hoon; Hwang, In Koo; Seong, Je Kyung; Yoon, Yeo Sung

    2016-06-30

    Aluminum (Al) accumulation increases with aging, and long-term exposure to Al is regarded as a risk factor for Alzheimer's disease. In this study, we investigated the effects of Al and/or D-galactose on neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons in the hippocampal dentate gyrus. AlCl3 (40 mg/kg/day) was intraperitoneally administered to C57BL/6J mice for 4 weeks. In addition, vehicle (physiological saline) or D-galactose (100 mg/kg) was subcutaneously injected to these mice immediately after AlCl3 treatment. Neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons were detected using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN, respectively, via immunohistochemistry. Subchronic (4 weeks) exposure to Al in mice reduced neural stem cells, proliferating cells, and differentiating neuroblasts without causing any changes to mature neurons. This Al-induced reduction effect was exacerbated in D-galactose-treated mice compared to vehicle-treated adult mice. Moreover, exposure to Al enhanced lipid peroxidation in the hippocampus and expression of antioxidants such as Cu, Zn- and Mn-superoxide dismutase in D-galactose-treated mice. These results suggest that Al accelerates the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in D-galactose-treated mice via oxidative stress, without inducing loss in mature neurons. PMID:26243606

  15. Effects of aluminum on the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in the dentate gyrus of D-galactose-treated mice via increasing oxidative stress

    PubMed Central

    Nam, Sung Min; Kim, Jong Whi; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Choi, Jung Hoon; Hwang, In Koo; Seong, Je Kyung

    2016-01-01

    Aluminum (Al) accumulation increases with aging, and long-term exposure to Al is regarded as a risk factor for Alzheimer's disease. In this study, we investigated the effects of Al and/or D-galactose on neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons in the hippocampal dentate gyrus. AlCl3 (40 mg/kg/day) was intraperitoneally administered to C57BL/6J mice for 4 weeks. In addition, vehicle (physiological saline) or D-galactose (100 mg/kg) was subcutaneously injected to these mice immediately after AlCl3 treatment. Neural stem cells, proliferating cells, differentiating neuroblasts, and mature neurons were detected using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN, respectively, via immunohistochemistry. Subchronic (4 weeks) exposure to Al in mice reduced neural stem cells, proliferating cells, and differentiating neuroblasts without causing any changes to mature neurons. This Al-induced reduction effect was exacerbated in D-galactose-treated mice compared to vehicle-treated adult mice. Moreover, exposure to Al enhanced lipid peroxidation in the hippocampus and expression of antioxidants such as Cu, Zn- and Mn-superoxide dismutase in D-galactose-treated mice. These results suggest that Al accelerates the reduction of neural stem cells, proliferating cells, and differentiating neuroblasts in D-galactose-treated mice via oxidative stress, without inducing loss in mature neurons. PMID:26243606

  16. Saffron (Crocus sativus L.) extract prevents and improves D-galactose and NaNO2 induced memory impairment in mice

    PubMed Central

    Dashti-r, M.H.; Zeinali, F.; Anvari, M.; Hosseini, S.M.

    2012-01-01

    This study was conducted to examine the effects of saffron extract on preventing D-galactose and NaNO2 induced memory impairment and improving learning and memory deficits in amnestic mice. In this study, the learning and memory functions in ovariectomized mice were examined by the one way passive and active avoidance tests. In active avoidance test, training in amnestic treated (AT) and amnestic prophylaxis (AP) groups, was improved so that there was a significant difference between them and the amnestic control (AC) group. In passive avoidance test, animal's step through latency, as an index for learning, in all test groups was significantly greater than control group. Total time spent in dark room (DS), which opposes the memory retention ability, in AC was significantly greater than AT group at 1 and 2 hours after full training, while there was not any significant difference between this index in AP and AT as compared with normal control (NC) group. Our findings indicate that saffron hydro-alcoholic extract prevents and improves amnesia induced by D-galactose and NaNO2 in mice.

  17. The extent of co-metabolism of glucose and galactose by Lactococcus lactis changes with the expression of the lacSZ operon from Streptococcus thermophilus.

    PubMed

    Solem, Christian; Koebmann, Brian; Jensen, Peter R

    2008-05-01

    The lactose transporter and beta-galactosidase from Streptococcus thermophilus, encoded by the lacSZ operon, were introduced into the lactose-negative strain Lactococcus lactis MG1363 and the expression of the lacSZ operon was modulated by substitution of the native promoter with randomized synthetic promoters. A series of strains with various expression levels of lacSZ were examined for their fermentation of lactose. Strains with a high expression level were found to metabolize lactose in a similar manner to S. thermophilus, i.e. the galactose moiety of lactose was excreted to the growth medium and only glucose was metabolized in glycolysis. Interestingly, strains with low expression of the operon showed a mixed acid metabolism and co-metabolism of galactose and glucose. The lactose flux increased gradually with increasing expression of the lacSZ operon until an optimum was observed at intermediate beta-galactosidase activities of 2000-3000 Miller units. At higher expression levels, the flux decreased. These strains had a glycolytic flux comparable with those of reference strains with the standard lactococcal PTS(lac) (lactose phosphotransferase transport system) lactose transporter, which indicates that lactose transport is not rate-limiting for glycolysis in Lactococcus. Finally, an additional ATP drain was introduced into the fastest growing strain, CS2004, to test whether the ATP demand controlled glycolysis under these conditions, but in fact no increase in glycolytic flux was observed. PMID:17822381

  18. Molecular cloning and expression of a new α-neoagarobiose hydrolase from Agarivorans gilvus WH0801 and enzymatic production of 3,6-anhydro-l-galactose.

    PubMed

    Liu, Nan; Yang, Meng; Mao, Xiangzhao; Mu, Bozhong; Wei, Dongzhi

    2016-03-01

    A new α-neoagarobiose hydrolase (NABH) called AgaWH117 was cloned from Agarivorans gilvus WH0801. The gene encoding this hydrolase consists of 1,086 bp and encodes a protein containing 361 amino acids. This new NABH showed 74% amino acid sequence identity with other known NABHs. The molecular mass of the recombinant AgaWH117 was estimated to be 41 kDa. Purified AgaWH117 showed endolytic activity during neoagarobiose degradation, yielding 3,6-anhydro-l-galactose (l-AHG) and d-galactose as products. It showed a maximum activity at a temperature of 30 °C and a pH of 6.0 and was stable at temperatures below 30 °C. Its Km and Vmax values were 2.094 mg/mL and 6.982 U/mg, respectively. The cloning strategy used and AgaWH117 isolated in this study will provide information on the saccharification process of marine biomass. This study provides a method to produce l-AHG from agarose by using AgaWH117 without an acid and describes its one-step purification by using Bio-Gel P2 chromatography. PMID:25676340

  19. Lower cell wall pectin solubilisation and galactose loss during early fruit development in apple (Malus x domestica) cultivar 'Scifresh' are associated with slower softening rate.

    PubMed

    Ng, Jovyn K T; Schröder, Roswitha; Brummell, David A; Sutherland, Paul W; Hallett, Ian C; Smith, Bronwen G; Melton, Laurence D; Johnston, Jason W

    2015-03-15

    Substantial differences in softening behaviour can exist between fruit even within the same species. Apple cultivars 'Royal Gala' and 'Scifresh' soften at different rates despite having a similar genetic background and producing similar amounts of ethylene during ripening. An examination of cell wall metabolism from the fruitlet to the ripe stages showed that in both cultivars pectin solubilisation increased during cell expansion, declined at the mature stage and then increased again during ripening. This process was much less pronounced in the slower softening 'Scifresh' than in 'Royal Gala' at every developmental stage examined, consistent with less cell separation and softening in this cultivar. Both cultivars also exhibited a progressive loss of pectic galactan and arabinan side chains during development. The cell wall content of arabinose residues was similar in both cultivars, but the galactose residue content in 'Scifresh' remained higher than that of 'Royal Gala' at every developmental stage. The higher content of cell wall galactose residue in 'Scifresh' cell walls correlated with a lower β-galactosidase activity and more intense immunolabelling of RG-I galactan side chains in both microscopy sections and glycan microarrays. A high cell wall galactan content has been associated with reduced cell wall porosity, which may restrict access of cell wall-modifying enzymes and thus maintain better structural integrity later in development. The data suggest that the composition and structure of the cell wall at very early development stages may influence subsequent cell wall loosening, and may even predispose the wall's ensuing properties. PMID:25602611

  20. Enzymatic production of 3,6-anhydro-L-galactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities.

    PubMed

    Yun, Eun Ju; Lee, Saeyoung; Kim, Ji Hye; Kim, Bo Bae; Kim, Hee Taek; Lee, Sun Hee; Pelton, Jeffrey G; Kang, Nam Joo; Choi, In-Geol; Kim, Kyoung Heon

    2013-04-01

    3,6-Anhydro-L-galactose (L-AHG) constitutes 50% of agarose, which is the main component of red macroalgae. No information is currently available on the mass production, metabolic fate, or physiological effects of L-AHG. Here, agarose was converted to L-AHG in the following three steps: pre-hydrolysis of agarose into agaro-oligosaccharides by using acetic acid, hydrolysis of the agaro-oligosaccharides into neoagarobiose by an exo-agarase, and hydrolysis of neoagarobiose into L-AHG and galactose by a neoagarobiose hydrolase. After these three steps, L-AHG was purified by adsorption and gel permeation chromatographies. The final product obtained was 95.6% pure L-AHG at a final yield of 4.0% based on the initial agarose. In a cell proliferation assay, L-AHG at a concentration of 100 or 200 μg/ mL did not exhibit any significant cytotoxicity. In a skin whitening assay, 100 μg/ mL of L-AHG showed significantly lower melanin production compared to arbutin. L-AHG at 100 and 200 μg/ mL showed strong anti-inflammatory activity, indicating the significant suppression of nitrite production. This is the first report on the production of high-purity L-AHG and its physiological activities. PMID:22678025

  1. D-glucose, D-galactose, and D-lactose non-enzyme quantitative and qualitative analysis method based on Cu foam electrode.

    PubMed

    Jiaojiao, Jin; Yangyang, Ge; Gangying, Zheng; Yanping, Cai; Wei, Liu; Guohua, Hui

    2015-05-15

    Here, D-glucose, D-galactose, and D-lactose non-enzyme quantitative and qualitative analysis method using Cu foam electrode had been investigated. Porous Cu foam material was prepared by electrodeposition strategy, and used as working electrode. Cyclic voltammetry (CV) explained sweetener electro-oxidation process occurring on Cu foam electrode. Amperometric i-t scanning results demonstrated that Cu foam electrode fast responded to D-glucose, D-galactose, and D-lactose in linear concentration range between 0.18 mM and 3.47 mM with significant sensitivity of 1.79 mA cm(-2)mM(-1), 0.57 mA cm(-2)mM(-1), and 0.64 mA cm(-2)mM(-1), respectively. Limit of detection (LOD) was 9.30 μM, 29.40 μM, and 26 μM respectively (S/N=3). Sweetener species was decided by stochastic resonance (SR) signal-to-noise ratio (SNR) eigen peak located noise intensities. Interference experiment results demonstrated that Cu foam electrode selectively responded to sweeteners against interference chemicals. The proposed method provides a promising way for sweetener non-enzyme quantitative and qualitative analysis. PMID:25577110

  2. Biodegradable Self-Assembled Nanoparticles of Galactose-Containing Amphiphilic Triblock Copolymers for Targeted Delivery of Paclitaxel to HepG2 Cells.

    PubMed

    Wang, Tieshi; Tang, Xinde; Han, Jingtian; Ding, Yuanyuan; Guo, Wenjuan; Pei, Meishan

    2016-05-01

    Biodegradable self-assembled polymeric nanoparticles (NPs) composed of poly(6-O-methacryloyl-D-galactopyranose)-b-poly(L-lactide)-b-poly(6-O-methacryloyl-D-galactopyranose) (PMAGP-b-PLA-b-PMAGP) are prepared as carriers for the hydrophobic anticancer drug paclitaxel (PTX), to achieve target delivery to hepatoma cells. PTX can be encapsulated by the NPs with various molar ratios of L-lactide (LA) and 6-O-methacryloyl-D-galactopyranose (MAGP) during the process of self-assembly, and the resulting NPs exhibit high drug loading efficacy and substantial stability in aqueous solution. The size, size distribution, and morphology of the NPs are characterized using a Zetasizer Nano ZS and transmission electron microscopy. The hemolysis assay and cell cytotoxicity assay indicate that the polymeric NPs are biocompatible and non-toxic. The cellular uptake assay demonstrates that the galactose-containing NPs can be selectively recognized and subsequently accumulate in HepG2 cells. All of these results demonstrate that galactose-containing polymeric NPs are potential carriers for hepatoma-targeted drug delivery and liver cancer therapy in clinical medicine. PMID:26833548

  3. Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase.

    PubMed

    Brando-Lima, Aline C; Saldanha-Gama, Roberta F; Henriques, Maria das Graças M O; Monteiro-Moreira, Ana C O; Moreira, Renato A; Barja-Fidalgo, Christina

    2005-10-15

    Several lectin-like molecules have been shown as potent activators of leukocytes. Galactose-binding lectins are of special interest since they could interact with several endogenous molecules involved in the innate and specific immune responses. The effects of Frutalin (FTL), an alpha-D-galactose (Gal)-binding plant lectin, on the modulation of neutrophil (PMN) functions were investigated. FTL induced a dose-dependent PMN migration in mice pleural cavity. Moreover, FTL was also a potent direct chemotactic for human PMN, in vitro, and triggered oxidative burst in these cells. These effects were accompanied by a rearrangement of the actin cytoskeleton dynamic, activation of tyrosine kinase (TK) pathways, increase in focal adhesion kinase (FAK) phosphorylation, and its subsequent association to phosphoinositide3-kinase (PI3K). All those effects were inhibited in the presence of Gal, suggesting specific carbohydrate recognition for FTL effects. The activations of TK and PI3K pathways are essential events for FTL-induced chemotaxis, since inhibitors of these pathways, genistein and LY294002, inhibited neutrophil migration in vitro. The data indicate that sugar-protein interactions between a soluble lectin and galacto-components on neutrophil surface trigger the TK pathway, inducing FAK and PI3K activation, interfering with cell motility and oxidative response. PMID:16183388

  4. A new case of UDP-galactose transporter deficiency (SLC35A2-CDG): molecular basis, clinical phenotype, and therapeutic approach.

    PubMed

    Dörre, K; Olczak, M; Wada, Y; Sosicka, P; Grüneberg, M; Reunert, J; Kurlemann, G; Fiedler, B; Biskup, S; Hörtnagel, K; Rust, S; Marquardt, T

    2015-09-01

    Congenital disorders of glycosylation (CDG) are a group of hereditary metabolic diseases characterized by abnormal glycosylation of proteins and lipids. Often, multisystem disorders with central nervous system involvement and a large variety of clinical symptoms occur. The main characteristics are developmental delay, seizures, and ataxia. In this paper we report the clinical and biochemical characteristics of a 5-year-old girl with a defective galactosylation of N-glycans, resulting in developmental delay, muscular hypotonia, epileptic seizures, inverted nipples, and visual impairment. Next generation sequencing revealed a de novo mutation (c.797G > T, p.G266V) in the X-chromosomal gene SLC35A2 (solute carrier family 35, UDP-galactose transporter, member A2; MIM 300896). While this mutation was found heterozygous, random X-inactivation of the normal allele will lead to loss of normal SLC35A2 activity in respective cells. The functional relevance of the mutation was demonstrated by complementation of UGT-deficient MDCK-RCA(r) and CHO-Lec8 cells by normal UGT-expression construct but not by the mutant version. The effect of dietary galactose supplementation on glycosylation was investigated, showing a nearly complete normalization of transferrin glycosylation. PMID:25778940

  5. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

    PubMed

    Wohlers, T M; Christacos, N C; Harreman, M T; Fridovich-Keil, J L

    1999-02-01

    Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes. PMID:9973283

  6. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

    PubMed Central

    Wohlers, T M; Christacos, N C; Harreman, M T; Fridovich-Keil, J L

    1999-01-01

    Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes. PMID:9973283

  7. The Macrophage Galactose-Type Lectin-1 (MGL1) Recognizes Taenia crassiceps Antigens, Triggers Intracellular Signaling, and Is Critical for Resistance to This Infection

    PubMed Central

    Montero-Barrera, Daniel; Valderrama-Carvajal, Héctor; Terrazas, César A.; Rojas-Hernández, Saúl; Ledesma-Soto, Yadira; Vera-Arias, Laura; Carrasco-Yépez, Maricela; Gómez-García, Lorena; Martínez-Saucedo, Diana; Becerra-Díaz, Mireya; Terrazas, Luis I.

    2015-01-01

    C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1) recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1−/− mice showed less binding ability toward parasite antigens than their wild-type (WT) counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1−/− macrophages. Following T. crassiceps infection, MGL1−/− mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1−/− mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1−/− macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance. PMID:25664320

  8. Late blowing of Cheddar cheese induced by accelerated ripening and ribose and galactose supplementation in presence of a novel obligatory heterofermentative nonstarter Lactobacillus wasatchensis.

    PubMed

    Ortakci, Fatih; Broadbent, Jeffery R; Oberg, Craig J; McMahon, Donald J

    2015-11-01

    Lactobacillus wasatchensis sp. nov. has been studied for growth and gas formation in a control Cheddar cheese and in cheese supplemented with 0.5% ribose, 0.5% galactose, or 0.25% ribose plus 0.25% galactose using regular and accelerated cheese ripening temperatures of 6 and 12°C, respectively. Milk was inoculated with (1) Lactococcus lactis starter culture, or (2) Lc. lactis starter culture plus Lb. wasatchensis (10(4) cfu/mL). In the control cheese with no added Lb. wasatchensis, starter numbers decreased from 10(7) initially to ~10(4) cfu/g over 23 wk of ripening at 6°C. When the cheese was ripened at 12°C, or if Lb. wasatchensis was added, the final starter counts were 1 log lower. In contrast, nonstarter lactic acid bacteria in the cheese increased from <10(2) cfu/g at press to 10(6) to 10(7) cfu/g after 23 wk, with higher numbers being observed with ripening at 12°C. In cheese with no added Lb. wasatchensis, levels of Lb. wasatchensis were initially below the enumeration threshold but counts of up to 10(3) cfu/g were detected after 23 wk. When the cheese was inoculated with Lb. wasatchensis, it could be enumerated throughout ripening, with final levels at 23 wk being dependent on whether ribose had been added to the cheese curd. With added ribose (with or without added galactose), Lb. wasatchensis grew to 10(7) to 10(8) cfu/g after 23 wk, whereas without added ribose it was 1 log lower. In all cheeses with added Lb. wasatchensis, greater gas formation was observed at 12°C, with most gas production occurring after ~16 wk. Very little gas production was detected in cheese without added Lb. wasatchensis ripened at 12°C or in cheese with added Lb. wasatchensis ripened at 6°C. Adding a combination of ribose and galactose caused more gas formation, putatively because of the ability of Lb. wasatchensis to co-utilize both sugars and grow to high numbers, and then produce gas from galactose as ribose levels were depleted. Even without sugar supplementation, gas

  9. Isolation, purification, and physicochemical characterization of a D-galactose-binding lectin from seeds of Erythrina speciosa.

    PubMed

    Konozy, Emadeldin H E; Bernardes, Emerson S; Rosa, Cesar; Faca, Vitor; Greene, Lewis Joel; Ward, Richard John

    2003-02-15

    present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination. PMID:12573281

  10. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    PubMed

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-10-25

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1. PMID:7971267

  11. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    PubMed Central

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-01-01

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1. Images PMID:7971267

  12. Using a Personal Glucose Meter and Alkaline Phosphatase for Point-of-Care Quantification of Galactose-1-Phosphate Uridyltransferase in Clinical Galactosemia Diagnosis.

    PubMed

    Zhang, Jingjing; Xiang, Yu; Novak, Donna E; Hoganson, George E; Zhu, Junjie; Lu, Yi

    2015-10-01

    The personal glucose meter (PGM) was recently shown to be a general meter to detect many targets. Most studies, however, focus on transforming either target binding or enzymatic activity that cleaves an artificial substrate into the production of glucose. More importantly, almost all reports exhibit their methods by using artificial samples, such as buffers or serum samples spiked with the targets. To expand the technology to even broader targets and to validate its potential in authentic, more complex clinical samples, we herein report expansion of the PGM method by using alkaline phosphatase (ALP) that links the enzymatic activity of galactose-1-phosphate uridyltransferase to the production of glucose, which allows point-of-care galactosemia diagnosis in authentic human clinical samples. Given the presence of ALP in numerous enzymatic assays for clinical diagnostics, the methods demonstrated herein advance the field closer to point-of-care detection of a wide range of targets in real clinical samples. PMID:26350570

  13. Resistance of Escherichia coli to Penicillins IX. Genetics and Physiology of Class II Ampicillin-Resistant Mutants That Are Galactose Negative or Sensitive to Bacteriophage C21, or Both

    PubMed Central

    Eriksson-Grennberg, Kerstin G.; Nordström, Kurt; Englund, Per

    1971-01-01

    Ampicillin-resistant mutants of class II are determined by a doubling of chromosomally and episomally mediated ampicillin resistance on agar plates. Several mutants were isolated from a female as well as from an Hfr strain. The mutants differed from each other in various properties such as response to colicin E2 and sodium cholate, response to the phages T4 and C21, and fermentation of galactose. By conjugation and transduction experiments, it was shown that mutations in at least four loci gave the class II phenotype. The mutations were found to be in the galU gene, the ctr gene, and two new genes close to mtl denoted lpsA and lpsB. The carbohydrate compositions of the lipopolysaccharides of the mutants were investigated and found to be changed compared to the parent strains. GalU mutants lacked rhamnose and galactose and had 11% glucose compared to the parent strain. The lpsA mutant also lacked rhamnose and had only traces of galactose and 58% glucose, whereas the lpsB mutant contained 14% rhamnose, traces of galactose, and 81% glucose compared to the parent strain. PMID:4945191

  14. Effects of the position of galactose units to Zn(II) phthalocyanine on the uptake and photodynamic activity towards breast cancer cells

    NASA Astrophysics Data System (ADS)

    Mantareva, V.; Kril, A.; Angelov, I.; Dimitrov, R.; Borisova, E.; Avramov, L.

    2012-06-01

    Zn(II)-phthalocyanines with tetra-substitution of D-galactose group on non-peripheral (nGalPc) and peripheral (pGalPc) positions have been studied as photodynamic sensitizers. The both complexes are water-soluble and highly aggregated in water and cell culture medium. The non-peripheral galactose units attached to the phthalocyanine macrocycle (nGalPc) lead to far red shift of absorbance maximum at 703 nm as compared to peripherally substituted pGalPc with maximum at 683 nm. The fluorescence maxima of the studied GalPcs were red shifted (8-14 nm) depending on the used solvent as compared to the absorption maxima. The relatively low fluorescence quantum yields in dimethylsulfoxide (0.06 for nGalPc and 0.21 for pGalPc) were determined. The singlet oxygen generation was determined with lower quantum yield for pGalPc (0.21) as compared to nGalPc (0.38). The lack of dark toxicity towards breast cancer cell line (MCF-7) in wide concentration range (0.125 - 10 μM) was observed. The uptake into the tumor cells and the subcellular localization in MCF-7 cells were determined with higher accumulation for pGalPc, compared to nGalPc. The in vitro photodynamic activity of GalPcs towards breast cancer cells was investigated for different dye concentrations and soft light parameters of 635 nm irradiation. The antitumor activity of nGalPc was superior to the pGalPc-induced cytotoxicity, due to higher generation of singlet oxygen and other reactive oxygen species.

  15. The tomato HD-Zip I transcription factor SlHZ24 modulates ascorbate accumulation through positive regulation of the D-mannose/L-galactose pathway.

    PubMed

    Hu, Tixu; Ye, Jie; Tao, Peiwen; Li, Hanxia; Zhang, Junhong; Zhang, Yuyang; Ye, Zhibiao

    2016-01-01

    Ascorbate (AsA) is an antioxidant that can scavenge the reactive oxygen species (ROS) produced when plants encounter stressful conditions. Here, it was revealed by a yeast one-hybrid assay that a tomato (Solanum lycopersicum) HD-Zip I family transcription factor, SlHZ24, binds to the promoter of an AsA biosynthetic gene encoding GDP-D-mannose pyrophosphorylase 3 (SlGMP3). Both the transient expression system and electrophoretic mobility shift assay (EMSA) showed that SlHZ24 binds to a regulatory cis-element in the SlGMP3 promoter, and further overexpression of SlHZ24 in transgenic tomato lines resulted in increased AsA levels. In contrast, suppressing expression of the gene using RNA interference (RNAi) had the opposite effect. These data suggest that SlHZ24 can positively regulate the accumulation of AsA, and in support of this it was shown that SlGMP3 expression increased in the SlHZ24-overexpressing lines and declined in SlHZ24-RNAi lines. SlHZ24 also affected the expression of other genes in the D-mannose/L-galactose pathway, such as genes encoding GDP-mannose-3',5'-epimerase 2 (SlGME2), GDP-L-galactose phosphorylase (SlGGP) and SlGMP4. The EMSA indicated that SlHZ24 bound to the promoters of SlGME2 and SlGGP, suggesting multi-targeted regulation of AsA biosynthesis. Finally, SlHZ24-overexpressing plants showed less sensitivity to oxidative stress; we therefore conclude that SlHZ24 promotes AsA biosynthesis, which in turn enhances oxidative stress tolerance. PMID:26610866

  16. The stacking tryptophan of galactose oxidase: a second coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis

    PubMed Central

    Rogers, Melanie S.; Tyler, Ejan M.; Akyumani, Nana; Kurtis, Christian R.; Spooner, R. Kate; Deacon, Sarah E.; Tamber, Sunita; Firbank, Susan J.; Mahmoud, Khaled; Knowles, Peter F.; Phillips, Simon E. V.; McPherson, Michael J.; Dooley, David M.

    2008-01-01

    The function of the stacking tryptophan, W290, a second coordination sphere residue in galactose oxidase has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and x -ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly lower in the W290 variants. The Km for D-galactose for W290H is similar to wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding/positioning via the –NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radical in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has similar properties to the wild type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semi-reduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes the variants are also closely similar to the wild type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function. PMID:17385891

  17. Reductively degradable α-amino acid-based poly(ester amide)-graft-galactose copolymers: facile synthesis, self-assembly, and hepatoma-targeting doxorubicin delivery.

    PubMed

    Lv, Jiaolong; Sun, Huanli; Zou, Yan; Meng, Fenghua; Dias, Aylvin A; Hendriks, Marc; Feijen, Jan; Zhong, Zhiyuan

    2015-07-01

    Novel reductively degradable α-amino acid-based poly(ester amide)-graft-galactose (SSPEA-Gal) copolymers were designed and developed to form smart nano-vehicles for active hepatoma-targeting doxorubicin (DOX) delivery. SSPEA-Gal copolymers were readily synthesized via solution polycondensation reaction of di-p-toluenesulfonic acid salts of bis-l-phenylalanine 2,2-thiodiethanol diester and bis-vinyl sulfone functionalized cysteine hexanediol diester with dinitrophenyl ester of adipic acid, followed by conjugating with thiol-functionalized galactose (Gal-SH) via the Michael addition reaction. SSPEA-Gal formed unimodal nanoparticles (PDI = 0.10 - 0.12) in water, in which average particle sizes decreased from 138 to 91 nm with increasing Gal contents from 31.6 wt% to 42.5 wt%. Notably, in vitro drug release studies showed that over 80% DOX was released from SSPEA-Gal nanoparticles within 12 h under an intracellular mimicking reductive conditions, while low DOX release (<20%) was observed for reduction-insensitive PEA-Gal nanoparticles under otherwise the same conditions and SSPEA-Gal nanoparticles under non-reductive conditions. Notably, SSPEA-Gal nanoparticles exhibited high specificity to asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells. MTT assays using HepG2 cells showed that DOX-loaded SSPEA-Gal had a low half maximal inhibitory concentration (IC50) of 1.37 μg mL(-1), approaching that of free DOX. Flow cytometry and confocal laser scanning microscopy studies confirmed the efficient uptake of DOX-loaded SSPEA-Gal nanoparticles by HepG2 cells as well as fast intracellular DOX release. Importantly, SSPEA-Gal and PEA-Gal nanoparticles were non-cytotoxic to HepG2 and MCF-7 cells up to a tested concentration of 1.0 mg mL(-1). These tumor-targeting and reduction-responsive degradable nanoparticles have appeared as an interesting multi-functional platform for advanced drug delivery. PMID:26221946

  18. Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts.

    PubMed

    Balakrishnan, Bijina; Chen, Wyman; Tang, Manshu; Huang, Xiaoping; Cakici, Didem Demirbas; Siddiqi, Anwer; Berry, Gerard; Lai, Kent

    2016-01-29

    The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels. PMID:26773505

  19. The human gene CGT encoding the UDP-galactose ceramide galactosyl transferase (cerebroside synthase): Cloning, characterization, and assignment to human chromosome 4, band q26

    SciTech Connect

    Bosio, A.; Binczek, E.; Stoffel, W.

    1996-05-15

    We have previously cloned the human UDP-galactose ceramide galactosyltransferase (CGT, E.C. 2.4.1.45) cDNA. Its open reading frame encodes the key enzyme in the biosynthesis of the glycosphingolipids, cerebrosides and sulfatides, essential constituents of the myelin membrane of the central nervous system (CNS) and PNS. Expression of the CGT gene and of the myelin-specific proteins in the terminal differentiated oligodendrocyte of CNS and in Schwann cells of PNS is cell-specific and highly time-regulated. The CGT gene therefore is important in the differentiation program of the oligodendrocyte lineage. Here we report the structural organization and the chromosomal localization of the human CGT gene. The coding sequence is separated into five exons, which are distributed over >40 kb. The CGT locus was mapped to the distal region of human chromosome 4, band q26. The organization of the CGT gene and of the UGT (uridylglucuronosyl-transferases) gene family suggests a correlation to functional domains of the encoded proteins. 19 refs., 4 figs., 1 tab.

  20. Characterization of a glucose-, xylose-, sucrose-, and D-galactose-stimulated β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125.

    PubMed

    Xu, Hu; Xiong, Ai-Sheng; Zhao, Wei; Tian, Yong-Sheng; Peng, Ri-He; Chen, Jian-Min; Yao, Quan-Hong

    2011-03-01

    The gene (Bhbgl) encoding a β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125 was synthesized chemically via the PCR-based two-step DNA synthesis (PTDS) method and expressed in Escherichia coli. Bhbgl contained an open reading frame (ORF) of 1359 bp encoding a 453-amino acid protein belonging to glycoside hydrolase family 1 (GHF1), and the deduced molecular mass of recombinant Bhbgl (52,488 Da) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a high specific activity with o-nitrophenyl-β-D-glucopyranoside (oNPGlu) and an apparent K (m) value of 0.32 mM. With oNPGlu as the substrate, Bhbgl displayed pH and temperature optima of ~7.0 and 50°C, respectively. The enzyme was relatively stable under alkaline conditions and >50% activity was retained after incubation at pH 9.5 for 24 h at 4°C. Recombinant Bhbgl activity was inhibited by 5 mM Zn(2+), Fe(3+), or Cd(2+), but was enhanced by 1 mM Mg(2+) and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose, D-galactose, xylose, glucose) at concentrations ranging from 50 to 800 mM. PMID:21046402

  1. Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin from Artocarpus incisa seeds.

    PubMed

    Monteiro-Moreira, Ana Cristina de Oliveira; D'Muniz Pereira, Humberto; Vieira Neto, Antonio Eufrasio; Mendes Batista Moreno, Frederico Bruno; Duarte Pinto Lobo, Marina; de Sousa, Felipe Domingos; Moreira, Renato de Azevedo

    2015-10-01

    Frutalin is an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å. The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b = 74.56, c = 118.98 Å, β = 96.56°. A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress. PMID:26457519

  2. Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of /sup 3/H-galactose

    SciTech Connect

    Michaels, J.E.; Hung, J.T.; Garfield, S.A.; Cardell, R.R. Jr.

    1984-05-01

    Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting /sup 3/H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.

  3. Coenzyme Q10 Inhibits the Aging of Mesenchymal Stem Cells Induced by D-Galactose through Akt/mTOR Signaling

    PubMed Central

    Zhang, Dayong; Yan, Bingxi; Yu, Shanshan; Zhang, Chong; Wang, Baoming; Wang, Yayan; Wang, Junbo; Yuan, Zhanggen; Zhang, Lihuang; Pan, Jianping

    2015-01-01

    Increasing evidences indicate that reactive oxygen species are the main factor promoting stem cell aging. Recent studies have demonstrated that coenzyme Q10 (CoQ10) plays a positive role in organ and cellular aging. However, the potential for CoQ10 to protect stem cell aging has not been fully evaluated, and the mechanisms of cell senescence inhibited by CoQ10 are still poorly understood. Our previous study had indicated that D-galactose (D-gal) can remarkably induce mesenchymal stem cell (MSC) aging through promoting intracellular ROS generation. In this study, we showed that CoQ10 could significantly inhibit MSC aging induced by D-gal. Moreover, in the CoQ10 group, the expression of p-Akt and p-mTOR was clearly reduced compared with that in the D-gal group. However, after Akt activating by CA-Akt plasmid, the senescence-cell number in the CoQ10 group was significantly higher than that in the control group. These results indicated that CoQ10 could inhibit D-gal-induced MSC aging through the Akt/mTOR signaling. PMID:25789082

  4. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    SciTech Connect

    Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu; Denda-Nagai, Kaori; Takada, Ayato; Irimura, Tatsuro

    2011-04-01

    Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

  5. NADPH oxidase 3-associated oxidative stress and caspase 3-dependent apoptosis in the cochleae of D-galactose-induced aged rats

    PubMed Central

    DU, ZHENGDE; LI, SHUO; LIU, LIN; YANG, QIONG; ZHANG, HONGWEI; GAO, CHUNSHENG

    2015-01-01

    Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D-galactose (D-gal), and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D-gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D-gal for 8 weeks. In addition, the levels of 8-hydroxy-2-deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling-positive cells were increased in the cochleae of D-gal-treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3-dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age-associated hearing loss, also termed presbycusis. PMID:26498835

  6. Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

    PubMed

    Shahbaz Mohammadi, Hamid; Mostafavi, Seyede Samaneh; Soleimani, Saeideh; Bozorgian, Sajad; Pooraskari, Maryam; Kianmehr, Anvarsadat

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family. PMID:25591389

  7. MALDI-TOF MS characterization of glycation products of whey proteins in a glucose/galactose model system and lactose-free milk.

    PubMed

    Carulli, Saverio; Calvano, Cosima D; Palmisano, Francesco; Pischetsrieder, Monika

    2011-03-01

    The major modifications induced by thermal treatment of whey proteins α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) in a model system mimicking lactose-free milk (L(-) sugar mix) were investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The analysis of the intact α-La revealed species with up to 7 and 14 adducts from lactose and sugar mix, respectively, whereas for β-Lg 3 and up to 5 sugar moieties were observed in the case of lactose and sugar mix experiments, respectively. A partial enzymatic hydrolysis with endoproteinase AspN prior to mass spectrometric analysis allowed the detection of further modifications and their localization in the amino acid sequence. Using α-cyano-4-chlorocinnamic acid as MALDI matrix, it could be shown that heating α-La and β-Lg with glucose or galactose led to the modification of lysine residues that are not glycated by lactose. The higher glycation degree of whey proteins in a lactose-free milk system relative to normal milk with lactose reflects the higher reactivity of monosaccharides compared to the parent disaccharide. Finally, the analysis of the whey extract of a commercial lactose-free milk sample revealed that the two whey proteins were present as three main forms (native, single, and double hexose adducts). PMID:21319853

  8. Chongcao-Shencha Attenuates Liver and Kidney Injury through Attenuating Oxidative Stress and Inflammatory Response in D-Galactose-Treated Mice

    PubMed Central

    Li, Cailan; Mo, Zhizhun; Xie, Jianhui; Xu, Lieqiang; Tan, Lihua; Luo, Dandan; Chen, Hanbin; Yang, Hongmei; Li, Yucui; Su, Ziren; Su, Zuqing

    2016-01-01

    The Chongcao-Shencha (CCSC), a Chinese herbal compound formula, has been widely used as food material and medicine for enhancing physical strength. The present study investigated the possible effect of CCSC in alleviating the liver and kidney injury in D-galactose- (D-gal-) treated mice and the underlying mechanism. Mice were given a subcutaneous injection of D-gal (200 mg/kg) and orally administered CCSC (200, 400, and 800 mg/kg) daily for 8 weeks. Results indicated that CCSC increased the depressed body weight and organ index induced by D-gal, ameliorated the histological deterioration, and decreased the levels of ALT, AST, BUN, and CRE as compared with D-gal group. Furthermore, CCSC not only elevated the activities of antioxidant enzymes SOD, CAT, and GPx but also upregulated the mRNA expression of SOD1, CAT, and GPx1, while decreasing the MDA level in D-gal-treated mice. Results of western blotting analysis showed that CCSC significantly inhibited the upregulation of expression of nuclear factor kappa B (NF-κB) p65, p-p65, p-IκBα, COX2, and iNOS and inhibited the downregulation of IκBα protein expression caused by D-gal. This study demonstrated that CCSC could attenuate the liver and kidney injury in D-gal-treated mice, and the mechanism might be associated with attenuating oxidative stress and inflammatory response. PMID:27340415

  9. The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding.

    PubMed

    Pey, Angel L; Padín-Gonzalez, Esperanza; Mesa-Torres, Noel; Timson, David J

    2014-11-15

    Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization. PMID:25150110

  10. Salidroside suppresses inflammation in a D-galactose-induced rat model of Alzheimer's disease via SIRT1/NF-κB pathway.

    PubMed

    Gao, Jin; Zhou, Rui; You, Xintong; Luo, Fen; He, He; Chang, Xiayun; Zhu, Lingpeng; Ding, Xuansheng; Yan, Tianhua

    2016-08-01

    Age-related inflammation is the predominant factor for neurodegenerative diseases like Alzheimer's disease (AD). In the present study, we examined memory performance and neuroinflammation in D-galactose (D-gal)-induced sub-acute aging model of rats. Our results demonstrated that chronic administration of D-gal (120 mg/kg) produced cognitive impairment as determined by Morris water maze (MWM) test and step-down passive avoidance test. D-gal also activated nuclear factor kappa B (NF-κB) p65/RelA by down-regulating the expression level of sirtuins 1 (SIRT1) in the hippocampus. Treatment with Salidroside (Sal, 20, 40 mg/kg) for 28 days ameliorated D-gal-induced memory deficits and inflammatory mediators including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Moreover, D-gal-induced activation of NF-κB signaling pathway in the brain was also inhibited by Sal via up-regulating SIRT1. These results suggest that D-gal-triggered memory impairment and inflammatory response may be associated with SIRT1/NF-κB signaling pathway, whereas treatment with Sal could positively affect these changes in hippocampus. PMID:26909502

  11. In Vivo Antioxidant and Anti-Skin-Aging Activities of Ethyl Acetate Extraction from Idesia polycarpa Defatted Fruit Residue in Aging Mice Induced by D-Galactose

    PubMed Central

    Jia, Ran-ran; Chen, Fang

    2014-01-01

    Two different concentrations of D-galactose (D-gal) induced organism and skin aging in Kunming mice were used to examine comprehensively the antioxidant and antiaging activities of ethyl acetate extraction (EAE) from Idesia polycarpa defatted fruit residue for the first time. The oxygen radical absorbance capacity (ORAC) of EAE was 13.09 ± 0.11 μmol Trolox equivalents (TE)/mg, which showed EAE had great in vitro free radical scavenging and antioxidant activity. Biochemical indexes and morphological analysis of all tested tissues showed that EAE could effectively improve the total antioxidant capacity (T-AOC) of the antioxidant defense system of the aging mice, enhance the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) of tissues and serum, increase glutathione (GSH) content and decrease the malondialdehyde (MDA) content, and maintain the skin collagen, elastin, and moisture content. Meanwhile, EAE could effectively attenuate the morphological damage in brain, liver, kidney, and skin induced by D-gal and its effect was not less than that of the well-known L-ascorbic acid (VC) and α-tocopherol (VE). Overall, EAE is a potent natural antiaging agent with great antioxidant activity, which can be developed as a new medicine and cosmetic for the treatment of age-related conditions. PMID:24971146

  12. Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana.

    PubMed

    Gao, Yongshun; Badejo, Adebanjo Ayobamidele; Shibata, Hitoshi; Sawa, Yoshihiro; Maruta, Takanori; Shigeoka, Shigeru; Page, Mike; Smirnoff, Nicholas; Ishikawa, Takahiro

    2011-01-01

    Arabidopsis thaliana contains two GDP-L-galactose phosphorylase genes, VTC2 and VTC5, which are critical for ascorbate (AsA) biosynthesis. We investigated the expression levels of both VTC2 and VTC5 genes in wild-type A. thaliana and the AsA deficient mutants during early seedling growth. Ascorbate accumulated to an equal extent in all genotypes up to 5 d post-germination (DPG). The transcript level of VTC2 was dominant, and increased in parallel with AsA accumulation in the wild type. On the other hand, the expression of VTC5 compensated for the reduced VTC2 transcription levels in the AsA deficient mutant vtc2-1 in young seedlings. A luciferase activity assay indicated that the VTC5 promoter was more active in young (2 DPG) cotyledons and that the VTC2 and VTC5 promoters drove a day-to-night variation in expression. The present work provides clues to the precise roles of VTC2 and VTC5 in AsA biosynthesis in A. thaliana at the young seedling stage. PMID:21897033

  13. Comparative Positron-Emission Tomography (PET) Imaging and Phototherapeutic Potential of 124I- Labeled Methyl- 3-(1′-iodobenzyloxyethyl) pyropheophorbide-a vs. the Corresponding Glucose- and Galactose-Conjugates

    PubMed Central

    Pandey, Suresh K.; Sajjad, Munawwar; Chen, Yihui; Zheng, Xiang; Yao, Rutao; Missert, Joseph R.; Batt, Carrie; Nabi, Hani A.; Oseroff, Allan R.; Pandey, Ravindra K.

    2009-01-01

    In our present study, 3-(1′-m-iodobenzyloxyethyl) pyropheophorbide-a methyl ester 1, 3-(1′-m-iodobenzyloxyethyl)-172-{(2-deoxy)glucose} pyropheophorbide-a 2, and 3-(1′-m-iodo benzyloxyethyl)-172-{(1-deoxy)galactose} pyropheophorbide-a 3 were synthesized and converted into the corresponding 124I- labeled analogs by reacting the intermediate trimethyltin analogs with Na124I. Photosensitizers 1–3 were evaluated for the PDT efficacy in C3H mice bearing RIF tumors at variable doses and showed a significant long-term tumor cure. Among the compounds investigated, the non-carbohydrate analog 1 was most effective. These results were in contrast to the in vitro data, where compared to the parent analog the corresponding galactose-and glucose derivatives showed enhanced cell kill. Among the corresponding 124I-labeled in analogs, excellent tumor images were obtained from compound 1 both tumor models (RIF and Colon-26) and the best tumor contrast was observed at 72 h post injection. Conjugating a glucose moiety to photosensitizer 1 diminished its tumor uptake, whereas with time the corresponding galactose analog showed improved tumor contrast. PMID:19090663

  14. Fluorescence Resonance Energy Transfer Glucose Sensor from Site-Specific Dual Labeling of Glucose/Galactose Binding Protein Using Ligand Protection

    PubMed Central

    Hsieh, Helen V.; Sherman, Douglas B.; Andaluz, Sandra A.; Amiss, Terry J.; Pitner, J. Bruce

    2012-01-01

    Background Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call “ligand protection.” Method In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents. The mutant E149C/A213C/L238S was first labeled at E149C in the presence of the ligand glucose. Following dialysis and removal of glucose, the protein was labeled with a second dye, either Texas Red (TR) C5 bromoacetamide or TR C2 maleimide, at the second site, A213C. Results Changes in glucose-dependent fluorescence were observed that were consistent with FRET between the nitrobenzoxadiazole and TR fluorophores. Comparison of models and spectroscopic properties of the C2 and C5 TR FRET constructs suggests the greater rigidity of the C2 linker provides more efficient FRET. Conclusions The ligand protection strategy provides a simple method for labeling GGBP with two different fluorophores to construct FRET-based glucose sensors with glucose affinity within the human physiological glucose range (1–30 mM). This general strategy may also have broad utility for other protein-labeling applications. PMID:23294773

  15. Fibroblast growth factor 21 protects mouse brain against D-galactose induced aging via suppression of oxidative stress response and advanced glycation end products formation.

    PubMed

    Yu, Yinhang; Bai, Fuliang; Wang, Wenfei; Liu, Yaonan; Yuan, Qingyan; Qu, Susu; Zhang, Tong; Tian, Guiyou; Li, Siming; Li, Deshan; Ren, Guiping

    2015-06-01

    Fibroblast growth factor 21 (FGF21) is a hormone secreted predominantly in the liver, pancreas and adipose tissue. Recently, it has been reported that FGF21-Transgenic mice can extend their lifespan compared with wild type counterparts. Thus, we hypothesize that FGF21 may play some roles in aging of organisms. In this study d-galactose (d-gal)-induced aging mice were used to study the mechanism that FGF21 protects mice from aging. The three-month-old Kunming mice were subcutaneously injected with d-gal (180mg·kg(-1)·d(-1)) for 8weeks and administered simultaneously with FGF21 (1, 2 or 5mg·kg(-1)·d(-1)). Our results showed that administration of FGF21 significantly improved behavioral performance of d-gal-treated mice in water maze task and step-down test, reduced brain cell damage in the hippocampus, and attenuated the d-gal-induced production of MDA, ROS and advanced glycation end products (AGEs). At the same time, FGF21 also markedly renewed the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total anti-oxidation capability (T-AOC), and decreased the enhanced total cholinesterase (TChE) activity in the brain of d-gal-treated mice. The expression of aldose reductase (AR), sorbitol dehydrogenase (SDH) and member-anchored receptor for AGEs (RAGE) declined significantly after FGF21 treatment. Furthermore, FGF21 suppressed inflamm-aging by inhibiting IκBα degradation and NF-κB p65 nuclear translocation. The expression levels of pro-inflammatory cytokines, such as TNF-α and IL-6, decreased significantly. In conclusion, these results suggest that FGF21 protects the aging mice brain from d-gal-induced injury by attenuating oxidative stress damage and decreasing AGE formation. PMID:25871519

  16. Identification of novel α1,3-galactosyltransferase and elimination of α-galactose-containing glycans by disruption of multiple α-galactosyltransferase genes in Schizosaccharomyces pombe.

    PubMed

    Ohashi, Takao; Fujiyama, Kazuhito; Takegawa, Kaoru

    2012-11-01

    The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of D-galactose (Gal) in addition to D-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1(+)-otg3(+) (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2(+) gene product had Gal transfer activity toward a pyridylaminated Man(9)GlcNAc(2) oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3(+) gene product exhibited Gal transfer activity toward the pyridylaminated Man(9)GlcNAc(2) oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, (1)H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides. PMID:22988247

  17. Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

    PubMed Central

    Séguin, R; Mann, B J; Keller, K; Chadee, K

    1995-01-01

    The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 PMID:8618866

  18. Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

    SciTech Connect

    Michaels, J.E.; Garfield, S.A.; Hung, J.T.; Cardell, R.R. Jr. )

    1990-08-01

    The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.

  19. Cloning and DNA Sequence Analysis of an Immunogenic Glucose-Galactose MglB Lipoprotein Homologue from Brachyspira pilosicoli, the Agent of Colonic Spirochetosis†

    PubMed Central

    Zhang, P.; Cheng, X.; Duhamel, G. E.

    2000-01-01

    Colonic spirochetosis (CS) is a newly emerging infectious disease of humans and animals caused by the pathogenic spirochete Brachyspira (formerly Serpulina) pilosicoli. The purpose of this study was to characterize an antigen that was recognized by antibodies present in sera of challenge-exposed pigs. The gene encoding the antigen was identified by screening a plasmid library of human B. pilosicoli strain SP16 (ATCC 49776) genomic DNA with hyperimmune and convalescent swine sera. The predicted amino acid sequence encoded by the cloned B. pilosicoli gene had a high degree of similarity and identity to glucose-galactose MglB lipoprotein. Located 106 bp downstream of the putative mglB gene was a 3′-truncated open reading frame with 73.8% similarity and 66.3% identity to mglA of Escherichia coli, suggesting a gene arrangement within an operon which is similar to those of other bacteria. A single copy of the gene was present in B. pilosicoli, and homologous sequences were widely conserved among porcine intestinal spirochetes Serpulina intermedia, Brachyspira innocens, Brachyspira murdochii, and the avian Brachyspira alvinipulli, but not in porcine Brachyspira hyodysenteriae, human Brachyspira aalborgi, and porcine Treponema succinifaciens. The deduced molecular weight of the mature MglB lipoprotein was consistent with expression by the cloned gene of a polypeptide with an apparent molecular weight of 36,000, as determined by Western blot analysis and [3H]palmitate labeling. Because mucin is the principal constituent of the colonic mucus gel and consists of glycoproteins that can serve as the substrate for growth and chemotaxis of B. pilosicoli in vitro, a role for MglB in mucosal localization of the spirochete appears consistent with the pathogenesis of CS. However, the presence of homologous sequences in closely related but nonpathogenic commensal spirochetes suggests that other virulence determinants may be required for pathogenesis. PMID:10899855

  20. Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family

    SciTech Connect

    Fridovich-Keil, J.L.; Langley, S.D.; Mazur, L.A.; Lennon, J.C.; Dembure, P.O.; Elsas, L.J. II

    1995-03-01

    We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system. A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neutral polymorphism. To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae. The results of these yeast-modeling studies were fully consistent with the patient data, leading us to conclude that, at least within the context of the cell types studied, in the homozygous state Q188R is a mutation that eliminates GALT activity, and S135L and V151A are both mutations that impair GALT activity to <6% of wild-type values. 22 refs., 5 figs.

  1. Identification of a Novel Streptococcal Adhesin P (SadP) Protein Recognizing Galactosyl-α1–4-galactose-containing Glycoconjugates

    PubMed Central

    Kouki, Annika; Haataja, Sauli; Loimaranta, Vuokko; Pulliainen, Arto T.; Nilsson, Ulf J.; Finne, Jukka

    2011-01-01

    Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1–4-galactose (Galα1–4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1–4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1–4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1–4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO3 (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1–4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure. PMID:21908601

  2. Identification of Novel α1,3-Galactosyltransferase and Elimination of α-Galactose-containing Glycans by Disruption of Multiple α-Galactosyltransferase Genes in Schizosaccharomyces pombe*

    PubMed Central

    Ohashi, Takao; Fujiyama, Kazuhito; Takegawa, Kaoru

    2012-01-01

    The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of d-galactose (Gal) in addition to d-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1+–otg3+ (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2+ gene product had Gal transfer activity toward a pyridylaminated Man9GlcNAc2 oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3+ gene product exhibited Gal transfer activity toward the pyridylaminated Man9GlcNAc2 oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, 1H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides. PMID:22988247

  3. Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium*

    PubMed Central

    Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

    2012-01-01

    A gene from the thermophilic archaeon Thermoplasma volcanium encoding an l-threonine dehydrogenase (l-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr137 was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: l-ThrDH-NAD+, l-ThrDH in complex with NAD+ and pyruvate, Y137F mutant in complex with NAD+ and l-threonine, and Y137F in complex with NAD+ and l-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like l-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of l-threonine via direct proton transfer driven by Tyr137. The factors contributing to the thermostability of T. volcanium l-ThrDH were analyzed by comparing its structure to that of F. frigidimaris l-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium l-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like l-ThrDH. PMID:22374996

  4. Allelic variation in paralogs of GDP-L-galactose phosphorylase is a major determinant of vitamin C concentrations in apple fruit.

    PubMed

    Mellidou, Ifigeneia; Chagné, David; Laing, William A; Keulemans, Johan; Davey, Mark W

    2012-11-01

    To identify the genetic factors underlying the regulation of fruit vitamin C (L-ascorbic acid [AsA]) concentrations, quantitative trait loci (QTL) studies were carried out in an F1 progeny derived from a cross between the apple (Malus × domestica) cultivars Telamon and Braeburn over three years. QTL were identified for AsA, glutathione, total antioxidant activity in both flesh and skin tissues, and various quality traits, including flesh browning. Four regions on chromosomes 10, 11, 16, and 17 contained stable fruit AsA-QTL clusters. Mapping of AsA metabolic genes identified colocations between orthologs of GDP-L-galactose phosphorylase (GGP), dehydroascorbate reductase (DHAR), and nucleobase-ascorbate transporter within these QTL clusters. Of particular interest are the three paralogs of MdGGP, which all colocated within AsA-QTL clusters. Allelic variants of MdGGP1 and MdGGP3 derived from the cultivar Braeburn parent were also consistently associated with higher fruit total AsA concentrations both within the mapping population (up to 10-fold) and across a range of commercial apple germplasm (up to 6-fold). Striking differences in the expression of the cv Braeburn MdGGP1 allele between fruit from high- and low-AsA genotypes clearly indicate a key role for MdGGP1 in the regulation of fruit AsA concentrations, and this MdGGP allele-specific single-nucleotide polymorphism marker represents an excellent candidate for directed breeding for enhanced fruit AsA concentrations. Interestingly, colocations were also found between MdDHAR3-3 and a stable QTL for browning in the cv Telamon parent, highlighting links between the redox status of the AsA pool and susceptibility to flesh browning. PMID:23001142

  5. Multiple sequence variations in SLC5A1 gene are associated with glucose-galactose malabsorption in a large cohort of Old Order Amish.

    PubMed

    Xin, B; Wang, H

    2011-01-01

    Glucose-galactose malabsorption (GGM) is an autosomal recessive disease with life-threatening newborn diarrhea caused by mutations in the Na(+) /glucose cotransporter gene SLC5A1. Because of its rarity, the clinical course of the disease has not been well studied. Here, we report 33 patients with GGM from a large Old Order Amish pedigree and the associated mutations in SLC5A1 gene. Clinically, all affected individuals presented with classic watery diarrhea and dehydration. The increased bowel sounds, distended abdomen, vigorous nursing regardless of their illness, and irritability and apathy were also noted as part of the initial presentation. Patients underwent a dramatic turnaround with an immediate cease of the diarrhea and a quick rehydration if they were correctly diagnosed and adequately managed, followed by a normal growth and development pattern afterwards; whereas a prolonged clinical course would follow if the disease was not recognized. Sequence analysis of the 15 protein-coding exons and the corresponding exon-intron boundaries of SLC5A1 gene revealed four homozygous missense mutations, c.152A>G (p.N51S), c.1231G>A (p.A411T), c.1673G>A (p.R558H), and c.1845C>G (p.H615Q), that co-segregate with the GGM phenotype in all of the affected individuals. These findings suggest that founder effect of the SLC5A1 mutations associated with the disease in Amish and a population specific genetic testing is in need to pursue an early diagnosis which is critical for a favorable outcome. PMID:20486940

  6. Protective effects of perindopril on d-galactose and aluminum trichloride induced neurotoxicity via the apoptosis of mitochondria-mediated intrinsic pathway in the hippocampus of mice.

    PubMed

    Yang, Weina; Shi, Lili; Chen, Lianji; Zhang, Bingyi; Ma, Kaige; Liu, Yong; Qian, Yihua

    2014-10-01

    Perindopril, an angiotensin converting enzyme inhibitor, has been reported to improve learning and memory in a mouse or rat model of Alzheimer's disease (AD) induced by injection of beta-amyloid protein. However, the exact mechanism of perindopril on the cognitive deficits is not fully understood. Our previous data have indicated that perindopril improves learning and memory in a mouse model of AD induced by D-galactose (D-gal) and aluminum trichloride (AlCl₃) via inhibition of acetylcholinesterase activity and oxidative stress. Whether perindopril also inhibit apoptosis to prevent cognitive decline remains unknown in mice. Therefore, the present study explored the protective effects of perindopril in the hippocampus of mice further. Perindopril (0.5 mg/kg/day) was administered intragastrically for 60 days after the mice were given a D-gal (150 mg/kg/day) and AlCl₃ (10 mg/kg/day) intraperitoneally for 90 days. Then the expression of Bcl-2, Bax, Fas, FasL, caspase-3, caspase-8 and caspase-9 were analyzed by RT-PCR and western blotting in the hippocampus. Perindopril significantly decreased caspase-3 and caspase-9 activities, and elevated Bcl-2/Bax ratio in the hippocampus. However, the expression of Fas, FasL and caspase-8 did not change in the hippocampus whether treatment with d-gal and AlCl₃ or perindopril. Taken together, the above findings indicated that perindopril inhibited apoptosis in the hippocampus may be another mechanism by which perindopril improves learning and memory functions in d-gal and AlCl₃ treated mice. PMID:25290208

  7. Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs

    PubMed Central

    Reynard, Olivier; Jacquot, Frédéric; Evanno, Gwénaëlle; Mai, Hoa Le; Martinet, Bernard; Duvaux, Odile; Bach, Jean-Marie; Conchon, Sophie; Judor, Jean-Paul; Perota, Andrea; Lagutina, Irina; Duchi, Roberto; Lazzari, Giovanna; Le Berre, Ludmilla; Perreault, Hélène; Lheriteau, Elsa; Raoul, Hervé; Volchkov, Viktor; Galli, Cesare; Soulillou, Jean-Paul

    2016-01-01

    Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1–3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1–3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1–3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection. PMID:27280712

  8. Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs.

    PubMed

    Reynard, Olivier; Jacquot, Frédéric; Evanno, Gwénaëlle; Mai, Hoa Le; Salama, Apolline; Martinet, Bernard; Duvaux, Odile; Bach, Jean-Marie; Conchon, Sophie; Judor, Jean-Paul; Perota, Andrea; Lagutina, Irina; Duchi, Roberto; Lazzari, Giovanna; Le Berre, Ludmilla; Perreault, Hélène; Lheriteau, Elsa; Raoul, Hervé; Volchkov, Viktor; Galli, Cesare; Soulillou, Jean-Paul

    2016-01-01

    Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection. PMID:27280712

  9. Amphiphilic Diblock Terpolymer PMAgala-b-P(MAA-co-MAChol)s with Attached Galactose and Cholesterol Grafts and Their Intracellular pH-Responsive Doxorubicin Delivery.

    PubMed

    Wang, Zhao; Luo, Ting; Sheng, Ruilong; Li, Hui; Sun, Jingjing; Cao, Amin

    2016-01-11

    In this work, a series of diblock terpolymer poly(6-O-methacryloyl-D-galactopyranose)-b-poly(methacrylic acid-co-6-cholesteryloxy hexyl methacrylate) amphiphiles bearing attached galactose and cholesterol grafts denoted as the PMAgala-b-P(MAA-co-MAChol)s were designed and prepared, and these terpolymer amphiphiles were further exploited as a platform for intracellular doxorubicin (DOX) delivery. First, employing a sequential RAFT strategy with preliminarily synthesized poly(6-O-methacryloyl-1,2:3,4-di-O-isopropylidene-d-galactopyranose) (PMAIpGP) macro-RAFT initiator and a successive trifluoroacetic acid (TFA)-mediated deprotection, a series of amphiphilic diblock terpolymer PMAgala-b-P(MAA-co-MAChol)s were prepared, and were further characterized by NMR, Fourier transform infrared spectrometer (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and a dynamic contact angle testing instrument (DCAT). In aqueous media, spontaneous micellization of the synthesized diblock terpolymer amphiphiles were continuously examined by critical micellization concentration assay, dynamic light scattering (DLS), and transmission electron microscopy (TEM), and the efficacies of DOX loading by these copolymer micelles were investigated along with the complexed nanoparticle stability. Furthermore, in vitro DOX release of the drug-loaded terpolymer micelles were studied at 37 °C in buffer under various pH conditions, and cell toxicities of as-synthesized diblock amphiphiles were examined by MTT assay. Finally, with H1299 cells, intracellular DOX delivery and localization by the block amphiphile vectors were investigated by invert fluorescence microscopy. As a result, it was revealed that the random copolymerization of MAA and MAChol comonomers in the second block limited the formation of cholesterol liquid-crystal phase and enhanced DOX loading efficiency and complex nanoparticle stability, that ionic interactions between the DOX and MAA comonomer

  10. Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli.

    PubMed Central

    Becker, P S; Akins, D R; Radolf, J D; Norgard, M V

    1994-01-01

    The recent discovery that abundant and immunogenic lipoproteins constitute the integral membrane proteins of Treponema pallidum has prompted efforts to investigate their importance in the physiology and ultrastructure of the organism and in immune responses during infection. Earlier studies identified a 38-kDa lipoprotein of T. pallidum believed to be specific to the pathogen. In the present study, monoclonal antibodies generated against the 38-kDa lipoprotein of T. pallidum reacted with cognate 37-kDa molecules in the nonpathogens Treponema phagedenis, Treponema denticola, and Treponema refringens. Cloning and expression of the 38-kDa-lipoprotein gene of T. pallidum in Escherichia coli revealed that the recombinant product displayed a slightly larger (39-kDa) apparent molecular mass but remained reactive with anti-38-kDa-protein monoclonal antibodies. The recombinant product was processed and acylated in E. coli. DNA and amino acid sequence analyses indicated an open reading frame encoding 403 amino acids, with the first 25 amino acids corresponding to a leader peptide terminated by a signal peptidase II processing site of Val-Val-Gly-Cys. The predicted mature protein is 378 amino acids in length with a deduced molecular weight of 40,422 (excluding acylation). Southern blotting failed to demonstrate in nonpathogenic treponemes genomic sequences homologous with the 38-kDa-lipoprotein gene of T. pallidum. Computer analysis revealed that the 38-kDa lipoprotein of T. pallidum had 34.2% identity and 58.9% similarity with the glucose/galactose-binding protein (MglB) of E. coli and Salmonella typhimurium. Furthermore, of the 19 amino acids of MglB involved in carbohydrate binding, the 38-kDa lipoprotein had identity with 11. These studies have allowed the first putative functional assignment (carbohydrate binding) to a T. pallidum integral membrane protein. Recognition of this potential physiological role for the 38-kDa lipoprotein underscores the possibility that the

  11. Phosphorescent cellular probes and uptake indicators derived from cyclometalated iridium(III) bipyridine complexes appended with a glucose or galactose entity.

    PubMed

    Law, Wendell Ho-Tin; Lee, Lawrence Cho-Cheung; Louie, Man-Wai; Liu, Hua-Wei; Ang, Tim Wai-Hung; Lo, Kenneth Kam-Wing

    2013-11-18

    A series of phosphorescent cyclometalated iridium(III) polypyridine complexes appended with a β-D-glucose moiety [Ir(N^C)2(bpy-TEG-ONCH3-β-D-glc)](PF6) [bpy-TEG-ONCH3-β-D-glc = 4-(10-N-methyl-N-(β-D-glucopyranosyl)-amino-oxy-2,5,8-trioxa-dec-1-yl)-4'-methyl-2,2'-bipyridine; HN^C = 2-((1,1'-biphenyl)-4-yl)benzothiazole) (Hbt) (1a), 2-phenylpyridine (Hppy) (2a), 2-phenylquinoline (Hpq) (3a), 7,8-benzoquinoline (Hbzq) (4a)] has been synthesized and characterized. The D-galactose counterparts [Ir(N^C)2(bpy-TEG-ONCH3-β-D-gal)](PF6) [bpy-TEG-ONCH3-β-D-gal = 4-(10-N-methyl-N-(β-D-galactopyranosyl)-amino-oxy-2,5,8-trioxa-dec-1-yl)-4'-methyl-2,2'-bipyridine; HN^C = Hbt (1b), Hppy (2b), Hpq (3b), Hbzq (4b)] and a sugar-free bt complex [Ir(bt)2(bpy-TEG-OMe)](PF6) [bpy-TEG-OMe = 4-(2,5,8,11-tetraoxa-dodec-1-yl)-4'-methyl-2,2'-bipyridine] (1c) have also been prepared. Upon photoexcitation, all the complexes displayed intense and long-lived triplet metal-to-ligand charge-transfer ((3)MLCT) [dπ(Ir) → π*(N^N)] or triplet intraligand ((3)IL) (π → π*) (N^C and N^N) emission. The lipophilicity, the cellular uptake efficiency, and cytotoxicity of the complexes toward human cervix epithelioid carcinoma cells (HeLa) have been examined. Temperature dependence and chemical inhibition experiments indicated that the transport of bt-glucose complex 1a across the cell membrane occurred through an energy-requiring process such as endocytosis, in additional to a pathway that was mediated by glucose transporters (GLUTs). Importantly, the cellular uptake efficiency of this complex was found to be strongly dependent on hormonal stimulation and inhibition, rendering it a new phosphorescent metabolic indicator. Additionally, laser-scanning confocal microscopy revealed that the complex was localized in the mitochondria and highly resistant to photobleaching compared to a fluorescent organic glucose derivative 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG

  12. Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene.

    PubMed

    Viggiano, E; Marabotti, A; Burlina, A P; Cazzorla, C; D'Apice, M R; Giordano, L; Fasan, I; Novelli, G; Facchiano, A; Burlina, A B

    2015-04-01

    Classical galactosemia is an autosomal recessive inborn error of metabolism due to mutations of the GALT gene leading to toxic accumulation of galactose and derived metabolites. With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. However, despite early diagnosis and treatment, the long term outcome for these patients is still unpredictable because they may go on to develop cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30years. No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications. A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of <1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype-phenotype correlation in classical galactosemia and underlines the importance of

  13. Bi- to tetravalent glycoclusters presenting GlcNAc/GalNAc as inhibitors: from plant agglutinins to human macrophage galactose-type lectin (CD301) and galectins.

    PubMed

    André, Sabine; O'Sullivan, Shane; Koller, Christiane; Murphy, Paul V; Gabius, Hans-Joachim

    2015-04-14

    Emerging insights into the functional spectrum of tissue lectins leads to identification of new targets for the custom-made design of potent inhibitors, providing a challenge for synthetic chemistry. The affinity and selectivity of a carbohydrate ligand for a lectin may immensely be increased by a number of approaches, which includes varying geometrical or topological features. This perspective leads to the design and synthesis of glycoclusters and their testing using assays of physiological relevance. Herein, hydroquinone, resorcinol, benzene-1,3,5-triol and tetra(4-hydroxyphenyl)ethene have been employed as scaffolds and propargyl derivatives obtained. The triazole-containing linker to the α/β-O/S-glycosides of GlcNAc/GalNAc presented on these scaffolds was generated by copper-catalysed azide-alkyne cycloaddition. This strategy was used to give a panel of nine glycoclusters with bi-, tri- and tetravalency. Maintained activity for lectin binding after conjugation was ascertained for both sugars in solid-phase assays with the plant agglutinins WGA (GlcNAc) and DBA (GalNAc). Absence of cross-reactivity excluded any carbohydrate-independent reactivity of the bivalent compounds, allowing us to proceed to further testing with a biomedically relevant lectin specific for GalNAc. Macrophage galactose(-binding C)-type lectin, involved in immune defence by dendritic cells and in virus uptake, was produced as a soluble protein without/with its α-helical coiled-coil stalk region. Binding to ligands presented on a matrix and on cell surfaces was highly susceptible to the presence of the tetravalent inhibitor derived from the tetraphenylethene-containing scaffold, and presentation of GalNAc with an α-thioglycosidic linkage proved favorable. Cross-reactivity of this glycocluster to human galectins-3 and -4, which interact with Tn-antigen-presenting mucins, was rather small. Evidently, the valency and spatial display of α-GalNAc residues is a key factor to design potent and

  14. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli.

    PubMed

    Jackson, D A; Symons, R H; Berg, P

    1972-10-01

    We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule. PMID:4342968

  15. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli

    PubMed Central

    Jackson, David A.; Symons, Robert H.; Berg, Paul

    1972-01-01

    We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3′ ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule. PMID:4342968

  16. Protective Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through Attenuating Oxidative Stress and the Wnt/β-Catenin Signaling Pathway in a Mouse Model of d-Galactose-induced Aging

    PubMed Central

    Li, Jing; Cai, Dachuan; Yao, Xin; Zhang, Yanyan; Chen, Linbo; Jing, Pengwei; Wang, Lu; Wang, Yaping

    2016-01-01

    Stem cell senescence is an important and current hypothesis accounting for organismal aging, especially the hematopoietic stem cell (HSC). Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng, which is a traditional Chinese medicine. This study explored the protective effect of ginsenoside Rg1 on Sca-1+ hematopoietic stem/progenitor cells (HSC/HPCs) in a mouse model of d-galactose-induced aging. The mimetic aging mouse model was induced by continuous injection of d-gal for 42 days, and the C57BL/6 mice were respectively treated with ginsenoside Rg1, Vitamin E or normal saline after 7 days of d-gal injection. Compared with those in the d-gal administration alone group, ginsenoside Rg1 protected Sca-1+ HSC/HPCs by decreasing SA-β-Gal and enhancing the colony forming unit-mixture (CFU-Mix), and adjusting oxidative stress indices like reactive oxygen species (ROS), total anti-oxidant (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA). In addition, ginsenoside Rg1 decreased β-catenin and c-Myc mRNA expression and enhanced the phosphorylation of GSK-3β. Moreover, ginsenoside Rg1 down-regulated advanced glycation end products (AGEs), 4-hydroxynonenal (4-HNE), phospho-histone H2A.X (r-H2A.X), 8-OHdG, p16Ink4a, Rb, p21Cip1/Waf1 and p53 in senescent Sca-1+ HSC/HPCs. Our findings indicated that ginsenoside Rg1 can improve the resistance of Sca-1+ HSC/HPCs in a mouse model of d-galactose-induced aging through the suppression of oxidative stress and excessive activation of the Wnt/β-catenin signaling pathway, and reduction of DNA damage response, p16Ink4a-Rb and p53-p21Cip1/Waf1 signaling. PMID:27294914

  17. Protective Effect of Ginsenoside Rg1 on Hematopoietic Stem/Progenitor Cells through Attenuating Oxidative Stress and the Wnt/β-Catenin Signaling Pathway in a Mouse Model of d-Galactose-induced Aging.

    PubMed

    Li, Jing; Cai, Dachuan; Yao, Xin; Zhang, Yanyan; Chen, Linbo; Jing, Pengwei; Wang, Lu; Wang, Yaping

    2016-01-01

    Stem cell senescence is an important and current hypothesis accounting for organismal aging, especially the hematopoietic stem cell (HSC). Ginsenoside Rg1 is the main active pharmaceutical ingredient of ginseng, which is a traditional Chinese medicine. This study explored the protective effect of ginsenoside Rg1 on Sca-1⁺ hematopoietic stem/progenitor cells (HSC/HPCs) in a mouse model of d-galactose-induced aging. The mimetic aging mouse model was induced by continuous injection of d-gal for 42 days, and the C57BL/6 mice were respectively treated with ginsenoside Rg1, Vitamin E or normal saline after 7 days of d-gal injection. Compared with those in the d-gal administration alone group, ginsenoside Rg1 protected Sca-1⁺ HSC/HPCs by decreasing SA-β-Gal and enhancing the colony forming unit-mixture (CFU-Mix), and adjusting oxidative stress indices like reactive oxygen species (ROS), total anti-oxidant (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA). In addition, ginsenoside Rg1 decreased β-catenin and c-Myc mRNA expression and enhanced the phosphorylation of GSK-3β. Moreover, ginsenoside Rg1 down-regulated advanced glycation end products (AGEs), 4-hydroxynonenal (4-HNE), phospho-histone H2A.X (r-H2A.X), 8-OHdG, p16(Ink4a), Rb, p21(Cip1/Waf1) and p53 in senescent Sca-1⁺ HSC/HPCs. Our findings indicated that ginsenoside Rg1 can improve the resistance of Sca-1⁺ HSC/HPCs in a mouse model of d-galactose-induced aging through the suppression of oxidative stress and excessive activation of the Wnt/β-catenin signaling pathway, and reduction of DNA damage response, p16(Ink4a)-Rb and p53-p21(Cip1/Waf1) signaling. PMID:27294914

  18. A novel strain of Lactobacillus mucosae isolated from a Gaotian villager improves in vitro and in vivo antioxidant as well as biological properties in D-galactose-induced aging mice.

    PubMed

    Yu, Xiaomin; Li, Shengjie; Yang, Dong; Qiu, Liang; Wu, Yaoping; Wang, Dengyuan; Shah, Nagendra P; Xu, Feng; Wei, Hua

    2016-02-01

    Twelve isolates isolated from the gastrointestinal tracts of Gaotian villagers in China, who had a lifespan of 92 yr, were examined for their antioxidants using free radical scavenging activity and 2,2-diphenyl-1-picrylhydrazyl. Three strains (i.e., Lactobacillus mucosae LMU1001, and Lactobacillus plantarum LPL0902 and LPL0302) were selected as candidates to prepare yogurt for testing their antioxidants in a model of d-galactose-induced aging mice, with vitamin C as a positive control. The results showed that L. mucosae LMU1001 was the best strain, which had similar in vivo antioxidant activity as vitamin C. A significant increase was found in the activities of glutathione peroxidase in serum and total superoxide dismutase in the liver, and a decrease in the level of malondialdehyde in serum. Regarding mRNA expression level detected quantitatively by real-time PCR, we observed that L. mucosae LMU1001 significantly upregulated antioxidant genes (i.e., MT1A and MT1M in HT-29 and Caco-2) and those genes (i.e., MT1, MT2, GPx1, and GPx2) in the intestinal tract of the model mice. Hence, this strain could be considered as a potential probiotic lactic acid bacterium for improving antioxidant levels in functional foods. PMID:26709186

  19. l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

    PubMed Central

    2014-01-01

    The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

  20. 'Click chemistry' synthesis of a library of 1,2,3-triazole-substituted galactose derivatives and their evaluation against Trypanosoma cruzi and its cell surface trans-sialidase.

    PubMed

    Carvalho, Ivone; Andrade, Peterson; Campo, Vanessa L; Guedes, Paulo M M; Sesti-Costa, Renata; Silva, João S; Schenkman, Sergio; Dedola, Simone; Hill, Lionel; Rejzek, Martin; Nepogodiev, Sergey A; Field, Robert A

    2010-04-01

    Trypanosoma cruzi trans-sialidase (TcTS) plays a key role in the recognition and invasion of host cells and in enabling the parasite to escape the human immune response. To explore this potential drug target, we have synthesized a small library of substrate analogues based on 1,4-disubstituted 1,2,3-triazole derivatives of galactose modified at either the C-1 or C-6 positions. This was achieved by coupling the appropriate azido-sugars with a panel of 23 structurally diverse terminal alkynes by using the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction, giving a library of 46 derivatives in good to excellent yield and with complete regioselectivity. The sugar triazoles showed weak inhibition towards TcTS-catalyzed hydrolysis of 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid in vitro (<40% inhibition at 1mM concentration); many of the compounds assessed proved to be acceptor substrates for the enzyme. Despite this modest inhibitory activity, in vitro trypanocidal activity assays against the trypomastigote form of T. cruzi Y strain revealed several compounds active in the low 100s of muM range. Further assessment of these compounds against cultured mouse spleen cells suggests a specific mode of anti-parasite action rather than a generic cytotoxic effect. PMID:20335038

  1. Differentiation of some positional and diastereomeric isomers of Boc-carbo-[beta]3 dipeptides containing galactose, xylose and mannose sugars by electrospray ionization tandem mass spectrometry (ESI MS/MS)

    NASA Astrophysics Data System (ADS)

    Reddy, P. Nagi; Ramesh, V.; Srinivas, R.; Sharma, G. V. M.; Nagendar, Pendem; Subash, V.

    2006-02-01

    Electrospray ionization (ESI) tandem mass spectrometry has been used to distinguish the positional and diastereomeric isomers of Boc-C-linked carbo-[beta]3 dipeptides (1-38) synthesized from glycine (Gly), [beta]-h-glycine ([beta]-hGly), [beta]-h-alanine ([beta]-hAla) and C-linked carbo-[beta]3-amino acid (Caa) that contain galactose, xylose and mannose sugars as side chains with "R" and "S" configurations at the amine center. The major fragmentation of [M + H]+ of the dipeptides (1-38) yields mainly two ions: (i) [M + H-C(CH3)3 + H]+ ([`]a') and (ii) [M + H-Boc + H]+ ([`]b') corresponding to losses of 2-methyl-prop-1-ene and -Boc moiety from [M + H]+ ions, respectively. The diastereomeric dipeptide isomers with Caa (R) and (S) configurations at the N-terminus can easily be distinguished by the difference in the abundance of ion [`]a' and [`]b'. The isomeric peptides with Caa (R) at the N-terminus gives prominent [M + H-C(CH3)3 + H]+ ([`]a') where as it is insignificant or totally absent for peptides which have Caa (S) at the N-terminus. This is presumably due to the different steric crowdings around the Boc-group in the different diastereomers. The positional isomers of dipeptides can also be differentiated by the difference in the abundance of ion [`]a' and [`]b' in the CID of [M + H]+ ions. The CID of [M + H-Boc + H]+ ions of the isomeric peptides also show y1+ ions at different m/z values. All these results suggest that the CID of [M + H]+ ions is highly useful for distinguishing the Boc-NH-Caa-[beta]3 dipeptide isomers with Caa of "S" configuration from the isomers with Caa of "R" configuration and the positional isomers.

  2. Close-up of the Immunogenic α1,3-Galactose Epitope as Defined by a Monoclonal Chimeric Immunoglobulin E and Human Serum Using Saturation Transfer Difference (STD) NMR

    PubMed Central

    Plum, Melanie; Michel, Yvonne; Wallach, Katharina; Raiber, Tim; Blank, Simon; Bantleon, Frank I.; Diethers, Andrea; Greunke, Kerstin; Braren, Ingke; Hackl, Thomas; Meyer, Bernd; Spillner, Edzard

    2011-01-01

    Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen α1,3-Gal (α-Gal) are associated with a delayed type of anaphylaxis, providing evidence for the clinical relevance of carbohydrate epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction, and fine epitope of α-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety of an α-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed by mediator release assays, surface plasmon resonance (SPR), and saturation transfer difference NMR analyses. The α-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The α1,3-Gal epitope fine structures of both the recombinant IgE and affinity-purified serum were defined by saturation transfer difference NMR, revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying α1,3-Gal-mediated anaphylaxis. The complementary data of affinity and fine specificity may help to elucidate the recognition of carbohydrates by the adaptive immune response and the molecular requirements of carbohydrate-based anaphylaxis. PMID:21990360

  3. Galactose-1-phosphate uridyltransferase blood test

    MedlinePlus

    Galactosemia screen; GALT; Gal-1-PUT ... which is found in milk and dairy products. 1 out of 65,000 newborns lack a substance ( ... Veins and arteries vary in size from one infant to another and from ... other. Obtaining a blood sample from some infants may be more ...

  4. Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

    PubMed Central

    Thomas, Suzanne; Brochu, Denis; Vadeboncoeur, Christian

    2001-01-01

    In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for α-galactosidase, β-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of α-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and

  5. Premature Bioprosthetic Aortic Valve Degeneration Associated with Allergy to Galactose-Alpha-1,3-Galactose.

    PubMed

    Hawkins, Robert B; Frischtak, Helena L; Kron, Irving L; Ghanta, Ravi K

    2016-07-01

    We present the cases of two patients with bioprosthetic aortic valves who developed an allergy to alpha-gal. Each had premature degeneration of their bioprosthesis and demonstrated rapidly increasing transvalvular gradients after development of their allergy. Each underwent successful replacement with a mechanical aortic valve within 1-2 years of symptom onset. doi: 10.1111/jocs.12764 (J Card Surg 2016;31:446-448). PMID:27238083

  6. A biologically active peptide mimetic of N-acetylgalactosamine/galactose

    PubMed Central

    Eggink, Laura L; Hoober, J Kenneth

    2009-01-01

    Background Glycosylated proteins and lipids are important regulatory factors whose functions can be altered by addition or removal of sugars to the glycan structure. The glycans are recognized by sugar-binding lectins that serve as receptors on the surface of many cells and facilitate initiation of an intracellular signal that changes the properties of the cells. We identified a peptide that mimics the ligand of an N-acetylgalactosamine (GalNAc)-specific lectin and asked whether the peptide would express specific biological activity. Findings A 12-mer phage display library was screened with a GalNAc-specific lectin to identify an amino acid sequence that binds to the lectin. Phage particles that were eluted from the lectin with free GalNAc were considered to have been bound to a GalNAc-binding site. Peptides were synthesized with the selected sequence as a quadravalent structure to facilitate receptor crosslinking. Treatment of human peripheral blood mononuclear cells for 24 h with the peptide stimulated secretion of interleukin-8 (IL-8) but not of IL-1β, IL-6, IL-10, or tumor necrosis factor-α (TNF-α). The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-γ. Conclusion The data indicate that the quadravalent peptide has biological activity with a degree of specificity. These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range. PMID:19284521

  7. Galactose engineered solid lipid nanoparticles for targeted delivery of doxorubicin.

    PubMed

    Jain, Ashay; Kesharwani, Prashant; Garg, Neeraj K; Jain, Atul; Jain, Som Akshay; Jain, Amit Kumar; Nirbhavane, Pradip; Ghanghoria, Raksha; Tyagi, Rajeev Kumar; Katare, Om Prakash

    2015-10-01

    The present investigation reports the preparation, optimization, and characterization of surface engineered solid lipid nanoparticles (SLNs) encapsulated with doxorubicin (DOX). Salient features such as biocompatibility, controlled release, target competency, potential of penetration, improved physical stability, low cost and ease of scaling-up make SLNs viable alternative to liposomes for effective drug delivery. Galactosylation of SLNs instructs some gratifying characteristic, which leads to the evolution of promising delivery vehicles. The impendence of lectin receptors on different cell surfaces makes the galactosylated carriers admirable for targeted delivery of drugs to ameliorate their therapeutic index. Active participation of some lectin receptors in immune responses to antigen overlaid the application of galactosylated carriers in delivery of antigen and immunotherapy for treatment of maladies like cancer. These advantages revealed the promising potential of galactosylated carriers in each perspective of drug delivery. The developed DOX loaded galactosylated SLNs formulation was found to have particle size 239 ± 2.40 nm, PDI 0.307 ± 0.004, entrapment efficiency 72.3 ± 0.9%. Higher cellular uptake, cytotoxicity, and nuclear localization of galactosylated SLNs against A549 cells revealed higher efficiency of the formulation. In a nutshell, the galactosylation strategy with SLNs could be a promising approach in improving the delivery of DOX for cancer therapy. PMID:26142628

  8. Syntheses and insulin-like activity of phosphorylated galactose derivatives.

    PubMed

    Caro, H N; Martín-Lomas, M; Bernabé, M

    1993-02-24

    The syntheses of the poly-phosphorylated galactosides 6, 8, 10, 13, 16, and 20, isolated as sodium salts, have been performed. The non-phosphorylated disaccharide 17 and trisaccharide 21 have been prepared via glycosylation of the 2-(trimethylsilyl)ethyl galactosides 3 and 2, respectively, and subsequent complete deprotection. Preliminary insulin-like activity of the phosphorylated derivatives is reported. PMID:8458006

  9. Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and (15)NH(3).

    PubMed Central

    Venter, Henrietta; Ashcroft, Alison E; Keen, Jeffrey N; Henderson, Peter J F; Herbert, Richard B

    2002-01-01

    The molecular mass of the galactose-H(+) symport protein GalP, as its histidine-tagged derivative GalP(His)(6), has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni(2+)-nitrilotriacetate affinity purification was essential to obtain GalP(His)(6) suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)(6) protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein; the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein/peptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-(13)C]Histidine was incorporated into GalP(His)(6) in vivo, and ESI-MS analysis enabled the measurement of a high (80%) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by (15)NH(3) (93% enrichment) and [(19)F]tryptophan (83% enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques. PMID:11931651

  10. Salidroside ameliorates cognitive impairment in a d-galactose-induced rat model of Alzheimer's disease.

    PubMed

    Gao, Jin; He, He; Jiang, Wenjiao; Chang, Xiayun; Zhu, Lingpeng; Luo, Fen; Zhou, Rui; Ma, Chunhua; Yan, Tianhua

    2015-10-15

    The purpose of the present study was to investigate possible preventive effects of salidroside (sal) on a rat model of Alzheimer's disease and to explore its possible mechanism. Sub-acute aging was induced in male SD rats by subcutaneous injection of d-gal (120mg/kg) for 42 days, and the rats were treated with sal (20, 40mg/kg) or normal saline for 28 days after 14 days of d-gal injection. Morris water maze (MWM) test and step-down passive avoidance test were conducted to evaluate the cognitive function of the rats. The levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in hippocampus were assayed by enzyme-linked immunosorbent assay (ELISA) to assess the anti-inflammatory effect of sal. Further, we estimated the expression levels of thioredoxin (Trx), thioredoxin interacting protein (Txnip/vitamin D3 up-regulated protein/thioredoxin binding protein-2), Bax, Bcl-2, caspase-9 and related-proteins of nuclear factor kappa B (NF-κB) signaling pathway by western blot assay. It showed that administration of sal significantly attenuated all the d-gal-induced changes in the hippocampus, including cognitive impairment and neuroinflammation. These analytical results provides evidence that sal can improve cognitive capacity by inhibiting neuroinflammation and affecting apoptosis-related proteins in hippocampus. PMID:26192909

  11. Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

    SciTech Connect

    Flentke, G.R.; Frey, P.A. )

    1990-03-06

    UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

  12. Neuroprotective effects of purslane herb aquenous extracts against D-galactose induced neurotoxicity.

    PubMed

    Hongxing, Zhang; Nancai, Yu; Guofu, Huang; Jianbo, Shao; Yanxia, Wu; Hanju, Huang; Qian, Liu; Wei, Ma; Yandong, Yi; Hao, Huang

    2007-12-15

    In order to evaluate mechanisms of natural plant purslane herb aquenous extracts (PHAS) for neuroprotective, we assessed neuroprotective effects of PHAS at doses of 2.5, 5 and 10 mg/(kg day) on SD mice injected daily with D-gal (50 mg/(kg day)) by behavioral tests. PHAS-fed mice showed higher activity upon induction by new environmental stimuli, lower anxiety and higher novelty-seeking behavior in the open field tasks, and significantly improved learning and memory ability in step-through compared with D-gal-treated mice. We further examined the mechanisms involved in neuroprotective effects of PHAS on mouse brain. PHAS significantly increased superoxide dismutase (SOD) activity and decreased the malondialdehyde (MDA) level. Meanwhile, PHAS also could up-regulate telomere lengths and telomerase activity in PHAS-fed groups. Furthermore, we examined the expression of p21(waf1) and p53 mRNA and protein in mouse brain by western blot analysis and real-time RT-PCR. We found that p21(waf1)was down-regulated by PHAS without changing the expression of p53. The results of this study suggested that the PHAS might be a primary target of p21(waf1)and the neuroprotective effect of PHAS might be carried out through a p21(waf1)-dependent and p53-independent pathway. PMID:17764668

  13. Polymerization-Induced Self-Assembly of Galactose-Functionalized Biocompatible Diblock Copolymers for Intracellular Delivery

    PubMed Central

    2013-01-01

    Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition–fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo. PMID:23941545

  14. Polymerization-induced self-assembly of galactose-functionalized biocompatible diblock copolymers for intracellular delivery.

    PubMed

    Ladmiral, Vincent; Semsarilar, Mona; Canton, Irene; Armes, Steven P

    2013-09-11

    Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition-fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo. PMID:23941545

  15. Chromatin transitions during activation and repression of galactose-regulated genes in yeast.

    PubMed Central

    Cavalli, G; Thoma, F

    1993-01-01

    To study the fate of nucleosomes during transcription, a yeast gene 'GAL-URARIB' was constructed which is tightly regulated by the GAL1 promoter and shows in its inactive state a series of positioned nucleosomes that are sensitive for monitoring structural changes by micrococcal nuclease. Upon transcriptional activation, nucleosome positions were lost, but a residual nucleosomal repeat with an altered repeat length and no changes in psoralen accessibility measured by a band shift assay indicated that nucleosomes were present but rearranged on the transcribed gene. When chromatin was prepared 10 or 50 min after glucose repression, nucleosomes were repositioned in a large fraction of the population by a rapid process which most likely did not depend on histone synthesis or DNA replication. However, complete regeneration of the inactive structure and repeat length was observed after one cell generation (2.5 h) suggesting that in this step some missing histones were replaced. The results are consistent with a local dissociation of nucleosomes at the site of the polymerase followed by a rapid reassembly into nucleosomes behind it. The data are further supported by analysis of the chromosomal GAL1, GAL7 and GAL10 genes. Images PMID:8223470

  16. Yam (Dioscorea pseudojaponica Yamamoto) ameliorates cognition deficit and attenuates oxidative damage in senescent mice induced by D-galactose.

    PubMed

    Chiu, Chuan-Sung; Deng, Jeng-Shyan; Hsieh, Ming-Tsuen; Fan, Ming-Jen; Lee, Min-Min; Chueh, Fu-Shin; Han, Chien-Kuo; Lin, Ying-Chih; Peng, Wen-Huang

    2009-01-01

    This study attempted to access the neuroprotective effect of yam (Dioscorea pseudojaponica Yamamoto) on the senescent mice induced by D-gal. The mice in the experiments were administered orally with yam (20, 100 or 500 mg/kg for 4 weeks, from the sixth week). The learning and memory abilities of the mice in Morris water maze test and the mechanisms involved in the neuroprotective effect of yam on the mice brain tissue were investigated. The content of diosgenin in the yam was also detected by using HPLC. Mice treated with yam were found to significantly improve their learning and memory abilities in Morris water maze test compared to those treated with D-gal (200 mg/kg for 10 weeks). In addition, yam was also found to increase the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and decrease the malondialdehyde (MDA) level on the brains of D-gal treated mice. Finally, the amount of diosgenin in the yam was 5.49 mg/g extract. To sum up, these results indicate that yam had the potential to be a useful treatment for cognitive impairment in TCM. Its beneficial effect may be partly mediated via enhancing endogenous antioxidant enzymatic activities. PMID:19885949

  17. Normal coordinates analyses of disaccharides constituted by D-glucose, D-galactose and D-fructose units.

    PubMed

    Mahdad-Benzerdjeb, A; Taleb-Mokhtari, I N; Sekkal-Rahal, M

    2007-10-01

    A thorough study of the vibrational spectra of two disaccharides, melibiose and turanose, is presented in this work. The infrared and Raman spectra of these two compounds have been first recorded in the mid infrared range then in the far infrared region (for Raman only). These spectra constitute the main experimental support for the calculations of the modified Urey-Bradley-Shimanouchi force fields of these two disaccharides. Good agreements have been obtained between the observed and calculated frequencies. In a second time, spectra of O-deuterated derivatives have been recorded and used in order to confirm some of the frequency assignments and to test the validity of the obtained force fields. PMID:17350882

  18. Gene regulation of UDP-galactose synthesis and transport: Potential rate limiting processes in initiation of milk production in humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lactose synthesis is believed to be rate-limiting for milk production. However, understanding the molecular events controlling lactose synthesis in humans is still rudimentary. We have utilized our established model of the RNA isolated from breast milk fat globule from 7 healthy exclusively breastfe...

  19. alpha-D-Glucuronosyl-(1-->3)-L-galactose, an unusual disaccharide from polysaccharides of the hornwort Anthoceros caucasicus.

    PubMed

    Popper, Zoë A; Sadler, Ian H; Fry, Stephen C

    2003-09-01

    Acid hydrolysis of cell wall-rich material from thalli of the hornwort Anthoceros caucasicus yielded substantial amounts of an unusual disaccharide (1). Hydrolysis of 1 yielded only GlcA, Gal and unhydrolysed 1. Compound 1 was identified as alpha-D-GlcpA-(1-->3)-L-Gal by 1H and 13C NMR spectroscopic analysis and by the susceptibility of its monosaccharide units to phosphorylation by enantiomer-specific kinases. Compound 1 was not detected in acid hydrolysates of other land plants including mosses, leafy and thalloid liverworts, lycopodiophytes and euphyllophytes; it was also absent from charophytes. The Anthoceros polysaccharide that yields 1 was partially extractable in cold aqueous buffer (pH 4.7) and Na(2)CO(3), but not in EDTA or NaOH, suggesting that it was not a typical pectin or hemicellulose. The yield of 1 from various polysaccharide fractions correlated with the yields of Xyl, suggesting a previously unreported polymer containing D-GlcA, L-Gal and Xyl. The existence of a unique polysaccharide in an evolutionarily isolated plant (Anthoceros) supports the view that major steps in plant phylogeny were accompanied by significant changes in cell wall composition. PMID:12946430

  20. Differential Effects of Estrogen Exposure on Arylsulfatase B, Galactose-6-Sulfatase, and Steroid Sulfatase in Rat Prostate Development

    PubMed Central

    Feferman, Leo; Bhattacharyya, Sumit; Birch, Lynn; Prins, Gail S.; Tobacman, Joanne K.

    2014-01-01

    Sulfatase enzymes remove sulfate groups from sulfated steroid hormones, including estrone-sulfate and dehydroepiandrosterone-sulfate, and from sulfated glycosaminoglycans (GAGs), including chondroitin sulfates and heparan sulfate. The enzymes N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) and N-acetylgalactosamine-6-sulfatase (GALNS), which remove sulfate groups from the sulfated GAGs chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate, respectively, have not been studied in prostate development previously. In this report, the endogenous variation and the impact of exogenous estradiol benzoate on the immunohistochemistry and activity of ARSB and GALNS in post-natal (days 1–30) ventral rat prostate are presented, as well as measurements of steroid sulfatase activity (STS), C4S, total sulfated GAGs, and versican, an extracellular matrix proteoglycan with chondroitin sulfate attachments on days 5 and 30. Findings demonstrate distinct and reciprocal localization of ARSB and GALNS, with ARSB predominant in the stroma and GALNS predominant in the epithelium. Control ARSB activity increased significantly between days 5 and 30, but following estrogen exposure (estradiol benzoate 25 µg in 25 µl sesame oil subcutaneously on days 1, 3, and 5), activity was reduced and the observed increase on day 30 was inhibited. However, estrogen treatment did not inhibit the increase in GALNS activity between days 5 and 30, and reduced STS activity by 50% on both days 5 and 30 compared to vehicle control. Sulfated GAGs, C4S, and the extracellular matrix proteoglycan versican declined between days 5 and 30 in the control, but these declines were inhibited following estrogen. Study findings indicate distinct variation in expression and activity of sulfatases, sulfated GAGs, C4S, and versican in the process of normal prostate development, and disruption of these events by exogenous estrogen. PMID:24508597

  1. Synthesis and evaluation of novel galactose-carbocyanine fluorescent contrast agents with enhanced hydrophilicity and rigid molecular constraint

    NASA Astrophysics Data System (ADS)

    Zhang, Zongren; Bloch, Sharon; Achilefu, Samuel I.

    2004-06-01

    A new carbocyanine optical molecular probe with enhanced water solubility and constrained structural conformations was designed and synthesized. The near infrared (NIR) fluorescent probe contains a nonionic D-galactopyranose, which could improve water solubility of the probe and enhance uptake in tumors mediated by glucose transporter. The possibility of multiple attachment points provides the potential to conjugate diverse bioactive molecules to the probe. We developed an efficient synthetic method that is optimized for large-scale synthesis. Preliminary in vivo biodistribution studies show that the probe is rapidly cleared from blood and localize in the liver as early as 5 minutes post-injection of the probe in nude mice. Additional studies to evaluate the tumor uptake of the probe and its bioactive peptide conjugates are in progress.

  2. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, but the acyl-galactose acyl composition varies with the plant species and applied stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Head group acylation of monogalactosyldiacylglycerol is a plant lipid modification occurring during bacterial infection. Little is known about the range of stresses that induce this lipid modification, the molecular species induced, and the function of the modification. Lipidomic analysis using trip...

  3. Crystal structure of MytiLec, a galactose-binding lectin from the mussel Mytilus galloprovincialis with cytotoxicity against certain cancer cell types

    PubMed Central

    Terada, Daiki; Kawai, Fumihiro; Noguchi, Hiroki; Unzai, Satoru; Hasan, Imtiaj; Fujii, Yuki; Park, Sam-Yong; Ozeki, Yasuhiro; Tame, Jeremy R. H.

    2016-01-01

    MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Galα(1,4)Galβ(1,4)Glcα1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Galα(1,6)Glc, and the trisaccharide globotriose, Galα(1,4)Galβ(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt’s lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the β-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding, and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects. PMID:27321048

  4. Click chemistry oligomerisation of azido-alkyne-functionalised galactose accesses triazole-linked linear oligomers and macrocycles that inhibit Trypanosoma cruzi macrophage invasion

    PubMed Central

    Campo, Vanessa L.; Ivanova, Irina M.; Carvalho, Ivone; Lopes, Carla D.; Carneiro, Zumira A.; Saalbach, Gerhard; Schenkman, Sergio; da Silva, João Santana; Nepogodiev, Sergey A.; Field, Robert A.

    2015-01-01

    Reaction of 2-(2-(2-azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-β-d-galactopyranoside (7) under CuAAC conditions gives rise to mixed cyclic and linear triazole-linked oligomers, with individual compounds up to d.p. 5 isolable, along with mixed larger oligomers. The linear compounds resolve en bloc from the cyclic materials by RP HPLC, but are separable by gel permeation chromatography. The triazole-linked oligomers—pseudo-galactooligomers—were demonstrated to be acceptor substrates for the multi-copy cell surface trans-sialidase of the human parasite Trypanosoma cruzi. In addition, these multivalent TcTS ligands were able to block macrophage invasion by T. cruzi. PMID:26435551

  5. Crystal structure of MytiLec, a galactose-binding lectin from the mussel Mytilus galloprovincialis with cytotoxicity against certain cancer cell types.

    PubMed

    Terada, Daiki; Kawai, Fumihiro; Noguchi, Hiroki; Unzai, Satoru; Hasan, Imtiaj; Fujii, Yuki; Park, Sam-Yong; Ozeki, Yasuhiro; Tame, Jeremy R H

    2016-01-01

    MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Galα(1,4)Galβ(1,4)Glcα1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Galα(1,6)Glc, and the trisaccharide globotriose, Galα(1,4)Galβ(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt's lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the β-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding, and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects. PMID:27321048

  6. Density functional conformational study of 2-O-sulfated 3,6 anhydro-α-D-galactose and of neo-κ- and ι-carrabiose molecules in gas phase and water.

    PubMed

    Bestaoui-Berrekhchi-Berrahma, Noreya; Derreumaux, Philippe; Sekkal-Rahal, Majda; Springborg, Michael; Sayede, Adlane; Yousfi, Noureddine; Kadoun, Abd-Ed-Daim

    2013-02-01

    We examined the conformational preferences of the 2-O-sulfated-3,6-α-D-anhydrogalactose (compound I) and two 1,3 linked disaccharides constituting-κ or ι-carrageenans using density functional and ab initio methods in gas phase and aqueous solution. Systematic modifications of two torsion angles leading to 324 and 144 starting geometries for the compound I and each disaccharide were used to generate adiabatic maps using B3LYP/6-31G(d). The lower energy conformers were then fully optimized using B3LYP, B3PW91 and MP2 with several basis sets. Overall, we discuss the impact of full relaxation on the energy and structure of the dominant conformations, present the performance comparison with previous molecular mechanics calculations if available, and determine whether our results are impacted, when polarization and diffuse functions are added to the 6-31G(d) basis set, or when the MP2 level of theory is used. PMID:23086461

  7. Brief report: a new profile of terminal N-acetyllactosamines glycans on pig red blood cells and different expression of alpha-galactose on Sika deer red blood cells and nucleated cells.

    PubMed

    Tan, Yingxia; Gong, Feng; Li, Subo; Ji, Shouping; Lu, Yanping; Gao, Hongwei; Xu, Hua; Zhang, Yangpei

    2010-05-01

    It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galbeta1-4GlcNAc-R) and alpha-Gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galbeta1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack alpha-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that alpha-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-alpha-Gal epitopes. Whereas, the expression of the alpha-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution. PMID:20422448

  8. The ortholog of human solute carrier family 35 member B1 (UDP-galactose transporter-related protein 1) is involved in maintenance of ER homeostasis and essential for larval development in Caenorhabditis elegans

    PubMed Central

    Dejima, Katsufumi; Murata, Daisuke; Mizuguchi, Souhei; Nomura, Kazuko H.; Gengyo-Ando, Keiko; Mitani, Shohei; Kamiyama, Shin; Nishihara, Shoko; Nomura, Kazuya

    2009-01-01

    Although the solute carrier 35B1 (SLC35B1) is evolutionarily conserved, its functions in metazoans remain unknown. To elucidate its function, we examined developmental roles of an SLC35B1 family gene (HUT-1: homolog of UDP-Gal transporter) in Caenorhabditis elegans. We isolated a deletion mutant of the gene and characterized phenotypes of the mutant and hut-1 RNAi-treated worms. GFP-HUT-1 reporter analysis was performed to examine gene expression patterns. We also tested whether several nucleotide sugar transporters can compensate for hut-1 deficiency. The hut-1 deletion mutant and RNAi worms showed larval growth defect and lethality with disrupted intestinal morphology. Inactivation of hut-1 induced chronic endoplasmic reticulum (ER) stress, and hut-1 showed genetic interactions with the atf-6, pek-1, and ire-1 genes involved in unfolded protein response signaling. ER ultrastructure and ER marker distribution in hut-1-deficient animals showed that HUT-1 is required for maintenance of ER structure. Reporter analysis revealed that HUT-1 is an ER protein ubiquitously expressed in tissues, including the intestine. Lethality and the ER stress phenotype of the mutant were rescued with the human hut-1 ortholog UGTrel1. These results indicate important roles for hut-1 in development and maintenance of ER homeostasis in C. elegans. PMID:19270184

  9. Genetics Home Reference: galactosemia

    MedlinePlus

    ... mutations in a particular gene and affect different enzymes involved in breaking down galactose. Classic galactosemia , also ... cause galactosemia . These genes provide instructions for making enzymes that are essential for processing galactose obtained from ...

  10. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...-galactoside galactohydrase (CAS Reg. No. CBS 683), which converts lactose to glucose and galactose. It is... in § 170.3(o)(9) of this chapter to convert lactose to glucose and galactose. (2) The ingredient...

  11. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...-galactoside galactohydrase (CAS Reg. No. CBS 683), which converts lactose to glucose and galactose. It is... in § 170.3(o)(9) of this chapter to convert lactose to glucose and galactose. (2) The ingredient...

  12. Galactosemia

    MedlinePlus

    ... other foods that contain galactose, for life. Read product labels to make sure you or your child with the condition are not eating foods that contain galactose. Infants can ... hydrolysate formula) Calcium supplements are recommended.

  13. Deviations from Michaelis-Menten kinetics. The possibility of complicated curves for simple kinetic schemes and the computer fitting of experimental data for acetylcholinesterase, acid phosphatase, adenosine deaminase, arylsulphatase, benzylamine oxidase, chymotrypsin, fumarase, galactose dehydrogenase, beta-galactosidase, lactate dehydrogenase, peroxidase and xanthine oxidase.

    PubMed Central

    Bardsley, W G; Leff, P; Kavanagh, J; Waight, R D

    1980-01-01

    The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:6821369

  14. Galactokinase activity in Streptococcus thermophilus

    SciTech Connect

    Hutkins, R.; Morris, H.A.; McKay, L.L.

    1985-10-01

    ATP-dependent phosphorylation of (/sup 14/C)galactose by 11 strains of streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal/sup +/) strains than in galactose-nonfermenting (Gal/sup -/) strains. Lactose-grown, Gal/sup -/ cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal/sup +/ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted, growth on galactose commenced, and galK increased from 0.025 to 0.22 ..mu..mol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal/sup +/ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal/sup -/ strains.

  15. Cancer therapy improvement with mesoporous silica nanoparticles combining targeting, drug delivery and PDT.

    PubMed

    Gary-Bobo, Magali; Hocine, Ouahiba; Brevet, David; Maynadier, Marie; Raehm, Laurence; Richeter, Sébastien; Charasson, Virginie; Loock, Bernard; Morère, Alain; Maillard, Philippe; Garcia, Marcel; Durand, Jean-Olivier

    2012-02-28

    The synthesis of mesoporous silica nanoparticles (MSN) covalently encapsulating fluoresceine or a photosensitizer, functionalized with galactose on the surface is described. Confocal microscopy experiments demonstrated that the uptake of galactose-functionalized MSN by colorectal cancer cells was mediated by galactose receptors leading to the accumulation of the nanoparticles in the endosomal and lysosomal compartments. The MSN functionalized with a photosensitizer and galactose were loaded with the anti-cancer drug camptothecin. Those MSN combining drug delivery and photodynamic therapy were tested on three cancer cell lines and showed a dramatic enhancement of cancer cell death compared to separate treatments. PMID:22178618

  16. The structural characterization of the O-polysaccharide antigen in the lipopolysaccharide of Escherichia coli serotype O118 and its relationship to the O-antigens of Salmonella enterica O47 and Escherichia coli serotype O151

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mild acid hydrolysis of the lipopolysaccharide produced by Escherichia coli O118:H16 (standard reference strain; NRCC 6613) contained an O-polysaccharide (O-PS) composed of D-galactose, 2-acetimidoylamino-2,6-dideoxy-L-galactose (L-FucNAm), 2-acetamido-2-deoxy-D-glucose, ribitol, and phosphate (1:1...

  17. 21 CFR 184.1387 - Lactase enzyme preparation from Candida pseudotropicalis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., which are incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are....2.1.23), which converts lactose to glucose and galactose. It is prepared from yeast that has been... defined in § 170.3(o)(9) of this chapter, to convert lactose to glucose and galactose. (2) The...

  18. 21 CFR 184.1387 - Lactase enzyme preparation from Candida pseudotropicalis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., which are incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are....2.1.23), which converts lactose to glucose and galactose. It is prepared from yeast that has been... defined in § 170.3(o)(9) of this chapter, to convert lactose to glucose and galactose. (2) The...

  19. 75 FR 8920 - Grant of Authority for Subzone Status; Danisco USA, Inc., Sweeteners Division (Xylitol, Xylose...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-26

    ... (74 FR 6856-6857, 2/11/2009) and the application has been processed pursuant to the FTZ Act and the... (Xylitol, Xylose, Galactose and Mannose); Thomson, IL Pursuant to its authority under the Foreign-Trade... Board for authority to establish a special-purpose subzone at the xylitol, xylose, galactose and...

  20. The separation of chondroitin sulfate disaccharides and hyaluronan oligosaccharides by capillary zone electrophoresis.

    PubMed

    Carney, S L; Osborne, D J

    1991-05-15

    We have developed techniques for the separation of unsulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo- hex-4-enopyranosyluronicacid)-D-galactose and -D-glucose), monosulfated (2-acetamido-2-deoxy-3- O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-D-galactose and 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex- 4-enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose),disulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4- enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex-4-enopy- ranosyluronic acid)-4,6-di-O-sulfo-D-galactose), and trisulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O- sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-4,6-di-O-sulfo-D-galactose) isomers of chondroitin using capillary zone electrophoresis. In addition, it is possible to separate oligomers of hyaluronan by similar protocols. These techniques represent a rapid, sensitive, and reproducible technique for the assay of these molecules from digests of connective tissues. PMID:1909507

  1. Acute and chronic exposure to ethanol and the electrophysiology of the brush border membrane of rat small intestine.

    PubMed Central

    al-Balool, F; Debnam, E S; Mazzanti, R

    1989-01-01

    In this study we have investigated the effects of (a) chronic ethanol intake on glucose and galactose absorption across the rat jejunum in vivo and on the potential difference across the isolated brush border membrane (Vm) and (b) acute exposure to ethanol (4% or 8%) and acetaldehyde (0.25%) on changes in Vm associated with Na(+)-dependent galactose absorption across the jejunum and ileum. Chronic ethanol intake was associated with hyperpolarization of Vm and an enhanced galactose but not glucose transport. Acute ethanol and acetaldehyde were without effect on Vm whether or not galactose was present. We conclude that while a greater electrochemical gradient across the brush border membrane is a likely explanation for the stimulation of galactose absorption induced by ethanol feeding, factors other than changes in Vm are responsible for the inhibitory effects of acute ethanol. PMID:2612984

  2. Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity☆

    PubMed Central

    Dott, William; Mistry, Pratibha; Wright, Jayne; Cain, Kelvin; Herbert, Karl E

    2014-01-01

    Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR) was significantly increased whereas extracellular acidification rate (ECAR), a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2•– level compared to cells in the glucose model. An antimycin A (AA) dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a galactose media model

  3. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  4. Advanced glycation endproduct changes to Bruch's membrane promotes lipoprotein retention by lipoprotein lipase.

    PubMed

    Cano, Marisol; Fijalkowski, Natalia; Kondo, Naoshi; Dike, Sonny; Handa, James

    2011-08-01

    Lipoprotein particles accumulate in Bruch's membrane before the development of basal deposits and drusen, two histopathologic lesions that define age-related macular degeneration (AMD). We therefore, sought to determine which molecules could participate in lipoprotein retention. Wild-type or lipoprotein lipase-deficient mice were injected with low-dose D-galactose or PBS subcutaneously for 8 weeks to induce advanced glycation endproduct (AGE) formation. Some mice were also injected with the AGE breaker phenacylphiazolium bromide and D-galactose. Rhodamine-labeled low-density lipoproteins were injected into mice, and the fluorescence was measured up to 72 hours later. AGEs, proteoglycans, and other lipid-retaining molecules were evaluated by IHC. Lipoprotein lipase distribution was assessed in AMD samples by IHC. D-galactose-treated mice retained lipoproteins in the retinal pigment epithelial and Bruch's membrane to a greater extent than either PBS- or phenacylphiazolium bromide/D-galactose-treated mice at 24 and 72 hours after injection (P ≤ 0.04). Immunolabeling for carboxymethyllysine, biglycan, and lipoprotein lipase was found in D-galactose-treated mice only. Mice deficient for lipoprotein lipase treated with D-galactose did not retain lipoproteins to any measureable extent. Human AMD samples had lipoprotein lipase labeling within drusen, basal deposits, and the choroid. Mice treated with D-galactose to induce AGE formation in Bruch's membrane retain intravenously injected lipoproteins. Our results suggest that lipoprotein retention in Bruch's membrane is mediated by lipoprotein lipase. PMID:21801873

  5. Polygenic evolution of a sugar specialization trade-off in yeast.

    PubMed

    Roop, Jeremy I; Chang, Kyu Chul; Brem, Rachel B

    2016-02-18

    The evolution of novel traits can involve many mutations scattered throughout the genome. Detecting and validating such a suite of alleles, particularly if they arose long ago, remains a key challenge in evolutionary genetics. Here we dissect an evolutionary trade-off of unprecedented genetic complexity between long-diverged species. When cultured in 1% glucose medium supplemented with galactose, Saccharomyces cerevisiae, but not S. bayanus or other Saccharomyces species, delayed commitment to galactose metabolism until glucose was exhausted. Promoters of seven galactose (GAL) metabolic genes from S. cerevisiae, when introduced together into S. bayanus, largely recapitulated the delay phenotype in 1% glucose-galactose medium, and most had partial effects when tested in isolation. Variation in GAL coding regions also contributed to the delay when tested individually in 1% glucose-galactose medium. When combined, S. cerevisiae GAL coding regions gave rise to profound growth defects in the S. bayanus background. In medium containing 2.5% glucose supplemented with galactose, wild-type S. cerevisiae repressed GAL gene expression and had a robust growth advantage relative to S. bayanus; transgenesis of S. cerevisiae GAL promoter alleles or GAL coding regions was sufficient for partial reconstruction of these phenotypes. S. cerevisiae GAL genes thus encode a regulatory program of slow induction and avid repression, and a fitness detriment during the glucose-galactose transition but a benefit when glucose is in excess. Together, these results make clear that genetic mapping of complex phenotypes is within reach, even in deeply diverged species. PMID:26863195

  6. Epimerase-Deficiency Galactosemia Is Not a Binary Condition

    PubMed Central

    Openo, Kimberly K.; Schulz, Jenny M.; Vargas, Claudia A.; Orton, Corey S.; Epstein, Michael P.; Schnur, Rhonda E.; Scaglia, Fernando; Berry, Gerard T.; Gottesman, Gary S.; Ficicioglu, Can; Slonim, Alfred E.; Schroer, Richard J.; Yu, Chunli; Rangel, Vanessa E.; Keenan, Jennifer; Lamance, Kerri; Fridovich-Keil, Judith L.

    2006-01-01

    Epimerase-deficiency galactosemia results from the impairment of UDP-galactose 4′-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign “peripheral” condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe “generalized” form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a “nonperipheral” tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%–64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the

  7. Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis

    PubMed Central

    2013-01-01

    Background L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this “L” enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. Results Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L-1 of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. Conclusions This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of “L” isomers precursors in the culture medium. We have engineered K. lactis

  8. Saccharomyces cerevisiae mutants resistant to catabolite repression: use in cheese whey hydrolysate fermentation

    SciTech Connect

    Bailey, R.B.; Benitez, T.; Woodward, A.

    1982-09-01

    Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant. (Refs. 23).

  9. 1-/sup 11/C-D-glucose and related compounds

    SciTech Connect

    Shiue, C.Y.; Wolf, A.P.

    1982-01-26

    The novel compounds 1-/sup 11/C-D-glucose, 1-/sup 11/C-D-mannose, 1-/sup 11/C-D-galactose, 2-/sup 11/C-D-glucose, 2-/sup 11/C-D-mannose and 2-/sup 11/C-D-galactose which can be used in nuclear medicine to monitor the metabolism of glucose and galactose can be rapidly prepared by reaction of the appropriate aldose substrate with an alkali metal /sup 11/C-labeled cyanide followed by reduction with a Raney alloy in formic acid.

  10. Developmental Defects in a Caenorhabditis elegans Model for Type III Galactosemia

    PubMed Central

    Brokate-Llanos, Ana M.; Monje, José M.; Murdoch, Piedad del Socorro; Muñoz, Manuel J.

    2014-01-01

    Type III galactosemia is a metabolic disorder caused by reduced activity of UDP-galactose-4-epimerase, which participates in galactose metabolism and the generation of various UDP-sugar species. We characterized gale-1 in Caenorhabditis elegans and found that a complete loss-of-function mutation is lethal, as has been hypothesized for humans, whereas a nonlethal partial loss-of-function allele causes a variety of developmental abnormalities, likely resulting from the impairment of the glycosylation process. We also observed that gale-1 mutants are hypersensitive to galactose as well as to infections. Interestingly, we found interactions between gale-1 and the unfolded protein response. PMID:25298520

  11. 1-.sup.11 C-D-Glucose and related compounds

    DOEpatents

    Shiue, Chyng-Yann; Wolf, Alfred P.

    1984-03-27

    The novel compounds 1-.sup.11 C-D-glucose, 1-.sup.11 C-D-mannose, 1-.sup.11 C-D-galactose, 2-.sup.11 C-D-glucose, 2-.sup.11 C-D-mannose and 2-.sup.11 C-D-galactose which can be used in nuclear medicine to monitor the metabolism of glucose and galactose can be rapidly prepared by reaction of the appropriate aldose substrate with an alkali metal .sup.11 C-labeled cyanide followed by reduction with a Raney alloy in formic acid.

  12. Galactosaemia: an unusual cause of chronic bilirubin encephalopathy.

    PubMed

    Sahoo, Tanushree; Thukral, Anu; Agarwal, Ramesh; Sankar, Mari Jeeva

    2015-01-01

    Galactosaemia is a disorder of galactose metabolism in which raised levels of galactose and galactose-1-phosphate damage various organs. Although galactosaemia is a common metabolic liver disease in childhood, it is a rare cause of neonatal hyperbilirubinemia requiring intervention. We report an unusual case of neonatal galactosaemia that at presentation had features of acute bilirubin encephalopathy requiring exchange transfusion and at discharge had features of chronic bilirubin encephalopathy. This case report emphasises the need for timely suspicion and diagnosis of this disease for prevention of chronic morbidity. PMID:25618877

  13. Developmental defects in a Caenorhabditis elegans model for type III galactosemia.

    PubMed

    Brokate-Llanos, Ana M; Monje, José M; Murdoch, Piedad Del Socorro; Muñoz, Manuel J

    2014-12-01

    Type III galactosemia is a metabolic disorder caused by reduced activity of UDP-galactose-4-epimerase, which participates in galactose metabolism and the generation of various UDP-sugar species. We characterized gale-1 in Caenorhabditis elegans and found that a complete loss-of-function mutation is lethal, as has been hypothesized for humans, whereas a nonlethal partial loss-of-function allele causes a variety of developmental abnormalities, likely resulting from the impairment of the glycosylation process. We also observed that gale-1 mutants are hypersensitive to galactose as well as to infections. Interestingly, we found interactions between gale-1 and the unfolded protein response. PMID:25298520

  14. Galactosemia

    MedlinePlus

    ... Blood culture for bacterial infection ( E coli sepsis ) Enzyme activity in the red blood cells Ketones in the urine Prenatal diagnosis by directly measuring the enzyme galactose-1-phosphate uridyl transferase "Reducing substances" in ...

  15. A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80.

    PubMed Central

    Parthun, M R; Jaehning, J A

    1992-01-01

    The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation. Images PMID:1406674

  16. 21 CFR 172.620 - Carrageenan.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... Gigartina stellata. (b) The food additive conforms to the following conditions: (1) It is a sulfated polysaccharide the dominant hexose units of which are galactose and anhydrogalactose. (2) Range of...

  17. 21 CFR 172.655 - Furcelleran.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (red seaweed). (b) The food additive conforms to the following: (1) It is a sulfated polysaccharide the dominant hexose units of which are galactose and anhydrogalactose. (2) Range of sulfate content: 8...

  18. 21 CFR 172.655 - Furcelleran.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... (red seaweed). (b) The food additive conforms to the following: (1) It is a sulfated polysaccharide the dominant hexose units of which are galactose and anhydrogalactose. (2) Range of sulfate content: 8...

  19. Galactosemia (image)

    MedlinePlus

    ... galactosemia is unable to use (metabolize) the simple sugar galactose, which reaches high levels in the body, causing damage to the liver, central nervous system and various other body systems. An infant with ...

  20. Systemic gene dysregulation in classical Galactosaemia: Is there a central mechanism?

    PubMed

    Coss, K P; Treacy, E P; Cotter, E J; Knerr, I; Murray, D W; Shin, Y S; Doran, P P

    2014-11-01

    Classical Galactosaemia is a rare disorder of carbohydrate metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT). The disease is life-threatening in the neonate, and the only treatment option is life-long dietary restriction of galactose. However, long-term complications persist in treated patients including cognitive impairments, speech and language abnormalities and premature ovarian insufficiency in females. Microarray analysis of T-lymphocytes from treated adult patients identified systemic dysregulation of numerous gene pathways, including the glycosylation, inflammatory and inositol pathways. Analysis of gene expression in patient-derived dermal fibroblasts of patients exposed to toxic levels of galactose, with immunostaining, has further identified the susceptibility of the glycosylation gene alpha-1,2-mannosyltransferase (ALG9) and the inflammatory gene annexin A1 (ANXA1) to increased galactose concentrations. These data suggest that Galactosaemia is a multi-system disorder affecting numerous signalling pathways. PMID:25174965

  1. L-Ascorbate biosynthesis in higher plants: the role of VTC2

    PubMed Central

    Linster, Carole L.; Clarke, Steven G.

    2008-01-01

    In the past year, the last missing enzyme of the L-galactose pathway, the linear form of which appears to represent the major biosynthetic route to L-ascorbate (vitamin C) in higher plants, has been identified as a GDP-L-galactose phosphorylase. This enzyme catalyzes the first committed step in the synthesis of that vital antioxidant and enzyme cofactor. Here, we discuss how GDP-L-galactose phosphorylase enzymes, encoded in Arabidopsis by the paralogous VTC2 and VTC5 genes, function in concert with the other enzymes of the L-galactose pathway to provide plants the appropriate levels of L-ascorbate. We hypothesize that regulation of L-ascorbate biosynthesis might occur at more than one step and warrants further investigation to allow for the manipulation of vitamin C levels in plants. PMID:18824398

  2. Fifty-six-year-old man with anaphylaxis: A novel delayed food hypersensitivity reaction.

    PubMed

    Wagner, Kendall D; Bell, Matthew C; Pesek, Robert D; Kennedy, Joshua L

    2015-11-01

    Anaphylaxis and urticaria are commonly seen in both primary care and allergy clinics. Foods, drugs, and insects are frequent culprits for immediate reactions; however, the trigger for recurring and/or chronic episodes is often unclear. We present a 56-year-old male with recurrent symptoms of urticaria, angioedema, and anaphylaxis found to be triggered by sensitization to galactose-alpha 1, 3-galactose (alpha-gal), a novel food allergen. PMID:26625604

  3. Electrocatalytic O2 Reduction at a Bio-inspired Mononuclear Copper Phenolato Complex Immobilized on a Carbon Nanotube Electrode.

    PubMed

    Gentil, Solène; Serre, Doti; Philouze, Christian; Holzinger, Michael; Thomas, Fabrice; Le Goff, Alan

    2016-02-12

    An original copper-phenolate complex, mimicking the active center of galactose oxidase, featuring a pyrene group was synthesized. Supramolecular pi-stacking allows its efficient and soft immobilization at the surface of a Multi-Walled Carbon Nanotube (MWCNT) electrode. This MWCNT-supported galactose oxidase model exhibits a 4 H(+)/4 e(-) electrocatalytic activity towards oxygen reduction at a redox potential of 0.60 V vs. RHE at pH 5. PMID:26748615

  4. Verbal dyspraxia and galactosemia.

    PubMed

    Webb, Amy Leigh; Singh, Rani H; Kennedy, Mary Jane; Elsas, Louis J

    2003-03-01

    Classical galactosemia is an autosomal recessive disorder resulting from deficient galactose-1-phosphateuridyl transferase (GALT) activity. Verbal dyspraxia is an unusual outcome in galactosemia. Here we validated a simplified breath test of total body galactose oxidation against genotype and evaluated five potential biochemical risk indicators for verbal dyspraxia in galactosemia: cumulative percentage dose (CUMPCD) of (13)CO(2) in breath, mean erythrocyte galactose-1-phosphate, highest erythrocyte galactose-1-phosphate, mean urinary galactitol, and erythrocyte GALT activity. Thirteen controls and 42 patients with galactosemia took a (13)C-galactose bolus, and the (CUMPCD) of (13)CO(2) in expired air was determined. Patients with <5% CUMPCD had mutant alleles that severely impaired human GALT enzyme catalysis. Patients with > or =5% CUMPCD had milder mutant human GALT alleles. Twenty-four patients consented to formal speech evaluation; 15 (63%) had verbal dyspraxia. Dyspraxic patients had significantly lower CUMPCD values (2.84 +/- 5.76% versus 11.51 +/- 7.67%; p < 0.008) and significantly higher mean erythrocyte galactose-1-phosphate (3.38 +/- 0.922 mg/dL versus 1.92 +/- 1.28 mg/dL; p = 0.019) and mean urinary galactitol concentrations (192.4 +/- 75.8 mmol/mol creatinine versus 122.0 +/- 56.4; p = 0.048) than patients with normal speech. CUMPCD values <5%, mean erythrocyte galactose-1-phosphate levels >2.7 mg/dL, and mean urinary galactitol levels >135 mmol/mol creatinine were associated with dyspraxic outcome with odds ratios of 21, 13, and 5, respectively. We conclude that total body oxidation of galactose to CO(2) in expired air reflects genotype and that this breath test is a sensitive predictor of verbal dyspraxia in patients with galactosemia. PMID:12595586

  5. Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii.

    PubMed

    Wong, T Y

    1990-07-01

    Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme. PMID:2167631

  6. Conversion of red-algae Gracilaria verrucosa to sugars, levulinic acid and 5-hydroxymethylfurfural.

    PubMed

    Jeong, Gwi-Taek; Ra, Chae Hun; Hong, Yong-Ki; Kim, Joong Kyun; Kong, In-Soo; Kim, Sung-Koo; Park, Don-Hee

    2015-02-01

    This study employed a statistical methodology to investigate the optimization of conversion conditions and evaluate the reciprocal interaction of reaction factors related to the process of red-algae Gracilaria verrucosa conversion to sugars (glucose, galactose), levulinic acid and 5-hydroxymethylfurfural (5-HMF) by acidic hydrolysis. Overall, the conditions optimized for glucose formation included a higher catalyst concentration than did those for galactose, and these conditions for galactose were similar to those for 5-HMF. Levulinic acid production, meanwhile, was optimized at a higher reaction temperature, a higher catalyst concentration, and a longer reaction time than was glucose, galactose or 5-HMF production. By this approach, the optimal yields (and reaction conditions) for glucose, galactose, levulinic acid, and 5-HMF were as follows: glucose 5.29 g/L (8.46 wt%) (reaction temperature 160 °C, catalyst concentration 1.92%, reaction time 20 min), galactose 18.38 g/L (29.4 wt%) (160 °C, 1.03%, 20 min), levulinic acid 14.65 g/L (18.64 wt%) (180.9 °C, 2.85%, 50 min), and 5-HMF 3.74 g/L (5.98 wt%) (160.5 °C, 1%, 20 min). PMID:25042893

  7. Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His∼P) and HPr(Ser-P)(His∼P) and Effects on Growth

    PubMed Central

    Lessard, Christian; Cochu, Armelle; Lemay, Jean-Dominique; Roy, Denis; Vaillancourt, Katy; Frenette, Michel; Moineau, Sylvain; Vadeboncoeur, Christian

    2003-01-01

    The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His∼P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIALacS) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIALacS was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His∼P) but also by HPr(Ser-P)(His∼P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIABLMan and IIABHMan, were unable to phosphorylate IIALacS. The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His∼P) or HPr(Ser-P)(His∼P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose. PMID:14617640

  8. Polygenic evolution of a sugar specialization tradeoff in yeast

    PubMed Central

    Roop, Jeremy I.; Chang, Kyu Chul; Brem, Rachel B.

    2016-01-01

    The evolution of novel traits can involve many mutations scattered throughout the genome1,2. Detecting and validating such a suite of alleles, particularly if they arose long ago, remains a key challenge in evolutionary genetics1-3. Here we dissect an evolutionary tradeoff of unprecedented genetic complexity between long-diverged species. When cultured in 1% glucose medium supplemented with galactose, Saccharomyces cerevisiae, but not S. bayanus or other Saccharomyces species, delayed commitment to galactose metabolism until glucose was exhausted. Promoters of seven galactose (GAL) metabolic genes from S. cerevisiae, when introduced together into S. bayanus, largely recapitulated the delay phenotype in 1% glucose-galactose medium, and most had partial effects when tested in isolation. Variation in GAL coding regions also contributed to the delay when tested individually in 1% glucose-galactose medium. When combined, S. cerevisiae GAL coding regions gave rise to profound growth defects in the S. bayanus background. In medium containing 2.5% glucose supplemented with galactose, wild-type S. cerevisiae repressed GAL gene expression and had a robust growth advantage relative to S. bayanus; transgenesis of S. cerevisiae GAL promoter alleles or GAL coding regions was sufficient for partial reconstruction of these phenotypes. S. cerevisiae GAL genes thus encode a regulatory program of slow induction and avid repression, and a fitness detriment during the glucose-galactose transition but a benefit when glucose is in excess. Together, these results make clear that genetic mapping of complex phenotypes is within reach, even in deeply diverged species. PMID:26863195

  9. Mechanisms of microbial movement in subsurface materials.

    PubMed Central

    Reynolds, P J; Sharma, P; Jenneman, G E; McInerney, M J

    1989-01-01

    The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2552920

  10. Manganese-Based Superoxide Dismutase Mimics Modify Both Acute and Long-Term Outcome Severity in a Drosophila melanogaster Model of Classic Galactosemia

    PubMed Central

    Jumbo-Lucioni, Patricia P.; Ryan, Emily L.; Hopson, Marquise L.; Bishop, Heather M.; Weitner, Tin; Tovmasyan, Artak; Spasojevic, Ivan; Batinic-Haberle, Ines; Liang, Yongliang; Jones, Dean P.

    2014-01-01

    Abstract Aims: The goal of this study was to use two manganese (Mn)-based superoxide dismutase (SOD) mimics to test the hypothesis that reactive oxygen species contribute to both acute and long-term outcomes in a galactose-1P uridylyltransferase (GALT)-null Drosophila melanogaster model of classic galactosemia. Results: We tested the impact of each of two Mn porphyrin SOD mimics, MnTnBuOE-2-PyP5+, and MnTE-2-PyP5+, (i) on survival of GALT-null Drosophila larvae reared in the presence versus absence of dietary galactose and (ii) on the severity of a long-term movement defect in GALT-null adult flies. Both SOD mimics conferred a significant survival benefit to GALT-null larvae exposed to galactose but not to controls or to GALT-null larvae reared in the absence of galactose. One mimic, MnTE-2-PyP5+, also largely rescued a galactose-independent long-term movement defect otherwise seen in adult GALT-null flies. The survival benefit of both SOD mimics occurred despite continued accumulation of elevated galactose-1P in the treated animals, and studies of thiolated proteins demonstrated that in both the presence and absence of dietary galactose MnTE-2-PyP5+ largely prevented the elevated protein oxidative damage otherwise seen in GALT-null animals relative to controls. Innovation and Conclusions: Our results confirm oxidative stress as a mediator of acute galactose sensitivity in GALT-null Drosophila larvae and demonstrate for the first time that oxidative stress may also contribute to galactose-independent adult outcomes in GALT deficiency. Finally, our results demonstrate for the first time that both MnTnBuOE-2-PyP5+ and MnTE-2-PyP5+ are bioavailable and effective when administered through an oral route in a D. melanogaster model of classic galactosemia. Antioxid. Redox Signal. 20, 2361–2371. PMID:23758052

  11. Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

    PubMed Central

    Adhikari, Hema; Cullen, Paul J.

    2014-01-01

    Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways. PMID:25356552

  12. Biosynthesis of Glycosyldiglycerides in Mycobacterium smegmatis

    PubMed Central

    Schultz, John C.; Elbein, Alan D.

    1974-01-01

    A particulate enzyme preparation from Mycobacterium smegmatis catalyzes the transfer of [14C]galactose from uridine 5′-diphosphate (UDP)-[14C]galactose and of [14C]glucose from UDP-[14C]glucose into chloroform-soluble products. The radioactive neutral lipids were purified by passage through diethylaminoethyl-cellulose, followed by thin-layer chromatography. When UDP-glucose was used as substrate, two major radioactive lipids were obtained; one had a hexose-glucose-glycerol ratio of 1:1:1. The second product had a hexose-glycerol ratio of 2:1 and, in addition to glucose, contained lesser amounts of mannose and galactose. With UDP-galactose as substrate, two radioactive products were observed that were chromatographically indistinguishable from the [14C]glucosyl-labeled mono- and diglycosyldiglyceride. Palmitate and oleate were the predominant fatty acid constituents in these lipids and were present in equimolar amounts in all of the products examined. The products have thus been identified as monoglycosyldiglyceride and a diglycosyldiglyceride containing glucose as the major hexose along with mannose and galactose. Properties of the galactosyl and glucosyl transferases are described. Images PMID:4808895

  13. Unimportance of Counterflux in the Energetics of “Downhill” Transport

    PubMed Central

    Koch, Arthur L.

    1974-01-01

    Adam Kepes suggested that the cellular transport and hydrolysis of orthonitrophenyl-β-d-galactopyranoside is powered by the counterflux of the d-galactose resulting from β-galactosidase action within the cell. His explanation would rationalize the unique insensitivity of this galactoside transport to energy poisons such as azide. But contrary to the predictions of this hypothesis, (i) there is no initial large inhibition that progressively lessens as galactose is produced. This was shown with a double wavelength stopped-flow spectrophotometer developed to eliminate interference from turbidity transients. (ii) The azide sensitivity does not increase with an external concentration of galactose sufficient to reverse the thermodynamic gradient. (iii) Mutation in galactose utilization or growth on highly catabolite-repressing regimens did not increase the azide sensitivity, and induction of galactose transport and metabolism did not decrease azide sensitivity. It was found that Kepes measurements must have contained two artifacts. One is that the control rate of hydrolysis decreases with time as the dense cell suspension becomes anaerobic. The other is that azide causes turbidity changes for some time after its introduction. If the former is avoided by magnetic stirring and the latter by double wavelength spectrophotometry or controls without substrate, the inhibition is constant from the earliest time that can be measured. It is therefore concluded that energy-unstarved cells, exposed to azide, still have adequate energy reserves to couple to the downhill transport, although their potential is not adequate to drive accumulation against a concentration gradient. PMID:4616952

  14. Evolutionary engineering of Saccharomyces cerevisiae for efficient conversion of red algal biosugars to bioethanol.

    PubMed

    Lee, Hye-Jin; Kim, Soo-Jung; Yoon, Jeong-Jun; Kim, Kyoung Heon; Seo, Jin-Ho; Park, Yong-Cheol

    2015-09-01

    The aim of this work was to apply the evolutionary engineering to construct a mutant Saccharomyces cerevisiae HJ7-14 resistant on 2-deoxy-D-glucose and with an enhanced ability of bioethanol production from galactose, a mono-sugar in red algae. In batch and repeated-batch fermentations, HJ7-14 metabolized 5-fold more galactose and produced ethanol 2.1-fold faster than the parental D452-2 strain. Transcriptional analysis of genes involved in the galactose metabolism revealed that moderate relief from the glucose-mediated repression of the transcription of the GAL genes might enable HJ7-14 to metabolize galactose rapidly. HJ7-14 produced 7.4 g/L ethanol from hydrolysates of the red alga Gelidium amansii within 12 h, which was 1.5-times faster than that observed with D452-2. We demonstrate conclusively that evolutionary engineering is a promising tool to manipulate the complex galactose metabolism in S. cerevisiae to produce bioethanol from red alga. PMID:25804535

  15. The size, shape and specificity of the sugar-binding site of the jacalin-related lectins is profoundly affected by the proteolytic cleavage of the subunits.

    PubMed Central

    Houlès Astoul, Corinne; Peumans, Willy J; van Damme, Els J M; Barre, Annick; Bourne, Yves; Rougé, Pierre

    2002-01-01

    Mannose-specific lectins with high sequence similarity to jacalin and the Maclura pomifera agglutinin have been isolated from species belonging to the families Moraceae, Convolvulaceae, Brassicaceae, Asteraceae, Poaceae and Musaceae. Although these novel mannose-specific lectins are undoubtedly related to the galactose-specific Moraceae lectins there are several important differences. Apart from the obvious differences in specificity, the mannose- and galactose-specific jacalin-related lectins differ in what concerns their biosynthesis and processing, intracellular location and degree of oligomerization of the protomers. Taking into consideration that the mannose-specific lectins are widely distributed in higher plants, whereas their galactose-specific counterparts are confined to a subgroup of the Moraceae sp. one can reasonably assume that the galactose-specific Moraceae lectins are a small-side group of the main family. The major change that took place in the structure of the binding site of the diverging Moraceae lectins concerns a proteolytic cleavage close to the N-terminus of the protomer. To corroborate the impact of this change, the specificity of jacalin was re-investigated using surface plasmon resonance analysis. This approach revealed that in addition to galactose and N -acetylgalactosamine, the carbohydrate-binding specificity of jacalin extends to mannose, glucose, N -acetylmuramic acid and N -acetylneuraminic acid. Owing to this broad carbohydrate-binding specificity, jacalin is capable of recognizing complex glycans from plant pathogens or predators. PMID:12169094

  16. Biosynthesis of T1 Antigen in Salmonella: Origin of d-Galactofuranose and d-Ribofuranose Residues

    PubMed Central

    Sarvas, Matti; Nikaido, Hiroshi

    1971-01-01

    The “T1 side chain” portion of cell wall lipopolysaccharide from T1 strains of Salmonella contains d-galactofuranose and d-ribofuranose residues. Isotope labeling studies, using intact cells of mutants each blocked at either of the two different steps of d-galactose metabolism (uridine diphosphate-glucose 4-epimerase and galactose-1-P uridylyl transferase) or at phosphoglucoisomerase, led to the following conclusions. (i) d-Galactofuranose residues are synthesized from d-galactopyranose or its derivatives, rather than by a direct conversion from other hexopyranoses or their derivatives. (ii) The pyranose-to-furanose conversion does not appear to take place at the level of the free d-galactose or d-galactose 1-phosphate. This result suggests that the conversion may occur at the stage of uridine diphosphate-d-galactose. (iii) In a mutant lacking phosphoglucoisomerase, d-ribofuranose residues in T1 side chains contained 14C derived from exogenous d-fructose-U-14C, but little 3H from exogenous d-glucose-1-3H. Thus, no evidence was found for a direct pathway of aldohexose-to-ribose conversion involving a loss of one of the carbons in the C2-C6 moiety of aldohexoses. This suggests, but does not prove, that the T1 ribofuranose residues are synthesized by conventional mechanisms involving hexose monophosphate shunt and transketolase-transaldolase reactions. PMID:4926677

  17. Inactivation of Metabolic Genes Causes Short- and Long-Range dys-Regulation in Escherichia coli Metabolic Network

    PubMed Central

    Barupal, Dinesh Kumar; Lee, Sang Jun; Karoly, Edward D.; Adhya, Sankar

    2013-01-01

    The metabolic network in E. coli can be severely affected by the inactivation of metabolic genes that are required to catabolize a nutrient (D-galactose). We hypothesized that the resulting accumulation of small molecules can yield local as well as systemic effects on the metabolic network. Analysis of metabolomics data in wild-type and D-galactose non-utilizing mutants, galT, galU and galE, reveal the large metabolic differences between the wild-type and the mutants when the strains were grown in D-galactose. Network mapping suggested that the enzymatic defects affected the metabolic modules located both at short- and long-ranges from the D-galactose metabolic module. These modules suggested alterations in glutathione, energy, nucleotide and lipid metabolism and disturbed carbon to nitrogen ratio in mutant strains. The altered modules are required for normal cell growth for the wild-type strain, explaining why the cell growth is inhibited in the mutants in the presence of D-galactose. Identification of these distance-based dys-regulations would enhance the systems level understanding of metabolic networks of microorganisms having importance in biomedical and biotechnological research. PMID:24363806

  18. Exopolysaccharide production by nitrogen-fixing bacteria within nodules of Medicago plants exposed to chronic radiation in the Chernobyl exclusion zone.

    PubMed

    Pawlicki-Jullian, Nathalie; Courtois, Bernard; Pillon, Michelle; Lesur, David; Le Flèche-Mateos, Anne; Laberche, Jean-Claude; Goncharova, Nadia; Courtois, Josiane

    2010-03-01

    Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family (Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family (Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 x 10(6) and 3 x 10(6) Da. Five different compositions of EPS were characterized by physico-chemical analyses and (1)H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs. PMID:20080178

  19. Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

    PubMed Central

    Isemura, M.; Schmid, K.

    1971-01-01

    Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma α1-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1→4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2→2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl→N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas–liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins. PMID:5135244

  20. Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency.

    PubMed

    Staubach, Simon; Pekmez, Murat; Hanisch, Franz-Georg

    2016-06-01

    Classical galactosemia is caused by a nearly complete deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism with galactose-1-phosphate and galactitol accumulation. Even on a galactose-restricted diet, patients develop serious long-term complications of the central nervous system and ovaries that may result from chronic cell-toxic effects exerted by endogenous galactose. To address the question of whether disease-associated cellular perturbations could affect the kidney function of the patients, we performed differential proteomics of detergent-resistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analyzed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group, the most striking finding was a drastic increase of abundant serum (glyco)proteins, like albumin, leucine-rich α-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 d-isomerase, and α-1-microglobulin protein (AMBP), pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in a heavy overload of exosomal membranes with adsorbed serum (glyco)proteins. Several of these proteins are connected to TBMN and IgAN, proteinuria, and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes. PMID:27103203

  1. Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

    SciTech Connect

    Michalska, Karolina; Cuff, Marianne E.; Tesar, Christine; Feldmann, Brian; Joachimiak, Andrzej

    2011-08-01

    The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.

  2. Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40

    PubMed Central

    Lee, Saeyoung; Lee, Jonas Yun; Ha, Sung Chul; Jung, Jina; Shin, Dong Hae; Kim, Kyoung Heon; Choi, In-Geol

    2009-01-01

    Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl-(1→3)-d-galactose] using various β-agarases. Neoagarobiose hydrolase is an enzyme that acts on the α-­1,3 linkage in neoagarobiose to yield d-galactose and 3,6-anhydro-l-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 Å, β = 101.86°. The crystals diffracted to 1.98 Å resolution and possibly contains two molecules in the asymmetric unit. PMID:20054134

  3. Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme.

    PubMed

    Cong, Qifei; Chen, Huanjun; Liao, Wenfeng; Xiao, Fei; Wang, Peipei; Qin, Yi; Dong, Qun; Ding, Kan

    2016-01-20

    A fucoidan FP08S2 was isolated from the boiling-water extract of Sargassum fusiforme, purified by CaCl2 precipitation and chromatography on DEAE-cellulose and Sephacryl S-300. FP08S2 contained fucose, xylose, galactose, mannose, glucuronic acid, and 20.8% sulfate. The sulfate groups were attached to diverse positions of fucose, xylose, mannose, and galactose residues. The backbone of FP08S2 consisted of alternate 1,2-linked α-D-Manp and 1,4-linked β-D-GlcpA. Sugar composition analysis and ESI-MS revealed that the oligosaccharides from branches contained fucose, xylose, galactose, glucuronic acid and sulfate. FP08S2 could significantly inhibit tube formation and migration of human microvascular endothelial cells (HMEC-1) dose-dependently. These results suggested that the fucoidan FP08S2 from brown seaweeds S. fusiforme could be a potent anti-angiogenic agent. PMID:26572427

  4. Fucose content of keratan sulphates from bovine articular cartilage.

    PubMed Central

    Tai, G H; Brown, G M; Morris, H G; Huckerby, T N; Nieduszynski, I A

    1991-01-01

    Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals). Nine keratan sulphate fractions of increasing molecular weight were prepared by gel-permeation chromatography on a calibrated column of TSK 30 XL. The samples were analysed for fucose and galactose contents (% by wt. of keratan sulphate) and fucose/galactose ratio. The fucose content increased with molecular size, but the galactose content remained constant. It was concluded that the alpha(1----3)-linked fucose [Thornton, Morris, Cockin, Huckerby, Nieduszynski, Carlstedt, Hardingham & Ratcliffe (1989) Biochem. J. 260, 277-282] was located within the poly-N-acetyl-lactosamine repeat sequence of articular-cartilage keratan sulphate. PMID:1991030

  5. Influence of Grape Maturity on Complex Carbohydrate Composition of Red Sparkling Wines.

    PubMed

    Martínez-Lapuente, Leticia; Apolinar-Valiente, Rafael; Guadalupe, Zenaida; Ayestarán, Belén; Pérez-Magariño, Silvia; Williams, Pascale; Doco, Thierry

    2016-06-22

    This paper studied how grape maturity affected complex carbohydrate composition during red sparkling wine making and wine aging. Grape ripening stage (premature and mature grapes) showed a significant impact on the content, composition, and evolution of polysaccharides and oligosaccharides of sparkling wines. Polysaccharides rich in arabinose and galactose, mannoproteins, rhamnogalacturonans II, and oligosaccharides in base wines increased with maturity. For both maturity stages, polysaccharides rich in arabinose and galactose, and the glucuronic acid glycosyl residue of the oligosaccharides were the major carbohydrates detected in all vinification stages. The total glycosyl content of oligosaccharides decreased during the whole period of aging on yeast lees. The reduction of polysaccharides rich in arabinose and galactose and rhamnogalacturonans type II during the aging was more pronounced in mature samples. To our knowledge, this is the first report of the polysaccharide and oligosaccharide composition of red sparkling wines. PMID:27226011

  6. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes.

    PubMed

    Solomon, B; Koppel, R; Schwartz, F; Fleminger, G

    1990-06-27

    Site-specific modification of monoclonal antibodies was achieved by oxidation of the carbohydrate moieties of antibodies which are located remote from the antigen binding sites. Sialic acid and galactose are terminal sugars of these carbohydrate chains. Concomitant treatment of the antibodies with neuraminidase and galactose oxidase generated aldehyde groups in the oligosaccharide moieties of immunoglobulins which reacted selectively with amino or hydrazide groups of the matrix. Subsequent immobilization of neuraminidase and galactose oxidase on Eupergit C-adipic dihydrazide proved to be an efficient and selective system for the enzymic oxidation of the monoclonal antibodies without impairing their immunological activity. Oriented immobilization of enzymically oxidized monoclonal antibodies on hydrazide or amino Eupergit C derivatives thus leads to the formation of antibody matrix conjugates which possess high antigen-binding activities. PMID:2119387

  7. Acid-catalyzed conversion of mono- and poly-sugars into platform chemicals: effects of molecular structure of sugar substrate.

    PubMed

    Hu, Xun; Wu, Liping; Wang, Yi; Song, Yao; Mourant, Daniel; Gunawan, Richard; Gholizadeh, Mortaza; Li, Chun-Zhu

    2013-04-01

    Hydrolysis/pyrolysis of lignocellulosic biomass always produces a mixture of sugars with distinct structures as intermediates or products. This study tried to elucidate the effects of molecular structure of sugars on their acid-catalyzed conversions in ethanol/water. Location of carbonyl group in sugars (fructose versus glucose) and steric configuration of hydroxyl groups (glucose versus galactose) significantly affected yields of levulinic acid/ester (fructose>glucose>galactose). The dehydration of fructose to 5-(hydroxymethyl)furfural produces much less soluble polymer than that from glucose and galactose, which results in high yields of levulinic acid/ester from fructose. Anhydrate sugar such as levoglucosan tends to undergo the undesirable decomposition to form less levulinic acid/ester. Catalytic behaviors of the poly-sugars (sucrose, maltose, raffinose, β-cyclodextrins) were determined much by their basic units. However, their big molecular sizes create the steric hindrance that significantly affects their followed conversion over solid acid catalyst. PMID:23454803

  8. Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

    PubMed Central

    Tanaka, Naotaka; Awai, Atsuro; Bhuiyan, M. Shah Alam; Fujita, Kiyotaka; Fukui, Hiroshi; Takegawa, Kaoru

    1999-01-01

    We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe. PMID:9973368

  9. Characterization of cellulose and other exopolysaccharides produced from Gluconacetobacter strains.

    PubMed

    Fang, Lin; Catchmark, Jeffrey M

    2015-01-22

    This study characterized the cellulosic and non-cellulosic exopolysaccharides (EPS) produced by four Gluconacetobacter strains. The yields of bacterial cellulose and water-soluble polysaccharides were dependent on both carbon source and Gluconacetobacter strain. The carbon substrate also affected the composition of the free EPS. When galactose served as an exclusive carbon source, Gluconacetobacter xylinus (G. xylinus) ATCC 53524 and ATCC 700178 produced a distinct alkaline stable crystalline product, which influenced the crystallization of cellulose. Gluconacetobacter hansenii (G. hansenii) ATCC 23769 and ATCC 53582, however, did not exhibit any significant change in cellulose crystal properties when galactose was used as the carbon source. Microscopic observation further confirmed significant incorporation of EPS into the cellulose composites. The cellulosic network produced from galactose medium showed distinctive morphological and structural features compared to that from glucose medium. PMID:25439946

  10. Mediated amperometry reveals different modes of yeast responses to sugars.

    PubMed

    Garjonyte, Rasa; Melvydas, Vytautas; Malinauskas, Albertas

    2016-02-01

    Menadione-mediated amperometry at carbon paste electrodes modified with various yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Pichia guilliermondii and Debaryomyces hansenii) was employed to monitor redox activity inside the yeast cells induced by glucose, fructose, sucrose, maltose or galactose. Continuous measurements revealed distinct modes (transient or gradually increasing) of the current development during the first 2 to 3 min after subjection to glucose, fructose and sucrose at electrodes containing S. cerevisiae and non-Saccharomyces strains. Different modes (increasing or decreasing) of the current development after yeast subjection to galactose at electrodes with S. cerevisiae or D. hansenii and at electrodes with C. pulcherrima and P. guilliermondii suggested different mechanisms of galactose assimilation. PMID:26523505

  11. A Lectin with Highly Potent Inhibitory Activity toward Breast Cancer Cells from Edible Tubers of Dioscorea opposita cv. Nagaimo

    PubMed Central

    Chan, Yau Sang; Ng, Tzi Bun

    2013-01-01

    A 70-kDa galactose-specific lectin was purified from the tubers of Dioscorea opposita cv. nagaimo. The purification involved three chromatographic steps: anion exchange chromatography on a Q-Sepharose column, FPLC-anion exchange chromatography on a Mono Q column, and FPLC-gel filtration on a Superdex 75 column. The purified nagaimo lectin presented as a single 35-kDa band in reducing SDS-PAGE while it exhibited a 70-kDa single band in non-reducing SDS-PAGE suggesting its dimeric nature. Nagaimo lectin displayed moderate thermostability, retaining full hemagglutinating activity after heating up to 62°C for 30 minutes. It also manifested stability over a wide pH range from pH 2 to 13. Nagaimo lectin was a galactose-specific lectin, as evidenced by binding with galactose and galactose-containing sugars such as lactose and raffinose. The minimum concentration of galactose, lactose and raffinose required to exert an inhibitory effect on hemagglutinating activity of nagaimo lectin was 20 mM, 5 mM and 40 mM, respectively. Nagaimo lectin inhibited the growth of some cancer cell lines including breast cancer MCF7 cells, hepatoma HepG2 cells and nasopharyngeal carcinoma CNE2 cells, with IC50 values of 3.71 µM, 7.12 µM and 19.79 µM, respectively, after 24 hour treatment with nagaimo lectin. The induction of phosphatidylserine externalization and mitochondrial depolarization indicated that nagaimo lectin evoked apoptosis in MCF7 cells. However, the anti-proliferative activity of nagaimo lectin was not blocked by application of galactose, signifying that the activity was not related to the carbohydrate binding specificity of the lectin. PMID:23349827

  12. Evidence for carrier-mediated uptake and efflux of sugars at the serosal side of the rat intestinal mucosa in vitro.

    PubMed Central

    Bronk, J R; Ingham, P A

    1976-01-01

    A modification of the everted sac technique is described which allows several sacs to be prepared rapidly and simultaneously from the same segment of rat intestine. 2. A method has been developed for comparing the transport of two sugars by measuring changes in the ratios of their concentrations as they pass across the intestinal wall. 3. With this method significant differences were observed between the D-[3H]galactose and L-[14C]glucose ratios in the mucosal epithelium, the serosal tissue and the serosal compartment. These results indicate that both the efflux of galactose from the serosal side of the mucosal epithelium and the uptake of the sugar into the mucosa are carrier-mediated processes. 4. The mediated efflux of galactose at the serosal side of the epithelial layer is inhibited by the presence of phlorizin on the mucosal side and to some extent by any reduction in the mucosal Na+ concentration. Both of these treatments inhibited galactose uptake at the brush border. Serosal efflux of the sugar appeared to be saturated at high concentrations of D-galactose. 5. Pre-treatment of the sacs with mercuric chloride considerably reduced D-galactose uptake from the luminal side, but did not affect its efflux relative to L-glucose at the serosal side of the mucosal epithelium. 6. Carrier-mediated sugar uptake into the mucosal epithelium from the serosal side was also examined. The role of the bidirectional, carrier-mediated sugar transport processes at the serosal pole of the mucosal epithelial cell in transintestinal transport is discussed. PMID:1255529

  13. Ethanol fermentation of a mixture of pentoses and hexoess by Pichia stipitis

    SciTech Connect

    Chung, Bong-Hwan; Ryu, Yeon-Woo; Seo, Jin-Ho

    1995-12-01

    Hydrolysis of cellulosic materials generates a mixture of hexoses (mannose, glucose, and galactose) and pentoses (xylose and arabinose). The research was carried out to study the pattern of carbon utilization by P. stipitis CBS5776 for ethanol production from cellulose hydrolyzates. The yeast utilized glucose and mannose first, and then galactose and xylose as carbon sources. Arabinose was not used at all, P. stipitis produced ethanol with a yield coefficient 0.33 g ethanol/g sugar in a mixture of sugars. The pattern for carbon utilization was also investigated in a continuous culture.

  14. A non-ionic water-soluble seed gum from Ipomoea campanulata.

    PubMed

    Singh, V; Pandey, M; Srivastava, A; Sethi, R

    2003-02-01

    A non-ionic water-soluble galactomannan, having galactose and mannose in 2:3 molar ratio was isolated from endosperm of the seeds of Ipomoea campanulata. The seed gum was found to have linear chain of beta (1-->4) linked mannopyranosyl units with D-galactose side chains attached through alpha (1-->6) linkage to the main chain. This structure is similar to many commercial gums like Guar, Carob and Locust bean gum. Various physical properties of the gum were studied in order to explore the possibility of commercial exploitation of the seed gum. PMID:12628393

  15. Synthesis of Galacto-oligosaccharide in Two-phase System.

    PubMed

    Gui, Li-Qiong; Wei, Dong-Zhi; Cui, Yu-Min; Yu, Jun-Tang

    1999-01-01

    35 of the total products of galacto-oligosaccharide (GOS) could be obtained from the two-phase system with cyclohexane and ethyl acetate as bulk organic phases and 15% phosphate buffer as aqueous phase. The effects of temperature pH of buffer lactose concentration galactose and glucose and the immobilization of enzyme on the synthesis of GOS were studied. It was found that the reaction temperature and initial lactose concentration didn'thave obvious effects while the addition of glucose and galactose somewhat affected the GOS yield and the GOS yields could reach 64.78% with lactase immobilized on resin D345. PMID:12136210

  16. Chemical Synthesis of the Tumor-Associated Globo H Antigen†

    PubMed Central

    Mandal, Satadru S.; Liao, Guochao; Guo, Zhongwu

    2015-01-01

    A derivative of the tumor-associated globo H antigen, a complex hexasaccharide, was synthesized by a convergent and efficient [3+2+1] strategy using various glycosylation methods. All glycosylation reactions afforded good to excellent yields and outstanding stereoselectivity, including the installation of cis α-linked D-galactose and L-fucose. The longest linear sequence for this synthesis was 11 steps from a galactose derivative 11 to give an overall yield of 2.6%. The synthetic target had a free and reactive amino group at the glycan reducing end, facilitating its conjugation with other molecules for various applications. PMID:26257889

  17. Seed mucilage from Ipomoea dasysperma.

    PubMed

    Singh, V; Pandey, M; Srivastava, A; Srivastava, V

    2007-04-01

    A non-ionic water-soluble galactomannan, having galactose and mannose in 1:6 molar ratio, was isolated from endosperm of the seeds of Ipomoea dasysperma. The seed mucilage was found to have a structure having a linear chain of beta (1-->4) linked mannopyranosyl units with D-galactose side chains attached through alpha (1-->6) linkage to the main chain. I. dasysperma seed gum possesses non-ionic characteristics of commercial seed gums and has potential to be used in food and pharmaceutical industries. PMID:17346904

  18. Expeditive synthesis of trithiotriazine-cored glycoclusters and inhibition of Pseudomonas aeruginosa biofilm formation

    PubMed Central

    Smadhi, Meriem; Gingras, Marc; Abderrahim, Raoudha

    2014-01-01

    Summary Readily accessible, low-valency glycoclusters based on a triazine core bearing D-galactose and L-fucose epitopes are able to inhibit biofilm formation by Pseudomonas aeruginosa. These multivalent ligands are simple to synthesize, are highly soluble, and can be either homofunctional or heterofunctional. The galactose-decorated cluster shows good affinity for Pseudomonas aeruginosa lectin lecA. They are convenient biological probes for investigating the roles of lecA and lecB in biofilm formation. PMID:25246957

  19. A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

    PubMed Central

    2012-01-01

    Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the

  20. Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group

    PubMed Central

    Arimitsu, Hideyuki; Sasaki, Keiko; Kojima, Hiroe; Yanaka, Tadashi; Tsuji, Takao

    2013-01-01

    Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease. PMID:24340102

  1. [IgE-induced anaphylactic reaction after eating lamb meat].

    PubMed

    Johansen, Kasper Lorenz; Rasmussen, Kirsten

    2014-08-25

    This article presents the case of a 54-year-old male, who was admitted to hospital after suffering a severe anaphylactic reaction after tasting a spoonful of chili con carne and four months later after eating lamb meat. Galactose-alpha-1,3-galactose allergy was suspected and the subsequent blood test showed highly elevated specific IgE levels. Delayed allergy to meat is a relatively new type of allergy and the delayed onset of allergic symptoms poses a diagnostic challenge to the physician. This type of allergy should be considered when there has been no obvious allergen exposition prior to the allergic reaction. PMID:25293709

  2. Purification and partial characterization of a mitogenic lectin from the latex of Euphorbia marginata.

    PubMed

    Stirpe, F; Licastro, F; Morini, M C; Parente, A; Savino, G; Abbondanza, A; Bolognesi, A; Falasca, A I; Rossi, C A

    1993-08-20

    A lectin was purified from the latex of Euphorbia marginata by affinity chromatography on acid-treated Sepharose 6B and elution with lactose. The lectin is a glycoprotein composed of two identical subunits with M(r) 30,000, approx. The haemagglutinating activity of the lectin is not specific for any human blood group, and is inhibited by galactose and galactose-containing sugars and by gentiobiose. The lectin is strongly mitogenic for human T-lymphocytes and induces the release of interleukin-1 beta and tumor necrosis factor-alpha from cultured mononuclear cells. PMID:8353129

  3. Effect of lectins on mouse peritoneal macrophage phagocytic activity.

    PubMed

    Maldonado, G; Porras, F; Fernández, L; Vázquez, L; Zenteno, E

    1994-11-01

    We studied the in vitro ability of lectin-treated murine peritoneal macrophages to attach and phagocytize particulate antigens. Glucose and mannose specific lectins such as Con-A and lentil lectin, as well as complex lactosamine residues specific lectins, such as Phaseolus vulgaris var. cacahuate and Phaseolus coccineus var. alubia, increased the macrophage phagocytic activity towards heterologous erythrocytes, whereas peanut agglutinin, a galactose-specific lectin, diminished the macrophage phagocytic activity. These results suggest that a galactose-N-acetyl-D galactosamine-containing structure could participate as negative modulator of the phagocytic activity. PMID:7851961

  4. Detection of Agar, by Analysis of Sugar Markers, Associated with Bacillus Anthracis Spores, After Culture

    SciTech Connect

    Wunschel, David S.; Colburn, Heather A.; Fox, Alvin; Fox, Karen F.; Harley, William M.; Wahl, Jon H.; Wahl, Karen L.

    2008-08-01

    Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-L-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artefactual background (reductive hydrolysis) or marker destruction (hydrolysis) lead to the search for alternative sugar markers. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.

  5. Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

    PubMed Central

    Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P.; Abramson, Jeff; Quick, Matthias; Shi, Lei

    2015-01-01

    The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

  6. Identification and characterization of DcUCGalT1, a galactosyltransferase responsible for anthocyanin galactosylation in purple carrot (Daucus carota L.) taproots

    PubMed Central

    Xu, Zhi-Sheng; Ma, Jing; Wang, Feng; Ma, Hong-Yu; Wang, Qiu-Xia; Xiong, Ai-Sheng

    2016-01-01

    Purple carrots (Daucus carota ssp. sativus var. atrorubens Alef.) accumulate large amounts of cyanidin-based anthocyanins in their taproots. Cyanidin can be glycosylated with galactose, xylose, and glucose in sequence by glycosyltransferases resulting in cyanidin 3-xylosyl (glucosyl) galactosides in purple carrots. The first step in the glycosylation of cyanidin is catalysis by UDP-galactose: cyanidin galactosyltransferase (UCGalT) transferring the galactosyl moiety from UDP-galactose to cyanidin. In the present study, a gene from ‘Deep purple’ carrot, DcUCGalT1, was cloned and heterologously expressed in E. coli BL21 (DE3). The recombinant DcUCGalT1 galactosylated cyanidin to produce cyanidin-3-O-galactoside and showed optimal activity for cyanidin at 30 °C and pH 8.6. It showed lower galactosylation activity for peonidin, pelargonidin, kaempferol and quercetin. It accepted only UDP-galactose as a glycosyl donor when cyanidin was used as an aglycone. The expression level of DcUCGalT1 was positively correlated with anthocyanin biosynthesis in carrots. The enzyme extractions from ‘Deep purple’ exhibited galactosylation activity for cyanidin, peonidin and pelargonidin, while those from ‘Kuroda’ (a non-purple cultivar) did not. PMID:27264613

  7. Genome sequence of Lactococcus garvieae IPLA 31405, a bacteriocin-producing, tetracycline-resistant strain isolated from a raw-milk cheese.

    PubMed

    Flórez, Ana Belén; Reimundo, Pilar; Delgado, Susana; Fernández, Elena; Alegría, Angel; Guijarro, José A; Mayo, Baltasar

    2012-09-01

    This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded. PMID:22933752

  8. [Determination of human serum galactosyltransferase using a kinetic spectrophotometric technic].

    PubMed

    Gauduchon, P; Baumann, J J; Bar, E; Le Talaër, J Y

    1985-01-01

    A kinetic spectrophotometric method in which galactose transfer is coupled to the production of NADH, has been adapted to the assay of galactosyltransferase activity in human serum. Under the described conditions, the rate of NADH production is linear with regard to enzyme concentration, and directly depends upon the various biochemical factors which control galactosyltransferase activity. PMID:3924359

  9. Structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae

    PubMed Central

    Michalska, Karolina; Cuff, Marianne E.; Tesar, Christine; Feldmann, Brian; Joachimiak, Andrzej

    2011-01-01

    In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-­glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-­oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein. PMID:21795809

  10. Carbon nanotube/biocompatible bola-amphiphile supramolecular biohybrid materials: preparation and their application in bacterial cell agglutination.

    PubMed

    Yu, Guocan; Li, Jinying; Yu, Wei; Han, Chengyou; Mao, Zhengwei; Gao, Changyou; Huang, Feihe

    2013-11-26

    Supramolecular biohybrid materials were successfully constructed driven by non-covalent interactions between three biocompatible bolaform amphiphiles and single walled carbon nanotubes (SWNTs). The existence of galactoses in these supramolecular systems endowed the hybrid materials with interesting bio-function. By introducing the SWNTs as semi-flexible platforms, these supramolecular biohybrid materials display excellent agglutination ability for E. coli. PMID:23996208

  11. Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide

    PubMed Central

    Wohlschlager, Therese; Buser, Reto; Skowyra, Michael L; Haynes, Brian C; Henrissat, Bernard; Doering, Tamara L; Künzler, Markus; Aebi, Markus

    2013-01-01

    The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom. PMID:23926231

  12. Genome Sequence of Lactococcus garvieae IPLA 31405, a Bacteriocin-Producing, Tetracycline-Resistant Strain Isolated from a Raw-Milk Cheese

    PubMed Central

    Flórez, Ana Belén; Reimundo, Pilar; Delgado, Susana; Fernández, Elena; Alegría, Ángel; Guijarro, José A.

    2012-01-01

    This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded. PMID:22933752

  13. Construction of ploidy series of Saccharomyces cerevisiae by the plasmid YCplac33-GHK.

    PubMed

    Hou, Lihua; Li, Xiaoyang; Wang, Cong; Cao, Xiaohong; Wang, Haiyong

    2013-04-01

    An effective approach, using the plasmid YCplac33-GHK, is developed to construct a ploidy series of Saccharomyces cerevisiae. YCplac33-GHK harbors the HO gene under the control of galactose-inducible promoter and KanMX4 as the selective marker. The simple method can solve the problem of industrial applications of strains with resistance genes. PMID:23430413

  14. HPAEC Coupled to an Evaporative Light Scattering Detector for Separation, Detection and Quantification of Galacturonic Acid Oligomers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A model homogalacturonan (HG); composed of galacturonic acid (94-97 %) and galactose (3-6 %), having a 94 % degree of esterification (DE) was used to produce a demethylated HG series by reacting it with the salt-independent pectin methylesterase (PME) from citrus fruit at pH 4.5 and 7.5. HGs with a...

  15. Structure elucidation, proliferation effect on macrophage and its mechanism of a new heteropolysaccharide from Lactarius deliciosus Gray.

    PubMed

    Hou, Yiling; Liu, Lu; Ding, Xiang; Zhao, Daqun; Hou, Wanru

    2016-11-01

    A new heteropolysaccharide was isolated from the fruiting bodies of Lactarius deliciosus Gray which had a molecular weight of 16kDa and was mainly composed of the galactose and glucose. Structural elucidation results indicated that Lactarius deliciosus Gray polysaccharide (LDG-B) had a backbone of (1,6)-linked d-galactose and (1, 2, 6)-linked d-galactose which branches were mainly composed of 4-linked d-glucose and 6-linked d-galactose residue. Cell cycle test results showed that LDG-B could promote the proliferation of B cells and macrophage cells by affecting G0/G1 phase, S phases and G2/M phases. The analysis of transcriptomes sequence of macrophages showed a total of 1839 genes were identified as DEGs, and approximately 708 genes were up-regulated, whereas 1131 genes were down-regulated in LDG-B group. KEGG pathway enrichment analysis showed that the MAPK, JAK-STAT and NF-κB signaling pathways are significantly enriched for DEGs in LDG-B group. Analysis of transcriptome resources enabled us to examine gene expression profiles, verify differential gene expression, and select candidate signaling pathways as the mechanisms of the immunomodulatory activity of LDG-B. PMID:27516315

  16. A Novel Agarolytic β-Galactosidase Acts on Agarooligosaccharides for Complete Hydrolysis of Agarose into Monomers

    PubMed Central

    Lee, Chan Hyoung; Kim, Hee Taek; Yun, Eun Ju; Lee, Ah Reum; Kim, Sa Rang; Kim, Jae-Han; Choi, In-Geol

    2014-01-01

    Marine red macroalgae have emerged to be renewable biomass for the production of chemicals and biofuels, because carbohydrates that form the major component of red macroalgae can be hydrolyzed into fermentable sugars. The main carbohydrate in red algae is agarose, and it is composed of d-galactose and 3,6-anhydro-l-galactose (AHG), which are alternately bonded by β1-4 and α1-3 linkages. In this study, a novel β-galactosidase that can act on agarooligosaccharides (AOSs) to release galactose was discovered in a marine bacterium (Vibrio sp. strain EJY3); the enzyme is annotated as Vibrio sp. EJY3 agarolytic β-galactosidase (VejABG). Unlike the lacZ-encoded β-galactosidase from Escherichia coli, VejABG does not hydrolyze common substrates like lactose and can act only on the galactose moiety at the nonreducing end of AOS. The optimum pH and temperature of VejABG on an agarotriose substrate were 7 and 35°C, respectively. Its catalytic efficiency with agarotriose was also similar to that with agaropentaose or agaroheptaose. Since agarotriose lingers as the unreacted residual oligomer in the currently available saccharification system using β-agarases and acid prehydrolysis, the agarotriose-hydrolyzing capability of this novel β-galactosidase offers an enormous advantage in the saccharification of agarose or agar in red macroalgae for its use as a biomass feedstock for fermentable sugar production. PMID:25038102

  17. C-phycocyanin protects against low fertility by inhibiting reactive oxygen species in aging mice

    PubMed Central

    Li, Yan-Jiao; Han, Zhe; Ge, Lei; Zhou, Cheng-Jie; Zhao, Yue-Fang; Wang, Dong-Hui; Ren, Jing; Niu, Xin-Xin; Liang, Cheng-Guang

    2016-01-01

    Women over 35 have higher rates of infertility, largely due to deterioration of oocyte quality characterized by fragmentation, abnormal meiotic spindle-chromosome complexes, and oxidative stress. C-phycocyanin (PC) is a biliprotein enriched in Spirulina platensis that is known to possess antioxidant, anti-inflammatory, and radical-scavenging properties. D-galactose-induced aging acceleration in mice has been extensively used to study aging mechanisms and for pharmaceutical screening. In this study, adult female B6D2F/1 mice injected with D-galactose were used as a model to test the age-reversing effects of PC on degenerated reproductive ability. Our results show that PC can prevent oocyte fragmentation and aneuploidy by maintaining cytoskeletal integrity. Moreover, PC can reverse the expression of antioxidant genes, increase superoxide dismutase (SOD) activity and decrease methane dicarboxylic aldehyde (MDA) content, and normalize mitochondria distribution. PC exerts its benefit by inhibiting reactive oxygen species (ROS) production, which decreases apoptosis. Finally, we observe a significant increase in litter size after PC administration to D-galactose-induced aging mice. Our study demonstrates for the first time that D-galactose-induced impaired female reproductive capability can be partially rescued by the antioxidant effects of PC. PMID:27008700

  18. Identification and characterization of DcUCGalT1, a galactosyltransferase responsible for anthocyanin galactosylation in purple carrot (Daucus carota L.) taproots.

    PubMed

    Xu, Zhi-Sheng; Ma, Jing; Wang, Feng; Ma, Hong-Yu; Wang, Qiu-Xia; Xiong, Ai-Sheng

    2016-01-01

    Purple carrots (Daucus carota ssp. sativus var. atrorubens Alef.) accumulate large amounts of cyanidin-based anthocyanins in their taproots. Cyanidin can be glycosylated with galactose, xylose, and glucose in sequence by glycosyltransferases resulting in cyanidin 3-xylosyl (glucosyl) galactosides in purple carrots. The first step in the glycosylation of cyanidin is catalysis by UDP-galactose: cyanidin galactosyltransferase (UCGalT) transferring the galactosyl moiety from UDP-galactose to cyanidin. In the present study, a gene from 'Deep purple' carrot, DcUCGalT1, was cloned and heterologously expressed in E. coli BL21 (DE3). The recombinant DcUCGalT1 galactosylated cyanidin to produce cyanidin-3-O-galactoside and showed optimal activity for cyanidin at 30 °C and pH 8.6. It showed lower galactosylation activity for peonidin, pelargonidin, kaempferol and quercetin. It accepted only UDP-galactose as a glycosyl donor when cyanidin was used as an aglycone. The expression level of DcUCGalT1 was positively correlated with anthocyanin biosynthesis in carrots. The enzyme extractions from 'Deep purple' exhibited galactosylation activity for cyanidin, peonidin and pelargonidin, while those from 'Kuroda' (a non-purple cultivar) did not. PMID:27264613

  19. Expanding Concepts in Mental Retardation; Scientific Symposium of the Joseph P. Kennedy, Jr., Foundation (3rd, Boston, Massachusetts, 1966).

    ERIC Educational Resources Information Center

    Jervis, George A., Ed.

    The genetics of mental retardation are discussed in terms of geographical isolates, prospects for prevention of trisomic conditions, population genetics, and cytogenetics of Down's syndrome; problems of neurogenesis described are anabolic pathways of galactose and glucose metabolism, abnormal cell migrations in developing brains, and genetic…

  20. The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

    PubMed Central

    Kemner, J M; Liang, X; Nester, E W

    1997-01-01

    The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. PMID:9079938

  1. Measurement of Enzyme Kinetics by Use of a Blood Glucometer: Hydrolysis of Sucrose and Lactose

    ERIC Educational Resources Information Center

    Heinzerling, Peter; Schrader, Frank; Schanze, Sascha

    2012-01-01

    An alternative analytical method for measuring the kinetic parameters of the enzymes invertase and lactase is described. Invertase hydrolyzes sucrose to glucose and fructose and lactase hydrolyzes lactose to glucose and galactose. In most enzyme kinetics studies, photometric methods or test strips are used to quantify the derivates of the…

  2. Carbohydrate Analysis Experiment Involving Mono- and Disaccharides with a Twist of Glycobiology: Two New Tests for Distinguishing Pentoses and Glycosidic Bonds

    ERIC Educational Resources Information Center

    Herdman, Chelsea; Diop, Lamine; Dickman, Michael

    2013-01-01

    Carbohydrate analysis is an excellent way to expose second-year biochemistry majors to the subtlety and nuance of sugar chemistry. In one, 3-h practical period, students must identify an unknown mono- or disaccharide from a collection of three hexoses (glucose, galactose, and mannose), two pentoses (ribose and arabinose), and three disaccharides…

  3. The Small Intestinal Epithelia of Beef Steers Differentially Express Sugar Transporter Messenger Ribonucleic Acid in Response to Abomasal Versus Ruminal Infusion of Starch Hydrolysate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In mammals, the absorption of mono¬saccharides from small intestinal lumen involves at least 3 sugar transporters (SugT): sodium-dependent glucose transporter 1 (SGLT1; gene SLC5A1) transports glucose and galactose, whereas glucose transporter (GLUT) 5 (GLUT5; gene SLC2A5) transports fructose, acros...

  4. Isolation and identification of biologically active compounds from Forsythia viridissima flowers.

    PubMed

    Tokar, Magdalena; Klimek, Barbara

    2004-01-01

    Flavonol glycosides, rutin and isoquercitrin, lignan glycosides, arctiin and matairesinoside, as well as phenylethanoid verbascoside (= acteoside), ursolic acid and beta-sitosterol have been isolated from the flowers of Forsythia viridissima. Two other isolated substances were characterized respectively as a wax and a hydrocoloidal polysaccharide consisting of galactose, galacturonic acid, arabinose, glucose, xylose, and rhamnose. PMID:15481244

  5. Modified pectin-based carrier for gene delivery: Cellular barriers in gene delivery course

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of biodegradable and biocompatible polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential liga...

  6. pH-Rate Profiles Support a General Base Mechanism for Galactokinase (Lactococcus lactis)

    PubMed Central

    Reinhardt, Laurie A.; Thoden, James B.; Peters, Greg S.; Holden, Hazel M.; Cleland, W.W.

    2013-01-01

    Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-D-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10,000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH–kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation. PMID:23872454

  7. The structure of a glycopeptide purified from porcine thyroglobulin

    PubMed Central

    Fukuda, Minoru; Egami, Fujio

    1971-01-01

    1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, α-l-fucosidase, β-d-galactosidase, β-N-acetyl-d-glucosaminidase and α-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid→galactose→N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose→N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, α-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide. PMID:4331180

  8. Investigating a Bio-Engineered Enzyme.

    ERIC Educational Resources Information Center

    Bullerwell, Lornie; And Others

    1994-01-01

    Describes science experiments with the enzyme lactose, which is available commercially as Lactaid and Dairy Ease. Experiments show how the rate of reaction of lactose converted to glucose and galactose is influenced by temperature, pH, and substrate concentration. (PR)

  9. Precursors of hexoneogenesis within the human mammary gland

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The human mammary gland is capable of de novo synthesis of glucose and galactose (hexoneogenesis); however, the carbon source is incompletely understood. In this study, we investigated the role of acetate, glutamine, lactate and glycerol as potential carbon sources for hexoneogenesis. Healthy breast...

  10. Conversion of Helminthosporium sacchari Toxin to Toxoids by beta-Galactofuranosidase from Helminthosporium.

    PubMed

    Livingston, R S; Scheffer, R P

    1983-06-01

    Helminthosporium sacchari produces a host-selective toxin and structurally related nontoxic compounds, here referred to as ;toxoids.' Toxin and the three toxoids were each isolated to a high level of purity and were hydrolyzed under acidic conditions. The released galactose was measured by a galactose oxidase/peroxidase assay. Toxin was found to contain four units of galactose per molecule, as previously reported. Toxoids I, II, and III contained one, two, and three units of galactose, respectively. In cultures of the fungus, toxin concentration peaked at 3 weeks, followed by a rapid decline; as toxin levels fell, the total amount of toxoids increased. An enzyme with beta-galactofuranosidase activity was found in small amounts in the cultures of H. sacchari; the enzyme converted toxin to the toxoids in vitro. beta-Galactofuranosidase was previously known from very few micro-organisms; therefore, several pathogenic Helminthosporia and other fungi were tested for production. beta-Galactofuranosidase activity in culture filtrates and mycelia of H. victoriae, H. maydis, H. carbonum, and H. turcicum was much greater than in filtrates and mycelium of H. sacchari. More work is needed to determine the significance of enzyme production by these fungi. No beta-galactofuranosidase was evident from Fusarium oxysporum and Cladosporium cucumerinum. PMID:16663037

  11. Structure of aldobiouronic acid and glucuronic acid from Agathis australis degraded gum polysaccharide.

    PubMed

    Singh, R B

    2007-04-01

    Agathis australis gum on acid hydrolysis with sulphuric acid yielded L-arabinose and D-galactose in 1:4 molar ratio with traces of L-fucose. The components of aldobiouronic acid and glucuronic acid were obtained by graded hydrolysis of degraded gum polysaccharide. The derivatives of aldobiouronic acid was obtained as methyl ester methyl glycoside. PMID:17915743

  12. Ethanol production from lignocellulosic biomass by recombinant Escherichia coli strain FBR5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignocellulosic biomass, upon pretreatment and enzymatic hydrolysis, generates a mixture of hexose and pentose sugars such as glucose, xylose, arabinose and galactose. Escherichia coli utilizes all these sugars well but it lacks the ability to produce ethanol from them. Recombinant ethanologenic E...

  13. Innovative Therapy for Classic Galactosemia - Tale of Two HTS

    PubMed Central

    Tang, M; Odejinmi, SI; Vankayalapati, H; Wierenga, K; Lai, K

    2011-01-01

    Classic Galactosemia is an autosomal recessive disorder caused by the deficiency of galactose-1-phosphate uridylyltransferase (GALT), one of the key enzymes in the Leloir pathway of galactose metabolism. While the neonatal morbidity and mortality of the disease are now mostly prevented by newborn screening and galactose restriction, long-term outcome for older children and adults with this disorder remains unsatisfactory. The pathophysiology of Classic Galactosemia is complex, but there is convincing evidence that galactose-1-phosphate (gal-1P) accumulation is a major, if not the sole pathogenic factor. Galactokinase (GALK) inhibition will eliminate the accumulation of gal-1P from both dietary sources and endogenous production, and efforts towards identification of therapeutic small molecule GALK inhibitors are reviewed in detail. Experimental and computational high-throughput screenings of compound libraries to identify GALK inhibitors have been conducted, and subsequent studies aimed to characterize, prioritize, as well as to optimize the identified positives have been implemented to improve the potency of promising compounds. Although none of the identified GALK inhibitors inhibit glucokinase and hexokinase, some of them cross-inhibit other related enzymes in the GHMP small molecule kinase superfamily. While this finding may render the on-going hit-to-lead process more challenging, there is growing evidence that such cross-inhibition could also lead to advances in antimicrobial and anti-cancer therapies. PMID:22018723

  14. Effects of glycowithanolides on lipid peroxidation and lipofuscinogenesis in male reproductive organs of mice

    PubMed Central

    Walvekar, Madhuri; Shaikh, Nilofar; Sarvalkar, Priti

    2013-01-01

    Background: Glycowithanolides (Withaferin A), is one of the main withanolides active principle isolated from plant Withania somnifera and is claimed that it possess the aphrodisiac, sedative, rejuvenate and life prolonging properties. Objective: In the present investigation, antioxidant activity of active principles of Withania somnifera was tested against D-galactose induced oxidative stress in mouse testes, epididymis and seminal vesicle. Materials and Methods: For the present investigation Swiss male albino mice Mus musculus (Linn) were used. They were grouped in to control (I), D-galactose treated (II), protective (III) and curative groups (IV). Oxidative stress was induced in six month old mice by injecting a low dose of D-galactose. Antioxidant effect of plant extract was studied in testes, epididymis, and seminal vesicle of oxidative stressed mice on Lipid peroxidation (LPO) and fluorescence product. Results: In the present study, both total as well as mitochondrial lipid peroxidation and fluorescence product in testes, epididymis and seminal vesicle were increased in D-galactose induced mice. After the treatment of glycowithanolides there was significantly decrease in total as well as mitochondrial lipid peroxidation and fluorescence product in protective and curative groups. Conclusion: Our results indicate that Withania somnifera has a capability of preventing oxidative stress and also combating stress induced infertility. PMID:24639810

  15. Activation of immobilized enzymes by acoustic wave resonance oscillation.

    PubMed

    Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu

    2014-12-01

    Acoustic wave resonance oscillation has been used successfully in the development of methods to activate immobilized enzyme catalysts. In this study, resonance oscillation effects were demonstrated for enzyme reactions on galactose oxidase (GAD), D-amino acid oxidase (DAAO), and L-amino acid oxidase (LAAO), all of which were immobilized covalently on a ferroelectric lead zirconate titanate (PZT) device that could generate thickness-extensional resonance oscillations (TERO) of acoustic waves. For galactose oxidation on immobilized GAD in a microreactor, TERO generation immediately increased enzyme activity 2- to 3-fold. Eliminating TERO caused a slight decrease in the activity, with ∼90% of the enhanced activity retained while the reaction proceeded. Contact of the enhanced enzyme with a galactose-free solution caused almost complete reversion of the activity to the original low level before TERO generation, indicating that, not only TERO-induced GAD activation, but also preservation of the increased activity, required a galactose substrate. Similar activity changes with TERO were observed for enzyme reactions on DAAO and LAAO. Kinetic analysis demonstrated that TERO helped strengthen the interactions of the immobilized enzyme with the reactant substrate and promoted formation of an activation complex. PMID:25442945

  16. MECHANISMS OF MICROBIAL MOVEMENT IN SUBSURFACE MATERIALS

    EPA Science Inventory

    The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster t...

  17. Characterizing the Glycocalyx of Poultry Spermatozoa: II. Low Temperature Storage of Turkey Semen and Sperm Mobility Phenotype Impact the Carbohydrate Component of Membrane Glycoconjugates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The turkey sperm glycocalyx is known to contain residues of sialic acid, alpha-mannose/alpha-glucose, alpha- and beta-galactose, alpha-fucose, alpha- and beta-N-acetyl-galactosamine, monomers and dimers of N-acetyl-glucosamine and N-acetyl-lactosamine. Potential changes in these carbohydrates during...

  18. Effects of Different Cryopreservation Methods on the Glyocalyx of Chicken Spermatozoa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The carbohydrate-rich zone on the sperm surface is essential for inmunoprotection in the female tract and early gamete interactions. We recently have shown the glycocalyx of chicken sperm to be extensively sialylated and contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine...

  19. Polysaccharide gel coating of the leaves of Brasenia schreberi lowers plasma cholesterol in hamsters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brasenia schreberi is an invasive aquatic weed in the U.S. but the plant has economic value in Asia where it is cultivated for food. The young leaves of B. schreberi are coated with gelatinous water-insoluble mucilage. This mucilage is a polysaccharide composed of galactose, mannose, fucose and ot...

  20. Gel coating of edible Brasenia schreberi leaves lowers plasma cholesterol in hamsters (abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The young leaves of B. schreberi are coated with gelatinous water-insoluble mucilage. This mucilage is a polysaccharide composed of galactose, mannose, fucose and other monosaccharides. Since some carbohydrate gels are hypocholesterolemic, we evaluated the cholesterol lowering properties in male h...

  1. Characterizing the glycocalyx of poultry spermatozoa; semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The carbohydrate-rich zone on the sperm surface is essential for inmunoprotection in the female tract and early gamete interactions. We recently have shown the glycocalyx of chicken sperm to be extensively sialylated and contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine...

  2. Gel coating of leaves of the water plant, Brasenia schreberi, lowers plasma cholesterol in hamsters on high fat diets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An edible, gelatinous water-insoluble coating surrounds the young leaves of the water plant, Brasenia schreberi. This mucilage is a polysaccharide of galactose, mannose, fucose and other monosaccharides. In order to determine if this edible gel has cholesterol lowering properties, we fed male hams...

  3. Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Yarrowia lipolytica lipase 2 gene (YlLIP2) was cloned into galactose- and fatty acid-inducible Saccharomyces cerevisiae expression vectors and used to generate yeast strains that secrete active LIP2 enzyme activity, as evidenced by results from gene expression analysis and tributyrin turbidity c...

  4. The role of gtcA in the pathogenesis of gastrointestinal listeriosis in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serotype 4b strains of Listeria monocytogenes have been responsible for most large outbreaks of listeriosis. In L. monocytogenes serotype 4b, gtcA and gltA have been implicated in serotype-specific glycosylation of the teichoic acid of the cell wall with galactose and glucose. In this study, we inve...

  5. Demethylation of a model homogalacturonan with the salt-independent pectin methylesterase from citrus: I. effect of pH on demethylated block size and distribution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A model homogalacturonan (HG); composed of galacturonic acid (94-97 %) and galactose (3-6 %), having a 94 % degree of esterification (DE) was used to produce a demethylated HG series by reacting it with the salt-independent pectin methylesterase (PME) from citrus fruit at pH 4.5 and 7.5. HGs with a...

  6. Carbohydrate composition of serum low and high density lipoproteins of nonhuman primate species.

    PubMed

    Pargaonkar, P S; Radhakrishnamurthy, B; Srinivasan, S R; Berenson, G S

    1977-01-01

    1. Carbohydrate composition of serum low and high density lipoproteins obtained from 5 nonhuman primate species (chimpanzee, patas, baboon, rhesus, and spider) and humans was studied. 2. Individual lipoproteins were isolated from pooled sera of each species by ultracentrifugal flotation between the densities 1.019-1.063 for LDL-2; 1.063-1.12 for HDL-2; and 1.12-1.21 for HDL-3. After delipidation, sialic acid, fucose, glucosamine, mannose, galactose, and glucose were determined on apo LDL-2, apo HDL-2, and apo HDL-3. 3. Glucosamine, galactose, and mannose constituted a major component of the sugars in apo LDL-2, with similar relative proportions in all species. Sialic acid, fucose, and glucose formed a minor component, the proportions of which varied greatly among the species. 4. Unlike apo LDL-2, sialic acid, fucose, and glucosamine constituted the bulk of the sugars in apo HDL-2 and apo HDL-3. Mannose, galactose, and glucose were minor components, with galactose predominating. 5. Qualitative differences were observed in electrophoretic mobilities of apo HDL-2 and apo HDL-3 on polyacrylamide gel. One faster moving band was unique to chimpanzee. 6. Intraspecies differences in the content of sialic acid and fucose of apolipoproteins may be related to lipoprotein metabolism and species susceptibility (or resistance) to either spontaneous or diet-induced atherosclerosis. PMID:233783

  7. Structure activity relationships of human galactokinase inhibitors.

    PubMed

    Liu, Li; Tang, Manshu; Walsh, Martin J; Brimacombe, Kyle R; Pragani, Rajan; Tanega, Cordelle; Rohde, Jason M; Baker, Heather L; Fernandez, Elizabeth; Blackman, Burchelle; Bougie, James M; Leister, William H; Auld, Douglas S; Shen, Min; Lai, Kent; Boxer, Matthew B

    2015-02-01

    Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor. PMID:25553891

  8. C-phycocyanin protects against low fertility by inhibiting reactive oxygen species in aging mice.

    PubMed

    Li, Yan-Jiao; Han, Zhe; Ge, Lei; Zhou, Cheng-Jie; Zhao, Yue-Fang; Wang, Dong-Hui; Ren, Jing; Niu, Xin-Xin; Liang, Cheng-Guang

    2016-04-01

    Women over 35 have higher rates of infertility, largely due to deterioration of oocyte quality characterized by fragmentation, abnormal meiotic spindle-chromosome complexes, and oxidative stress. C-phycocyanin (PC) is a biliprotein enriched in Spirulina platensis that is known to possess antioxidant, anti-inflammatory, and radical-scavenging properties. D-galactose-induced aging acceleration in mice has been extensively used to study aging mechanisms and for pharmaceutical screening. In this study, adult female B6D2F/1 mice injected with D-galactose were used as a model to test the age-reversing effects of PC on degenerated reproductive ability. Our results show that PC can prevent oocyte fragmentation and aneuploidy by maintaining cytoskeletal integrity. Moreover, PC can reverse the expression of antioxidant genes, increase superoxide dismutase (SOD) activity and decrease methane dicarboxylic aldehyde (MDA) content, and normalize mitochondria distribution. PC exerts its benefit by inhibiting reactive oxygen species (ROS) production, which decreases apoptosis. Finally, we observe a significant increase in litter size after PC administration to D-galactose-induced aging mice. Our study demonstrates for the first time that D-galactose-induced impaired female reproductive capability can be partially rescued by the antioxidant effects of PC. PMID:27008700

  9. Polysaccharides of Type 6 Klebsiella

    PubMed Central

    Gormus, B. J.; Wheat, R. W.

    1971-01-01

    Water-extractable type 6 Klebsiella antigens were separated into a type 6-specific acidic polysaccharide and a neutral polysaccharide. The neutral polymer was devoid of type 6 activity although it was serologically active. The type 6-specific polymer contained fucose, glucose, and mannose, and pyruvic, galacturonic, and possibly glucuronic acids. The neutral polymer contained glucose, galactose, and mannose. PMID:5003178

  10. Structure of triphosphonoglycosphingolipid containing N-acetylgalactosamine 6-O-2-aminoethylphosphonate in the nervous system of Aplysia kurodai.

    PubMed

    Abe, S; Araki, S; Satake, M; Fujiwara, N; Kon, K; Ando, S

    1991-05-25

    A phosphonoglycosphingolipid, named F-21, was found in the nervous system of Aplysia kurodai by two-dimensional thin-layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). F-21 was isolated from the nervous tissue of Aplysia in this study, and its chemical structure was characterized as follows, where 2-AEP is 2-aminoethylphosphonate. (Formula; see text) The major aliphatic components of the ceramide portion were palmitic acid (75%), stearic acid (22%), octadeca-4-sphingenine (43%), and anteisononadeca-4-sphingenine (54%). Some information on the steric interactions in the sugar moiety was obtained by NMR spectroscopy. The ring protons of the internal galactose, H1, H3, and H4 and the H3 of the side chain galactose were shifted, as compared to the corresponding protons of dephosphonylated F-21. This may indicate the interactions between the 2-AEP residue of N-acetylgalactosamine and the internal galactose and between the N-acetyl group of N-acetylgalactosamine and the side chain galactose, implying a sterically restricted and unique structure that may relate to some biological functions of F-21. PMID:2033079

  11. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    PubMed

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-01

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids. PMID:27074906

  12. Purification and partial characterization of a lectin from the seeds of Trichosanthes kirilowii Maximowicz.

    PubMed

    Falasca, A I; Abbondanza, A; Barbieri, L; Bolognesi, A; Rossi, C A; Stirpe, F

    1989-03-27

    A lectin was purified from the seeds of Trichosanthes kirilowii, belonging to the family Cucurbitaceae, growing in China. The lectin is a glycoprotein of 57 kDa, consists of two subunits with apparent molecular masses of 37 and 25 kDa, is specific for galactose, and is not mitogenic for human lymphocytes. PMID:2707434

  13. A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis

    SciTech Connect

    Gondolf, Vibe M.; Stoppel, Rhea; Ebert, Berit; Rautengarten, Carsten; Liwanag, April J.M.; Loqué, Dominique; Scheller, Henrik V.

    2014-12-10

    Background: Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose. Results: First it was tested if overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the β-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as β-1,4-galactan in secondary cell walls. Conclusions: This approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in

  14. A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis

    DOE PAGESBeta

    Gondolf, Vibe M.; Stoppel, Rhea; Ebert, Berit; Rautengarten, Carsten; Liwanag, April J.M.; Loqué, Dominique; Scheller, Henrik V.

    2014-12-10

    Background: Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose. Results: First it was tested ifmore » overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the β-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as β-1,4-galactan in secondary cell walls. Conclusions: This approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in fiber cells while

  15. Impact of T1r3 and Trpm5 on carbohydrate preference and acceptance in C57BL/6 mice.

    PubMed

    Zukerman, Steven; Glendinning, John I; Margolskee, Robert F; Sclafani, Anthony

    2013-06-01

    Knockout (KO) mice missing the sweet taste receptor subunit T1r3 or the signaling protein Trpm5 have greatly attenuated sweetener preferences but learn to prefer sucrose in 24-h tests. Here, we examined 24-h preferences of T1r3 KO, Trpm5 KO, and C57BL/6J wild-type (WT) mice for glucose, fructose, galactose, and corn starch. Unlike glucose, fructose has little postoral reward effect in WT mice, whereas conflicting data have been obtained with galactose. Naïve KO mice were initially indifferent to dilute glucose solutions (0.5-4%) but exhibited strong preferences for 8-32% concentrations. In a second test, they strongly preferred (~90%) all glucose concentrations although they drank less sugar than WT mice. Naïve KO mice were indifferent to 0.5-8% fructose and avoided 16-32% fructose. However, the glucose-experienced KO mice displayed significant preferences for all fructose solutions. Naïve KO mice preferred only 8% galactose, whereas WT mice preferred 4-16% galactose, and all mice avoided 32% galactose. Galactose experience enhanced the preference for this sugar in KO and WT mice. Naïve T1r3 KO and WT mice displayed similar preferences for 0.5-32% corn starch, which were enhanced by starch experience. Naïve Trpm5 KO mice did not prefer starch but did so after 1-bottle starch experience. The results confirm the sweet taste deficits of T1r3 KO and Trpm5 KO mice but demonstrate their ability to develop strong glucose and milder galactose preferences attributed to the postoral actions of these sugars. The acquired preference for the non-sweet flavor properties of glucose generalized to those of fructose. The findings further demonstrate that although Trpm5 (but not T1r3) signaling is essential for starch preference, Trpm5 KO mice can learn to prefer starch based on its postoral effects. PMID:23547138

  16. Processing difficulties and instability of carbohydrate microneedle arrays

    PubMed Central

    Donnelly, Ryan F.; Morrow, Desmond I.J.; Singh, Thakur R.R.; Migalska, Katarzyna; McCarron, Paul A.; O’Mahony, Conor; Woolfson, A. David

    2010-01-01

    Background A number of reports have suggested that many of the problems currently associated with the use of microneedle (MN) arrays for transdermal drug delivery could be addressed by using drug-loaded MN arrays prepared by moulding hot melts of carbohydrate materials. Methods In this study, we explored the processing, handling, and storage of MN arrays prepared from galactose with a view to clinical application. Results Galactose required a high processing temperature (160°C), and molten galactose was difficult to work with. Substantial losses of the model drugs 5-aminolevulinic acid (ALA) and bovine serum albumin were incurred during processing. While relatively small forces caused significant reductions in MN height when applied to an aluminium block, this was not observed during their relatively facile insertion into heat-stripped epidermis. Drug release experiments using ALA-loaded MN arrays revealed that less than 0.05% of the total drug loading was released across a model silicone membrane. Similarly, only low amounts of ALA (approximately 0.13%) and undetectable amounts of bovine serum albumin were delivered when galactose arrays were combined with aqueous vehicles. Microscopic inspection of the membrane following release studies revealed that no holes could be observed in the membrane, indicating that the partially dissolved galactose sealed the MN-induced holes, thus limiting drug delivery. Indeed, depth penetration studies into excised porcine skin revealed that there was no significant increase in ALA delivery using galactose MN arrays, compared to control (P value < 0.05). Galactose MNs were unstable at ambient relative humidities and became adhesive. Conclusion The processing difficulties and instability encountered in this study are likely to preclude successful clinical application of carbohydrate MNs. The findings of this study are of particular importance to those in the pharmaceutical industry involved in the design and formulation of

  17. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-04-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  18. Effect of monosaccharide sugars on LH-induced differentiation and sugar transport facilitator (SLC2A) expression in sheep theca cells in vitro.

    PubMed

    Campbell, B K; Kendall, N R; Onions, V; Guo, L; Scaramuzzi, R J

    2014-03-01

    The aim of the present study was to investigate the effects of glucose, galactose and fructose on the LH-induced differentiation and mRNA expression of sugar transport facilitators (SLC2A) by sheep thecal cells derived from small antral follicles cultured under serum-free conditions for 6 days. The dose and type of monosaccharide had a significant effect on LH-induced androstenedione production by theca cells and there was a significant interaction (P<0.001). Glucose and galactose were used with equal efficiency so that cell numbers and androstenedione production at the end of the culture were comparable. Pharmacological doses of glucose (16.7 mM) inhibited steroidogenesis (P<0.05). Cell numbers and androstenedione production by cells cultured with fructose were lower than for cells cultured with either glucose or galactose (P<0.001). None of the monosaccharides resulted in the production of lactate. Expression of SLC2A1, SLC2A4 and SLC2A8, but not SLC2A5, mRNA was detected in fresh and cultured theca cells. Large doses (16.7 mM) of glucose and fructose, but not galactose, suppressed (P<0.05) SLC2A expression. The results show that glucose and galactose, but not fructose, are readily metabolised via oxidative pathways to support LH-induced differentiation of sheep theca cells. Further work is required to determine the mechanisms resulting in these differences in relation to the established effects of nutrition on reproductive function. PMID:23711112

  19. Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

    NASA Technical Reports Server (NTRS)

    Komoszynski, M.; Bandurski, R. S.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

  20. Evidence that cell surface beta 1,4-galactosyltransferase spontaneously galactosylates an underlying laminin substrate during fibroblast migration.

    PubMed

    Begovac, P C; Shi, Y X; Mansfield, D; Shur, B D

    1994-12-16

    beta 1,4-Galactosyltransferase is unusual among the glycosyltransferases in that a subpopulation exists on the cell surface in addition to its traditional biosynthetic location within the Golgi complex. On the cell surface, galactosyltransferase is expressed in spatially restricted, cell type-specific domains, where it functions as a receptor for extracellular oligosaccharide ligands during selected cellular interactions. For example, galactosyltransferase is found on the leading and trailing edges of migrating cells, where it facilitates lamellipodia formation and cell spreading by binding to specific N-linked oligosaccharides within laminin. Although the ability of galactosyltransferase to serve as a laminin receptor is well documented, it is unclear whether it functions solely in a lectin-like capacity to bind laminin glycoside ligands or uses its intrinsic catalytic activity to release itself from and modify its oligosaccharide substrate. In this study, we determined whether cell surface galactosyltransferase spontaneously galactosylates laminin matrices during cell migration using endogenous galactose donors. Cells were prelabeled with [3H]galactose, washed, and transferred in small clusters onto laminin matrices. The prelabeled cells migrated out from the cell cluster, during which time they deposited covalently bound [3H]galactose residues onto the laminin matrix. The degree of galactosylation was both laminin- and time-dependent and required actively migrating, intact cells. The radioactivity released from the 3H-galactosylated laminin by acid hydrolysis comigrated with authentic galactose standards on paper chromatography. In parallel assays, there was no radioactivity deposited on laminin matrices when cells were prelabeled with [3H]fucose or [3H]leucine. Furthermore, [3H]galactosylation was dependent upon galactosyltransferase-mediated cell migration, since prelabeled cells did not deposit [3H]galactose when migrating on fibronectin, upon which migration

  1. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

    PubMed

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ. PMID:27112153

  2. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: Application for cell surface antigen detection

    PubMed Central

    Boeggeman, Elizabeth; Ramakrishnan, Boopathy; Pasek, Marta; Manzoni, Maria; Puri, Anu; Loomis, Kristin H.; Waybright, Timothy J.; Qasba, Pradman K.

    2012-01-01

    The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using β1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant β1,4 galactosyltransferase (β1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect Enzyme Linked Immunosorbent Assay (ELISA) and Fluorescence Activated Cell Sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods. PMID:19425533

  3. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  4. Energy metabolism and biotransformation as endpoints to pre-screen hepatotoxicity using a liver spheroid model

    SciTech Connect

    Xu Jinsheng . E-mail: jinsheng.xu@uwe.ac.uk; Purcell, Wendy M.

    2006-10-15

    The current study investigated liver spheroid culture as an in vitro model to evaluate the endpoints relevant to the status of energy metabolism and biotransformation after exposure to test toxicants. Mature rat liver spheroids were exposed to diclofenac, galactosamine, isoniazid, paracetamol, m-dinitrobenzene (m-DNB) and 3-nitroaniline (3-NA) for 24 h. Pyruvate uptake, galactose biotransformation, lactate release and glucose secretion were evaluated after exposure. The results showed that pyruvate uptake and lactate release by mature liver spheroids in culture were maintained at a relatively stable level. These endpoints, together with glucose secretion and galactose biotransformation, were related to and could reflect the status of energy metabolism and biotransformation in hepatocytes. After exposure, all of the test agents significantly reduced glucose secretion, which was shown to be the most sensitive endpoint of those evaluated. Diclofenac, isoniazid, paracetamol and galactosamine reduced lactate release (P < 0.01), but m-DNB increased lactate release (P < 0.01). Diclofenac, isoniazid and paracetamol also reduced pyruvate uptake (P < 0.01), while galactosamine had little discernible effect. Diclofenac, galactosamine, paracetamol and m-DNB also reduced galactose biotransformation (P < 0.01), by contrast, isoniazid did not. The metabolite of m-DNB, 3-NA, which served as a negative control, did not cause significant changes in lactate release, pyruvate uptake or galactose biotransformation. It is concluded that pyruvate uptake, galactose biotransformation, lactate release and glucose secretion can be used as endpoints for evaluating the status of energy metabolism and biotransformation after exposure to test agents using the liver spheroid model to pre-screen hepatotoxicity.

  5. Structural analysis of colanic acid from Escherichia coli by using methylation and base-catalysed fragmentation. Comparison with polysaccharides from other bacterial sources

    PubMed Central

    Lawson, C. J.; McCleary, C. W.; Nakada, Henry I.; Rees, D. A.; Sutherland, I. W.; Wilkinson, J. F.

    1969-01-01

    Essentially the same methanolysis products were obtained after methylation of the slime and capsular polysaccharides from Escherichia coli K12 (S53 and S53C sub-strains) and the slime polysaccharides from E. coli K12 (S61), Aerobacter cloacae N.C.T.C. 5290 and Salmonella typhimurium SL1543. These were the methyl glycosides of 2-O-methyl-l-fucose, 2,3-di-O-methyl-l-fucose, 2,3-di-O-methyl-d-glucuronic acid methyl ester, 2,4,6-tri-O-methyl-d-glucose, 2,4,6-tri-O-methyl-d-galactose and the pyruvic acid ketal, 4,6-O-(1′-methoxycarbonylethylidene)-2,3-O-methyl-d-galactose. All were identified as crystalline derivatives from an E. coli polysaccharide. The structure of the ketal was proved by proton-magnetic-resonance and mass spectrometry, and by cleavage to pyruvic acid and 2,3-di-O-methyl-d-galactose. All these polysaccharides are therefore regarded as variants on the same fundamental structure for which the name colanic acid is adopted. Although containing the same sugar residues, quite different methanolysis products were obtained after methylation of the extracellular polysaccharide from Klebsiella aerogenes (1.2 strain). The hydroxypropyl ester of E. coli polysaccharide, when treated with base under anhydrous conditions, underwent β-elimination at the uronate residues with release of a 4,6-O-(1′-alkoxycarbonylethylidene)-d-galactose. Together with the identification of 3-O-(d-glucopyranosyluronic acid)-d-galactose as a partial hydrolysis product, this establishes the nature of most, if not all, of the side chains as O-[4,6-O-(1′-carboxyethylidene)-d-galactopyranosyl]-(1→4)-O-(d-glucopyranosyluronic acid)-(1→3)-d-galactopyranosyl... PMID:4902692

  6. The mechanisms of sodium absorption in the human small intestine

    PubMed Central

    Fordtran, John S.; Rector, Floyd C.; Carter, Norman W.

    1968-01-01

    The present studies were designed to characterize sodium transport in the jejunum and ileum of humans with respect to the effects of water flow, sodium concentration, addition of glucose and galactose, and variations in aniomic composition of luminal fluid. In the ileum, sodium absorption occurred against very steep electrochemical gradients (110 mEq/liter, 5-15 mv), was unaffected by the rate or direction of water flow, and was not stimulated by addition of glucose, galactose, or bicarbonate. These findings led to the conclusion that there is an efficiently active sodium transport across a membrane that is relatively impermeable to sodium. In contrast, jejunal sodium (chloride) absorption can take place against only the modest concentration gradient of 13 mEq/liter, was dramatically influenced by water movement, and was stimulated by addition of glucose, galactose, and bicarbonate. The stimulatory effect of glucose and galactose was evident even when net water movement was inhibited to zero by mannitol. These observations led to the conclusion that a small fraction of jejunal sodium absorption was mediated by active transport coupled either to active absorption of bicarbonate or active secretion of hydrogen ions. The major part of sodium absorption, i.e. sodium chloride absorption, appeared to be mediated by a process of bulk flow of solution along osmotic pressure gradients. The stimulatory effect of glucose and galactose, even at zero water flow, was explained by a model in which the active transport of monosaccharide generates a local osmotic force for the absorption of solution (NaCl and water) from the jejunal lumen, which, in the presence of mannitol, is counterbalanced by a reverse flow of pure solvent (H2O) through a parallel set of channels which are impermeable to sodium. Support for the model was obtained by the demonstration that glucose and bicarbonate stimulated the absorption of the nonactively transported solute urea even when net water flow was

  7. Reversion reactions of beta-galactosidase (Escherichia coli).

    PubMed

    Huber, R E; Hurlburt, K L

    1986-04-01

    The reversion reactions of beta-galactosidase (Escherichia coli) produced beta-galactosyl-galactoses and beta-galactosyl-glucoses. About 10 beta-galactosyl-galactose and 10 beta-galactosyl-glucose gas-liquid chromatographic peaks were detected and it is thus very likely that every possible isomer of beta-galactosyl-galactose and beta-galactosyl-glucose was formed by the reversion reactions (taking into account both anomers for each isomer). The presence of lactose and allolactose among the beta-galactosyl-glucoses was confirmed with standards. An important finding relating to the role of allolactose as an inducer of the lac operon was that allolactose (beta-D-galactosyl-(1----6)-D-glucose) was the only disaccharide formed initially, and at equilibrium it was present in the largest amount (50%). Obviously the enzyme is specific in its ability to form allolactose, and allolactose is the most stable beta-galactosyl-glucose, both important inducer properties. The equilibrium constant (concentration of disaccharides divided by the concentration of reactants at equilibrium) of the reaction was about 9.5 mM-1. This is the first report of an equilibrium constant for the beta-galactosidase reaction. Of mechanistic significance is the fact that only three compounds were able to replace D-galactose as a reversion reactant. Two of these (L-arabinose and D-fucose) had alterations at carbon 6. The 6 position, therefore, is not essential for reactivity. The third compound was D-galactal. Any other sugars tested (even with very minor changes relative to D-galactose) did not react. Of special consequence is the 2 position. The results strongly suggest that there has to be either an equatorial hydroxyl at the 2 position of a sugar or a special reactivity (as with D-galactal) in order for the enzyme to catalyze the beta-galactosidase reaction. PMID:3083779

  8. Anti-Aging Effects of Some Selected Iranian Folk Medicinal Herbs-Biochemical Evidences

    PubMed Central

    Mohammadirad, Azadeh; Aghamohammadali-Sarraf, Fatemeh; Badiei, Simin; Faraji, Zakie; Hajiaghaee, Reza; Baeeri, Maryam; Gholami, Mahdi; Abdollahi, Mohammad

    2013-01-01

    Objective(s): In the current study, the effects of selected folk medicinal herbs were evaluated in D-galactose-induced aging in male mice. Materials and Methods: Male BALB/c mice were randomly divided into 12 groups composing sham, control, and treated groups. Aging was induced by administration of D-galactose (500 mg/kg/day for 6 weeks). A positive control group was assigned that received vitamin E (200 mg/kg/day). The extract of herbs was prepared, lyophilized, and used in this study. The herbs were administered by gavage for 4 weeks to D-galactose-aged animals at the selected doses (mg/kg/day) as follows: Zingiber officinale (250), Glycyrrhiza glabra (150), Rosmarinus officinalis (300), Peganum harmala (50), Aloe vera (150), Satureja hortensis (200), Teucrium scordium (200), Hypericum perforatum (135) and Silybum marianum (150). One group of animals was assigned as sham and not given D-galactose. Results: At the end of treatment, pro-inflammatory markers including tumor necrosis factor-α (TNF-α), interlukine-1β (IL-β), interlukine-6 (IL-6), NF-kappaB (NF-κb), total antioxidant power (TAP), thiobarbituric acid reactive substances (TBARS) as lipid peroxidation (LPO) marker and male sex hormones i.e. testosterone and dehydroepiandrosterone-sulfate (DHEA-S) were measured in the blood. Conclusion: These data for the first time indicate significant anti-aging potential of examined herbs. Results showed that D-galactose induces a significant oxidative stress and promotes proinflammatory cascade of aging while all herbs more or less recovered these changes. Among 9 herbal extracts, Silybum marianum showed the best effect in restoring aging changes. PMID:24494070

  9. Rapid screening of classic galactosemia patients: a proof-of-concept study using high-throughput FTIR analysis of plasma.

    PubMed

    Lacombe, Caroline; Untereiner, Valérie; Gobinet, Cyril; Zater, Mokhtar; Sockalingum, Ganesh D; Garnotel, Roselyne

    2015-04-01

    Classic galactosemia is an autosomal recessive metabolic disease involving the galactose pathway, caused by the deficiency of galactose-1-phosphate uridyltransferase. Galactose accumulation induces in newborns many symptoms, such as liver disease, cataracts, and sepsis leading to death if untreated. Neonatal screening is developed and applied in many countries using several methods to detect galactose or its derived product accumulation in blood or urine. High-throughput FTIR spectroscopy was investigated as a potential tool in the current screening methods. IR spectra were obtained from blood plasma of healthy, diabetic, and galactosemic patients. The major spectral differences were in the carbohydrate region, which was first analysed in an exploratory manner using principal component analysis (PCA). PCA score plots showed a clear discrimination between diabetic and galactosemic patients and this was more marked as a function of the glucose and galactose increased concentration in these patients' plasma respectively. Then, a support vector machine leave-one-out cross-validation (SVM-LOOCV) classifier was built with the PCA scores as the input and the model was tested on median, mean and all spectra from the three population groups. This classifier was able to discriminate healthy/diabetic, healthy/galactosemic, and diabetic/galactosemic patients with sensitivity and specificity rates ranging from 80% to 94%. The total accuracy rate ranged from 87% to 96%. High-throughput FTIR spectroscopy combined with the SVM-LOOCV classification procedure appears to be a promising tool in the screening of galactosemia patients, with good sensitivity and specificity. Furthermore, this approach presents the advantages of being cost-effective, fast, and straightforward in the screening of galactosemic patients. PMID:25622686

  10. The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

    PubMed

    Shin, Sun-Mi; Choi, Jin Myung; di Luccio, Eric; Lee, Yong-Jik; Lee, Sang-Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2015-11-01

    UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism. PMID:26344854

  11. N.m.r. spectroscopic studies of fucose-containing oligosaccharides derived from keratanase digestion of articular cartilage keratan sulphates. Influence of fucose residues on keratanase cleavage.

    PubMed Central

    Tai, G H; Huckerby, T N; Nieduszynski, I A

    1993-01-01

    Keratan sulphate chains from bovine articular cartilage were fully digested with keratanase from Pseudomonas sp. and the products were reduced with alkaline borohydride. The resultant fragments were fractionated on a Nucleosil 5SB column and the earliest eluting fucose-containing oligosaccharides were isolated. Structural analysis using 1H n.m.r. spectroscopy (600 MHz) showed the two least-charged species to have the following structure: GlcNAc(6S) beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S) beta 1- 3Gal beta 1-4GlcNAc(6S) beta 1-3Gal-ol and GlcNAc(6S) beta 1-3Gal beta 1- 4(Fuc alpha 1-3)GlcNAc(6S) beta 1-3Gal beta 1-4GlcNAc(6S) beta 1-6(Gal beta 1- 3)GalNAc-ol. Both galactoses adjacent to the fucosylated N-acetylglucosamine residue are unsulphated. Therefore, it can be deduced from these structures that the presence of fucose on N-acetylglucosamine residues in keratan sulphates protects both of the adjacent unsulphated galactose residues from keratanase cleavage. This result has implications for the interpretation of keratanase fingerprints, because in articular cartilage keratan sulphates the keratanase-resistant blocks are not solely those with fully sulphated galactose residues, but also include the fucosylated sequences, which have unsulphated galactoses. It is, therefore, not possible to estimate their galactose sulphation or the size of the fully sulphated disaccharide-repeat sequences from keratan sulphates that contain fucose. PMID:8489515

  12. Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine

    PubMed Central

    Bauer, Christian H.; Lukaschek, Rainer; Reutter, Werner G.

    1974-01-01

    1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100–120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose–N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-14C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of

  13. Purification and characterization of a novel small-molecule polysaccharide from the Maitake medicinal mushroom Grifola frondosa (higher Basidiomycetes).

    PubMed

    Zhou, Changyan; Qiao, Yanru; Tang, Qingjiu; Jia, Wei; Liu, Yanfang; Wu, Aizhong

    2013-01-01

    A novel small-molecule polysaccharide with a molecular mass of 2.6 kDa, was isolated from the culinary-medicinal Maitake mushroom Grifola frondosa. GFPS is composed of fucose, rhamnose, galactose, glucose, and mannose; galactose, glucose, and mannose were the dominant monosaccharides. Absorption peaks at 1077 cm-1, 1024 cm-1, and 873 cm-1, as revealed by infrared spectrum, suggesting that GFPS consists of pyranoside. GFPS significantly enhanced the production of nitric oxide and secretion of cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-δ) from macrophages in vitro. These results indicate that this novel small-molecule polysaccharide might be beneficial for immune defense. PMID:23557366

  14. Characterization of the O- and N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni

    SciTech Connect

    Nyame, A.K.

    1987-01-01

    The structures of the O- and N-linked oligosaccharides in glycoproteins synthesized by larval and adult schistosomes of Schistosoma mansoni have been investigated. Mechanically transformed schistosomula or adult schistosomes were incubated in media containing either (/sup 3/H)mannose, (/sup 3/H)glucosamine or (/sup 3/H)galactose for 48 and 24 hr, respectively, to radiolabel metabolically the oligosaccharide moieties of newly synthesized glycoproteins. Analyses of the radiolabeled glycoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and fluorography demonstrated that numerous glycoproteins from 48-hr old schistosomula and adult schistosomes were labeled by both the (/sup 3/H)mannose and (/sup 3/H)glucosamine precursors. The (/sup 3/H)galactose precursor was incorporated into numerous glycoproteins in adult schistosomes; however, few, if any, glycoproteins in schistosomula were labeled by this radioactive sugar precursor.

  15. Pentavalent pillar[5]arene-based glycoclusters and their multivalent binding to pathogenic bacterial lectins.

    PubMed

    Galanos, Nicolas; Gillon, Emilie; Imberty, Anne; Matthews, Susan E; Vidal, Sébastien

    2016-04-01

    Anti-adhesive glycoclusters offer potential as therapeutic alternatives to classical antibiotics in treating infections. Pillar[5]arenes functionalised with either five galactose or five fucose residues were readily prepared using CuAAC reactions and evaluated for their binding to three therapeutically relevant bacterial lectins: LecA and Lec B from Pseudomonas aeuruginosa and BambL from Burkholderia ambifaria. Steric interactions were demonstrated to be a key factor in achieving good binding to LecA with more flexible galactose glycoclusters showing enhanced activity. In contrast binding to the fucose-selective lectins confirmed the importance of topology of the glycoclusters for activity with the pillar[5]arene ligand proving a selective ligand for BambL. PMID:26972051

  16. Synthesis, characterisation and microbial utilisation of amorphous polysugars from lactose.

    PubMed

    Daines, Alison M; Smart, Zlatka; Sims, Ian M; Tannock, Gerald W; Hinkley, Simon F R

    2015-03-01

    The melt polymerisations of glucose, galactose, xylose and fucose with citric acid, and mixtures of sugars therein are reported. Characterisation of the citric-acid catalysed reaction products indicated similar degrees of branched polymerisation but differences in the overall molecular weight of the polymers produced. The dairy by-product lactose could not be polymerised in a similar fashion but was shown to be readily hydrolysed using microwave radiation and a polymer generated from the melt condensation of the resultant glucose and galactose monosaccharides. A preliminary assessment of the bifido-bacterial utilisation of the lactose-derived polymerised products demonstrated a significantly different growth profile compared to commercially utilised galactooligosaccharides (GOS). PMID:25498629

  17. Structural analysis and immunostimulatory activity of glycopeptides from Paecilomyces sinensis.

    PubMed

    Zhu, Zhen-Yuan; Meng, Meng; Sun, Huiqing; Li, Yang; Yu, Na; Zhang, Yong-Min

    2016-03-01

    The parasitic fungus, Paecilomyces sinensis, is used to produce Cordyceps materials as a succedaneum of natural Cordyceps sinensis (C. sinensis) in China. In this work, a glycopeptide (CPS-II) was isolated and purified from Paecilomyces sinensis. The result of HPLC indicated that CPS-II was a glycopeptide. The estimated average molecular weight of CPS-II was 2 × 10(6) Da. FTIR, methylation, periodate oxidation, Smith degradation, (1)H NMR, (13)C NMR and CD were used for its structural analysis. The glycopeptide CPS-II was mainly composed of (1 → 3), (1 → 4) connected glucose and galactose as the backbone, there are (1 → 2,3,6) connected glucose, (1 → 3,6) connected mannose, and (1 → 6) connected galactose. Cell proliferation assay and morphological observations indicated that in a certain range of concentrations and time, CPS-II can significantly improve the proliferation activity of RAW264.7 cells. PMID:26912165

  18. Enzymatic synthesis of cholecalciferol glycosides using β-glucosidase from sweet almond.

    PubMed

    Manohar, Balaraman; Divakar, Soundar

    2010-10-01

    β-Glucosidase from sweet almond (Amygdalus communis var. 'Dulcis') entrapped on to calcium alginate beads catalysed the synthesis of water soluble 17-O-(D-glucopyranosyl)cholecalciferol. Optimum conditions for the reaction were: 60% (w/w D-glucose) β-glucosidase, 0.12 mM pH 6 phosphate buffer and 30 h incubation period. β-Glucosidase also catalyzed the reaction with D-glucose 2, D-galactose 3, D-mannose 4 and D-fructose 5 with generally low yields in the range of 3-14%. Both α/β anomers of D-glucose 2, D-galactose 3 and D-mannose 4 reacted, of which the former two formed C6-O derivatives also. PMID:23572673

  19. Towards determination of the distribution of methyl esterification in pectin

    SciTech Connect

    Maness, N.O.; Mort, A.J. )

    1990-05-01

    A procedure for determining the distribution of methyl esterification in pectin will be described. The first step is quantitative and selective reduction of methyl esterified galacturonic acid to galactose. Treatment of the reduced pectin with anhydrous liquid HF at {minus}10C results in cleavage of the galactosyl linkages without cleaving the galacturonosyl linkages. From the resultant oligosaccharides the distribution of contiguous non-esterified galacturonic acid residues in the polymer can be determined. If the reduced pectin is instead treated with lithium in ethylenediamine, galacturonic acid residues are destroyed and oligomers containing contiguous galactose residues (methyl esterified galacturonic acid in the parent polymer) are obtained. The distribution of contiguously esterified and non-esterified galacturonic acid segments in the original polymer is determined by separation and quantitation of these oligomers by HPLC. Application to commercially available pectins and to pectins from cotton suspension culture cells walls will be presented.

  20. A new method for measuring degree of methyl esterification in pectin

    SciTech Connect

    Maness, N.O.; Ryan, J.D.; Mort, A.J. )

    1989-04-01

    A simple method to measure the degree of methyl esterification in small samples of pectins or isolated cell walls will be described. The method involves selective reduction of methyl esterified galacturonic acid to galactose with sodium borohydride or sodium borodeuteride in the presence of strong buffer at 4 C. Quantitative reduction of samples can be accomplished in 1 h using 20 mg borohydride per mg sample. The degree of pectin methyl esterification can then be determined by measuring an increase of galactose using gas chromatography (borohydride reduced samples) or gas chromatography/mass spectroscopy (borodeuteride reduced samples), or by measuring the decrease in galacturonic acid using conventional colorimetric methods. Pectin samples as small as 50 {mu}g have been analyzed using the reduction method with good results.