Hafnia alvei lipopolysaccharides: Isolation, sugar composition and SDS-PAGE analysis

Abstract Lipopolysaccharides (LPS) of 33 strains of Hafnia alvei were isolated and purified. LPS content of the dry bacterial mass ranged from 1.2 to 4.5%. All examined lipopolysaccharides contained glucose, glucosamine, heptose, 3-deoxy-octulosonic acid and often galactose. Rhamnose, mannose, galactosamine, mannosamine and unidentified amino sugars were found in some H. alvei strains. Sialic acid was present in LPS of one strain. d -3-Hydroxybutyryl groups also were identified in lipopolysaccharides of 5 strains of this genus.
SDS-PAGE of the lipopolysaccharides was presented in the paper. According to these results two core types exist in H. alvei .     

Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases

We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp. strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei. These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains.     

Biochemical Correlates of Epilepsy in the El Mouse: Analysis of Glial Fibrillary Acidic Protein and Gangliosides

The E1 (epileptic) mouse is considered a model for complex partial seizures in humans. Seizures in E1 mice begin around 7-8 weeks of age and persist throughout life. To determine if astrocytic gliosis was present in adult seizing E1 mice, the distribution of glial fibrillary acidic protein (GFAP) was studied in the hippocampus using an antibody to GFAP. The mean number of GFAP-positive cells per square millimeter of hippocampus was approximately 15- to 40-fold higher in adult E1 mice than in nonseizing control C57BL/6J (B6) mice or in young nonseizing E1 mice. Relative GFAP concentration (expressed per milligram of total tissue protein) in hippocampus and cerebellum was estimated by densitometric scanning of peroxidase-stained western blots. GFAP concentration was 2.7-fold greater in hippocampus of adult seizing E1 mice than in the control B6 mice. No differences in GFAP content were detected between the strains in the cerebellum. Because gangliosides can serve as cell surface markers for changes in neuronal cytoarchitecture, they were analyzed to determine if the gliotic response in E1 mice was associated with changes in neural composition. Although the total ganglioside concentration of hippocampus, cerebral cortex, and cerebellum was similar in adult E1 and control B6 mice, a synaptic membrane enriched ganglioside, GD1a, was elevated in the adult E1 cerebral cortex and hippocampus. The findings indicate that E1 mice express a type of gliosis that is not accompanied by obvious neuronal loss.     

Recombinant cytochrome rC557 obtained from Escherichia coli cells expressing a truncated Thermus thermophilus cycA gene. Heme inversion in an improperly matured protein

Cytochrome rC(557) is an improperly matured, dimeric cytochrome c obtained from expression of the "signal peptide-lacking" Thermus thermophilus cycA gene in the cytoplasm of Escherichia coli. It is characterized by its Q(00) (or alpha-) optical absorption band at 557 nm in the reduced form (Keightley, J. A., Sanders, D., Todaro, T. R., Pastuszyn, A., and Fee, J. A. (1998) J. Biol. Chem. 273, 12006-12016). We report results of a broad ranging, biochemical and spectral characterization of this protein that reveals the presence of a free vinyl group on the porphyrin and a disulfide bond between the protomers and supports His-Met ligation in both valence states of the iron. A 3-A resolution x-ray structure shows that, in comparison with the native protein, the heme moiety is rotated 180 degrees about its alpha,gamma-axis; cysteine 14 has formed a thioether bond with the 2-vinyl of pyrrole ring I instead of the 4-vinyl of pyrrole ring II, as occurs in the native protein; and a cysteine 11 from each protomer has formed an intermolecular disulfide bond. Numerous, minor perturbations exist within the structure of rC(557) in comparison with that of native protein, which result from heme inversion and protein-protein interactions across the dimer interface. The unusual spectral properties of rC(557) are rationalized in terms of this structure.     

Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome p-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential (Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, congruent with 630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.     

Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.     

Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.

Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5. We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads. We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis. A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems. We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies. We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads. Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range. The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution. We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications.     

Arachidonic Acid-Induced Oxidative Injury to Cultured Spinal Cord Neurons

Spinal cord trauma can cause a marked release of free fatty acids, in particular, arachidonic acid (AA), from cell membranes. Free fatty acids, and AA by itself, may lead to secondary damage to spinal cord neurons. To study this hypothesis, cultured spinal cord neurons were exposed to increasing concentrations of AA (0.01-10 microM). AA-induced injury to spinal cord neurons was assessed by measurements of cellular oxidative stress, intracellular calcium levels, activation of nuclear factor-KB (NF-kappaB), and cell viability. AA treatment increased intracellular calcium concentrations and decreased cell viability. Oxidative stress increased significantly in neurons exposed to 1 and 10 microM AA. In addition, AA treatment activated NF-kappaB and decreased levels of the inhibitory subunit, IKB. It is interesting that manganese superoxide dismutase protein levels and levels of intracellular total glutathione increased in neurons exposed to this fatty acid for 24 h, consistent with a compensatory response to increased oxidative stress. These results strongly support the hypothesis that free fatty acids contribute to the tissue injury observed following spinal cord trauma.     

Enhanced Aspartate Release from Hippocampal Slices of Epileptic (El) Mice

The release of putative neurotransmitters [aspartate, glutamate, and gamma-aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 microns thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium-dependent and calcium-independent potassium-induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatography with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice +/- SEM. No significant differences were found between the El and B6 mice for the calcium-dependent potassium-evoked release of glutamate (18.20 +/- 2.62 and 15.41 +/- 3.56), or GABA (17.28 +/- 2.90 and 12.73 +/- 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 +/- 0.69) than in the B6 mice (3.31 +/- 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.