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Berlin Brandenburg

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  • 1
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2013, Vol.1022, pp.173-83
    Description: In vitro assays are invaluable for the biochemical characterization of UDP-sugar:undecaprenyl-phosphate sugar-1-phosphate transferases. These assays typically involve the use of a radiolabeled substrate and subsequent extraction of the product, which resides in a lipid environment. Here, we describe the preparation of bacterial membranes containing these enzymes, a standard in vitro transferase assay with solvents containing chloroform and methanol, and two methods to measure product formation: scintillation counting and thin layer chromatography.
    Keywords: Bacteria -- Enzymology ; Chromatography, Thin Layer -- Methods ; Enzyme Assays -- Methods ; Polyisoprenyl Phosphates -- Metabolism ; Transferases -- Metabolism ; Uridine Diphosphate -- Metabolism
    E-ISSN: 1940-6029
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 2
    Language: English
    In: Journal of Bacteriology, May, 2012, Vol.194(9-10), p.2646(12)
    Description: Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.
    Keywords: Escherichia Coli -- Genetic Aspects ; Caulobacter -- Genetic Aspects ; Microbiological Synthesis -- Research ; Gas Chromatography -- Usage ; Glycosyltransferases -- Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 3
    Language: English
    In: Journal of Bacteriology, 2012, Vol. 194(10), p.2646
    Description: Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins. ; p. 2646-2657.
    Keywords: Gas-Chromatography-Mass Spectrometry ; Oligosaccharides ; Bioassays ; Glucose 1-Phosphate ; Lipopolysaccharides ; Salmonella Enterica Subsp. Enterica Serovar Typhimurium ; Transferases ; Proteins ; Escherichia Coli K12 ; Antigens ; Caulobacter Crescentus ; Mutants;
    ISSN: 1098-5530
    ISSN: 10985530
    ISSN: 00219193
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  • 4
    In: Scientific Reports, 2015, Vol.5
    Description: We recently demonstrated that incorporation of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to the lipid A moiety of lipopolysaccharide (LPS) is required for transport of LPS to the outer membrane and viability of the Gram-negative bacterium Burkholderia cenocepacia. ArnT is a membrane protein catalyzing the transfer of l-Ara4N to the LPS molecule at the periplasmic face of the inner membrane, but its topology and mechanism of action are not well characterized. Here, we elucidate the topology of ArnT and identify key amino acids that likely contribute to its enzymatic function. PEGylation assays using a cysteineless version of ArnT support a model of 13 transmembrane helices and a large C-terminal region exposed to the periplasm. The same topological configuration is proposed for the Salmonella enterica serovar Typhimurium ArnT. Four highly conserved periplasmic residues in B. cenocepacia ArnT, tyrosine-43, lysine-69, arginine-254 and glutamic acid-493, were required for activity. Tyrosine-43 and lysine-69 span two highly conserved motifs, (42)RYA(44) and (66)YFEKP(70), that are found in ArnT homologues from other species. The same residues in S. enterica ArnT are also needed for function. We propose these aromatic and charged amino acids participate in either undecaprenyl phosphate-l-Ara4N substrate recognition or transfer of l-Ara4N to the LPS.
    Keywords: Amino Acids -- Chemistry ; Amino Sugars -- Metabolism ; Bacterial Proteins -- Chemistry ; Burkholderia Cenocepacia -- Metabolism ; Lipopolysaccharides -- Metabolism ; Salmonella Enterica -- Metabolism;
    ISSN: 20452322
    E-ISSN: 20452322
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  • 5
    In: Glycobiology, 2012, Vol. 22(1), pp.116-122
    Description: Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP CT ) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP CT allowing us to obtain highly purified protein. We demonstrate that WbaP CT is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP CT domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.
    Keywords: Isoprenoid ; Lipopolysaccharide ; Membrane Protein ; O - Antigen ; Sugar Transferase
    ISSN: 0959-6658
    E-ISSN: 1460-2423
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  • 6
    In: Glycobiology, 2010, Vol. 20(11), pp.1389-1401
    Description: CT CT CT WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP CT ) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaP CT domain N-terminally fused to thioredoxin (TrxA–WbaP CT ) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be α-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.
    Keywords: Lipopolysaccharide ; Membrane Protein ; O Antigen ; Sugar Transferase
    ISSN: 0959-6658
    E-ISSN: 1460-2423
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  • 7
    Language: English
    Description: Lipopolysaccharide (LPS), a major component of the outer leaflet of the Gram-negative bacterial outer membrane [1], consists of lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen [1,2]. LPS is a surface molecule unique to Gram-negative bacteria that plays a key role as an elicitor of innate immune responses, ranging from localized inflammation to disseminated sepsis [3]. The O-antigen, which is the most surface-exposed LPS moiety, also contributes to pathogenicity by protecting invading bacteria from bactericidal host responses [2]. A detailed understanding of the biosynthesis of the LPS O-antigen may contribute to identifying new means to curtail infections by interfering with its assembly.
    Keywords: Chemistry ; Applied Microbiology ; Immunology ; Medical Biochemistry ; Carbohydrate Chemistry ; Pharmacology/Toxicology ; Medicinal Chemistry ; Biology ; Chemistry ; Anatomy & Physiology
    ISBN: 9783709107324
    ISBN: 3709107326
    Source: SpringerLink Books
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  • 8
    Language: English
    In: Indian Journal of Hematology and Blood Transfusion, 2015, Vol.31(1), pp.1-8
    Description: Fifteen patients, with a median age of 19 years having severe aplastic anaemia (SAA) underwent human leucocyte antigen (HLA) identical sibling donor hematopoietic stem cell transplantation (HSCT) using conditioning regimens containing cyclophosphamide with antithymocyte globulin (ATG) or a combination of fludarabine and cyclophosphamide with or without ATG during December 2007 to May 2013. Cyclosporine and mini methotrexate were used as graft versus host disease (GVHD) prophylaxis. Graft source included peripheral blood stem cells in 11, bone marrow in 3 and both in 1. One patient had primary graft failure while 14 patients were engrafted with a median neutrophil and platelet engraftment time of 13.5 days. One patient had secondary graft rejection. Acute GVHD occurred in 3 patients and chronic GVHD in 4. One year death rate in engrafted patients was 14.28 %. At a mean follow-up of 21.2 months, 12 (80 %) are alive and well. One of the donors was a patient of haemophilia but the disease did not occur in the recipient. The graft was successful and the recipient is alive till date.
    Keywords: Aplastic anaemia ; HLA-matched sibling-donor ; Allogeneic bone marrow transplantation ; Peripheral blood stem cell transplantation ; Gujarat
    ISSN: 0971-4502
    E-ISSN: 0974-0449
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  • 9
    Language: English
    In: Gene, 01 November 2015, Vol.572(1), pp.8-16
    Description: The aim of the present study is to identify functional non-synonymous SNPs of TRPC6 gene using various approaches. These SNPs are believed to have a direct impact on protein stability through conformation changes. Transient receptor potential cation channel-6 (TRPC6) is one of the proteins that plays a key role causing focal segmental glomerulosclerosis (FSGS) associated with the steroid-resistant nephritic syndrome (SRNS). Data of TRPC6 was collected from dbSNP and further used to investigate a damaging effect using SIFT, PolyPhen, PROVEAN, and PANTHER. The comparative analysis predicted that two functional SNPs “rs35857503 at position N157T and rs36111323 at position A404V” showed a damaging effect (score of 0.096–1.00).We modeled the 3D structure of TRPC6 using a SWISS-MODEL workspace and validated it PROCHECK to get a Ramachandran plot (83.0% residues in the most favored region, 12.7% in additionally allowed regions, 2.3% in a generously allowed region and 2.0% were in a disallowed region). QMEAN (0.311) and MUSTER (10.06) scores were under acceptable limits. Putative functional SNPs that may possibly undergo post-translation modifications were also identified in TRPC6 protein. It was found that mutation at N157T can lead to alteration in glycation whereas mutation at A404V was present at a ligand binding site. Additionally, I-Mutant showed a decrease in stability for these nsSNPs upon mutation, thus suggesting that the N157T and A404V variants of TRPC6 could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein to some extent.
    Keywords: Trpc6 Gene ; Genomic Variants ; In Silico Analysis ; Modeled Structure ; Post Translational Modification ; Ligand Binding Sites ; Engineering ; Biology ; Anatomy & Physiology
    ISSN: 0378-1119
    E-ISSN: 1879-0038
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  • 10
    Language: English
    In: Indian Journal of Hematology and Blood Transfusion, 2015, Vol.31(1), pp.9-13
    Description: Beta thalassemia major, one of the most prevalent hemoglobinopathy throughout the word, can be cured by allogenic stem cell transplantation (SCT) (Bone Marrow Transplant 36:971–975, 2005). Many patients, however, lack a suitably matched related sibling donor. Unrelated umbilical cord blood (UCB) can be used as an alternative stem cell source for these patients. This report describes SCT for nine children with beta-thalassemia major using partially HLA-matched unrelated UCB. Conditioning included oral busulfan 16 mg/kg (day −10 to −7), cyclophosphamide (Cy) 200 mg/kg (day −5 to −2), fludarabine 90 mg/kg (day −13 to −11), and antithymocyte globulin (rabbit) 7.5 mg/kg (day −3 to −1). The infused cell dose was 10.71 × 10 7 /kg total nucleated cells (TNC) (range 6.5–17 × 10 7 /kg TNC). The patients ranged in age from 1.5 to 7 years, in weight from 10.5 to 17 kg. A second transplant with two unrelated cord blood units was attempted in two patients who had primary graft failure. The retransplant recipients were preconditioned with i.v Cy 120 mg/kg (day −3 to −2). Five of the nine patients engrafted promptly with 50–100 % donor chimerism (56 %). They engrafted at a median of 17 days (range 12–19). One patient is transfusion free for 36 months; a second patient is transfusion free for 18 months and a third is transfusion free for 9 months. There was no transplant related mortality. Four of the nine children had autologous recovery without engraftment. Primary graft rejection is the major complication. Post transplant complications were mild hepatic veno-occlusive disease, acute GVHD grade II, and CMV interstitial pneumonia. The chronic GVHD was limited and could be controlled by Methylprednisolone combined with Mycophenolate. The lack of a marrow donor registry in India makes UCBT from related and unrelated donors a good alternative. Transplant should be delayed until the child is at least 18 months of age. The dose of UCB stem cells is the most important factor for engraftment. UCB has the advantages of rapid availability and low risk of severe GVHD despite donor–recipient HLA disparity (Transplant Proc 37:2667–2669, 2005). We demonstrate the feasibility of this procedure in the setting of a developing country.
    Keywords: Unrelated umbilical cord blood transplantation ; Transfusion dependent thalassemia ; Engraftment
    ISSN: 0971-4502
    E-ISSN: 0974-0449
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