Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 1. Silver-stained 16.4% (w/v) T (total acrylamide), 1.9% (w/v) C (bisacrylamide) Tricine SDS-polyacrylamide gel show- ing the LPS profiles of E. coli K12 strains. 1, x705; 2, x711; 3, x711(pJB2); 4, Y10; 5, D21e7 (rfa-1); 6, CS2051 (has a deletion elimi- nating rfaG, rfaP, rfaM, rfaN, and rfaB); 7, D31m4 (rfa-229, rfa-230).

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 2. Physical map of the lpcA region. Vector sequences are not shown. pE4021 is a cosmid clone containing chromosomal DNA from E. coli W3110, including the region from 4.9 to 5.8 min. RNHQ, PEPD, GPTA, PHOE, and PROAB indicate the location of sequenced genes. pJB1 contains a 14-kb EcoRI fragment cloned from pE4021. pJB2 and pJB8 contain a 3-kb BamHI fragment cloned from pJB1 into different vectors. pJB2-9 to pJB2-34 indicate the various deletions of pJB2 span- ning the lpcA region. pJB15 indicates the DNA insert used for construc- tion of DIG-labeled riboprobes. ORF1 and ORF2 are two open reading frames found on opposite strands of the DNA. The direction of tran- scription of lpcA is indicated by the arrow beneath ORF2. The comple- mentation of the novobiocin supersensitivity phenotype by the deletion clones is indicated: R, successful complementation; S, unsuccessful complementation. Restriction enzymes indicated are: A, AvaII; B, BamHI; Bs, BstEII; E, EcoRI; Ev, EcoRV; Hc, HincII; P, PvuI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Plasmid Preparation, Clone Assay, Labeling

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 4. A schematic representation of the proposed events leading to the chromosomal deletion of the lpcA locus in E. coli strain x711. Panel A, chromosomal map of E. coli K12 strain x705. RNHQ, LPCA, PROAB, IS30A, IS5A, IS1B, and IS30 indicate the location of sequenced genes. Panel B, Southern blot showing chromo- somal DNA profiles of E. coli strains x711 and x705 probed with a 14-kb EcoRI DIG-11-dUTP-labeled DNA probe. M, l HindIII molecular weight markers; 1, x711 DNA digested with EcoRI; 2, x705 DNA di- gested with EcoRI; 3, pJB2 digested with BamHI; 4, pJB1 digested with EcoRI. Panel C, restriction maps of pJB1 and pJB16 showing identical nucleotide sequence (hatched box) and the IS5 element (open box). Panel D, transposition of the IS5A insertion element from approximately 5.9 min to 5.2 min followed by replication of the element, and chromosomal map of E. coli strain x711 showing the resulting deletion of the lpcA locus. Restriction endonucleases indicated are: E, EcoRI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Southern Blot, Labeling, Molecular Weight, Sequencing

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 5. Reversed-phase high performance liquid chromatogra- phy analyses of carbohydrates synthesized by E. coli strains x711 and x711(pJB2) cell extracts following incubation with 1.0 mmol of sedoheptulose 7-phosphate. Panel A, x711 incubated 60 min. Panel B, x711(pJB2) incubated 2 min. Panel C, x711(pJB2) incu- bated 60 min without sedoheptulose 7-phosphate. Panel D, x711(pJB2) boiled extract incubated 60 min. Large arrow indicates the retention peak of the phosphorylated product. Small arrow indicates the reten- tion peak of sedoheptulose 7-phosphate.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Synthesized, Incubation

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 6. Effect of alkaline phosphatase treatment in the reac- tion products analyzed by reversed-phase high performance liquid chromatography. Upper panel, HPLC profile of x711(pJB2) extract incubated with 1.0 mmol of sedoheptulose 7-phosphate (SED- 7-P) and treated with alkaline phosphatase (4 units) prior to derivat- ization with ABEE. Arrow indicates the location of the reaction peak of the reaction product in the absence of alkaline phosphatase treatment. Lower panel, HPLC profile of authentic glyceromannoheptose derivat- ized with ABEE. ABEE, p-aminobenzoic ethyl ester; AP, alkaline phos- phatase; GMH, glyceromannoheptose.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: High Performance Liquid Chromatography, Incubation

Journal: The Journal of biological chemistry

Article Title: Lipid dependence and basic kinetics of the purified 1,2-diacylglycerol 3-glucosyltransferase from membranes of Acholeplasma laidlawii.

doi: 10.1074/jbc.272.2.929

Figure Lengend Snippet: FIG. 1. Purification of the 1,2-diacylglycerol 3-glucosyltrans- ferase in three column chromatography steps. Protein (E) and MGlcDAG synthesis activity (G) were eluted in three different column chromatography steps as described under “Materials and Methods.” A, IEC on SP-Sepharose (Pharmacia), pH 8. The solid black line is the applied NaCl concentration. B, gel filtration on Sephacryl S-100 HR (Pharmacia); one of the four best fractions from the former IEC step. C, HAC on ceramic hydroxyapatite Macro-Prep (Bio-Rad), pH 8. The solid black line is the applied KH2PO4/K2HPO4 concentration. The MGlcDAG synthesis activity was assayed as described under “Materials and Meth- ods” with 27 mM amphiphiles present (3.7 mol % DOG, 33 mol % DOPG, and 63 mol % CHAPS).

Article Snippet: Hydroxyapatite Chromatography (HAC)—A column (16 mm in diameter) packed with 6 ml of ceramic hydroxyapatite Macro-Prep (Bio-Rad) was equilibrated with HAC-buffer: 100 mM KH2PO4/K2HPO4, pH 8, 20% (v/v) glycerol, and 20 mM CHAPS.

Techniques: Purification, Column Chromatography, Activity Assay, Concentration Assay, Filtration

Journal: The Journal of biological chemistry

Article Title: Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

doi: 10.1074/jbc.272.5.3073

Figure Lengend Snippet: FIG. 2. Analysis of the fractions separated by size exclusion column chromatography. The arrow marks recombinant rat litho- stathine. Lane S represents the selectively concentrated media proteins as loaded onto the column. Proteins were separated by SDS-PAGE (15% acrylamide) and visualized by silver staining. Molecular mass markers are shown at the right (in kDa).

Article Snippet: The fractions were evaluated by SDS-PAGE, and those containing relevant amounts of rat lithostathine were concentrated to a final volume of 9 ml in a Centriprep 10 device and then loaded onto a size exclusion chromatography column (Econo column 25/1200; BioRad) with a P10 matrix (Bio-Rad) and a bed volume of 540 ml.

Techniques: Column Chromatography, Recombinant, SDS Page, Silver Staining

Journal: The Journal of biological chemistry

Article Title: Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

doi: 10.1074/jbc.272.5.3073

Figure Lengend Snippet: FIG. 3. Analysis of the fractions eluted from the anion ex- change column. The size exclusion chromatography fractions that were most enriched with lithostathine were pooled and loaded onto a DEAE-Sephacel column (lane S). Proteins were eluted with a gradient of 10–300 mM NaCl. Lane L contains lithostathine purified to apparent homogeneity. Proteins were separated by SDS-PAGE (15% acrylamide) and visualized by silver staining. Molecular mass markers are shown at the right (kDa).

Article Snippet: The fractions were evaluated by SDS-PAGE, and those containing relevant amounts of rat lithostathine were concentrated to a final volume of 9 ml in a Centriprep 10 device and then loaded onto a size exclusion chromatography column (Econo column 25/1200; BioRad) with a P10 matrix (Bio-Rad) and a bed volume of 540 ml.

Techniques: Size-exclusion Chromatography, Purification, SDS Page, Silver Staining

Journal: The Journal of biological chemistry

Article Title: Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

doi: 10.1074/jbc.272.5.3073

Figure Lengend Snippet: FIG. 4. Immunodetection of rat lithostathine. Proteins contained in the fractions of the anion exchange chromatography were separated by SDS-PAGE (see Fig. 3) and transblotted onto a polyvinylidene diflu- oride membrane. Baculovirus expressed rat lithostathine was detected using anti-rat lithostathine antibodies. L and S as in Fig. 3. Molecular mass markers are shown at the right (in kDa).

Article Snippet: The fractions were evaluated by SDS-PAGE, and those containing relevant amounts of rat lithostathine were concentrated to a final volume of 9 ml in a Centriprep 10 device and then loaded onto a size exclusion chromatography column (Econo column 25/1200; BioRad) with a P10 matrix (Bio-Rad) and a bed volume of 540 ml.

Techniques: Immunodetection, Chromatography, SDS Page, Membrane