PMC

Insertional mutagenesis of bgp in strain N40 clone D10/E9. (A, top) Structure of the wild-type N40 bgp locus. The open arrow represents bgp (nucleotides 607138 to 607935, as indicated below map), which can be specifically amplified using primers 5Bgp and 3Bgp (small arrows). Flanking shaded or solid arrows represent neighboring open reading frames. The region between nucleotides 605042 and 609410 (indicated above map) that encompasses bgp was amplified using the primers 5BgpL and 3BgpL (small arrows), cloned, and subjected to kan insertion mutagenesis. (A, bottom) Structure of the bgp locus of mutants NP1.3, NP1.4, and NP2.1, which carry an insertion of the kan gene in bgp. Vertical open arrowheads indicate HindIII sites (see panel C). (B) PCR analysis of NP1.3, NP1.4, and NP2.1, three kanamycin-resistant B. burgdorferi colonies. (Top agarose gel) PCR using primers that specifically amplify kan resulted in the expected 1.2-kb product (Kan^r→) from each Kan^r clone and from plasmid pNP1, which carries bgp::kan and was used as a positive control. No product was amplified from plasmid pbgpL, which carries wild-type bgp. (Bottom agarose gel) Amplification using primers 5Bgp and 3Bgp that flank bgp (see panel A) resulted in the expected 2.0-kb bgp::kan product (bgp::Kan^r→) from each Kan^r clone and from plasmid pNP1. In contrast, an 800-bp bgp product (bgp→) was amplified from plasmid pbgpL, which carries wild-type bgp. The leftmost and rightmost lanes of the ethidium-stained gels contain λ HindIII and 1-kb ladder markers, respectively. (C) Southern blot analysis of wild-type N40 D10/E9, NP1.3, NP1.4, and NP2.1. HindIII-digested genomic DNA from the parental strain and each Kan^r clone was subjected to Southern hybridization with a bgp probe. HindIII cleavage of the wild-type N40 bgp locus results in the predicted 800-bp bgp-containing fragment (bgp→), whereas HindIII cleavage of NP1.3, NP1.4, or NP2.1 results in the predicted 2.0-kb bgp::kan-containing fragment (bgp::Kan^r→).

Biochemical characterization of recombinant human bGP. a, SDS-PAGE analysis of purified bGP. 1, 2, and 5 μg of purified bGP were subjected to SDS-PAGE under reducing conditions and stained with Coomassie Blue. b, Western blotting analysis of untagged and His₆-tagged bGP. Western blotting analysis was first conducted using a polyclonal antibody against the six-histidine tag and then reprobed using a monoclonal antibody against bGP. c, plot of the activity of glycogenolysis as a function of the purified bGP concentration. Various concentrations of purified protein were assayed for enzymatic activity in the direction of glycogen breakdown in the presence of 1 mm AMP and 0.25% glycogen. The data represent the averages of initial velocities (expressed as mAbs/min) of three experiments ± S.D. The curve was fitted to the data using linear regression. d, glycogen saturation curve of purified bGP. Purified recombinant bGP (100 nm) activity was measured in the direction of glycogen breakdown in the presence of a saturating concentration of AMP (1 mm) and various concentrations of glycogen (0–0.35%). The data shown represent the means of initial velocities of three different experiments (expressed as mean Abs/min ± S.D.). e, activation of bGP by phosphorylation and AMP. Specific phosphorylation of bGP Ser¹⁴ was performed prior to the assay by incubating the recombinant enzyme with phosphorylase kinase in the presence of ATP for 2 h at room temperature. bGP and phosphorylated bGP was then assayed for activity with or without 1 mm AMP. Activity was expressed as the percentage of the control (bGP activity in the presence of AMP alone). The data shown represent the means of initial velocities of three different experiments ± S.D. ***, p < 0.001. f, Western blotting of phosphorylated and non-phosphorylated bGP. Recombinant bGP was incubated with (lane 1) or without (lane 2) phosphorylase kinase and subjected to Western blotting analysis using an anti-phosphoserine antibody. The presence of purified protein was assessed by Ponceau red staining. g, non-cooperative binding of AMP. bGP activity was measured in the presence of various concentrations (0–3 mm) of AMP and a saturating concentration of glycogen (0.25%). Initial velocities were determined and fitted to the Hill equation. The resulting n_Hill was equal to 0.994, indicating non-cooperative binding of AMP to bGP as previously reported (). MW, molecular weight.

Comparison of BGP levels in four groups of rats. * represents BGP level compared with normal group at the same time, P<0.001; # represents the BGP level compared with the control group at the same time, P<0.001; ∆ represents the BGP level compared with the model group at the same time, P<0.001; Ñ represents BGP level compared with the same group at T0, P<0.001; □ represents BGP level compared with the same group at T1, P<0.001; ☆ represents BGP level compared with the same group at T2, P<0.001.

a, Schematic illustration of the BGP update return intervals. Shown are the intervals τ ₁ and τ ₂ calculated for threshold q = 1 and q = 2 respectively. b, Typical sequence of 500 BGP update return intervals for NTT, where q = 4, calculated for (magenta) original and (black) shuffled data. c, The distribution function P _q(τ) of BGP update return intervals of the NTT, calculated for different values of q. The inset depicts the average return interval as a function of threshold q. d, P _q(τ) for BGP update return intervals of the NTT monitor calculated for q = 1. Original data is shown with red while shuffled data is shown with black. e, Scaled plots of the BGP return intervals for the NTT monitor. f, The mean conditional return interval as a function of preceding return interval τ ₀ for the NTT monitor. Both and τ ₀ are normalized with the mean return interval (). For BGP updates without memory we expect , as supported by the open symbols obtained for shuffled return interval data.