Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: p53 expression is regulated in a dose dependent manner by Dox . (A and B) Cells treated with Dox for 48 h were harvested and western blot for p53 was performed. (C) No induction of p53 in response to Dox was observed in HTet43GFP cells. (D) Fold induction of p53 was calculated by densitometric analysis of p53 western blot in HTet23p53, HTet26p53 and HTet43GFP cells taking control = 1with normalization to β-Actin or β -Tubulin.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Control

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: p53 expression is regulated in a time dependent manner . (A, B and C) HTet23p53, HTet26p53 and HTet43GFP cells were treated with 1000 ng/ml Dox for indicated time points and western blots were performed for p53. (D) Fold induction was calculated by densitometric analysis of p53 in HTet23p53, HTet26p53 and HTet43GFP cells taking 0 h = 1 with normalization to β -Tubulin or GAPDH.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: p53 over expression did not reduce colony number . Five thousand cells were plated in low-melting agarose containing complete medium and allowed to grow for 30 days. (A, B and C) Cells were stained with crystal violet and photographs were taken under microscope for HTet23p53, HTet26p53 and HTet43GFP plates. (D) Colonies were counted and average number of colonies was plotted vs Dox concentration. Bar represents average of colonies from two representative fields (± SE).

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Over Expression, Staining, Microscopy, Concentration Assay

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is stable . (A and B) p53 was overexpressed with 1000 ng/ml of Dox for 48 h or not overexpressed and western blotswere performed after indicated time of Chx treatment in HTet23p53 and HTet26p53 cells. (C) Western blot for p53 was performed with or without addition of 1000 ng/ml Dox for 48 h followed by Chx treatment for indicated time points in HTet43GFP cells.(D) Graphical representation of percentage of p53 protein remaining after indicated time points in HTet23p53 and HTet26p53 cells by densitometric analysis following normalization with β-Actin. Protein percentage for 0 h Chx was taken as 100.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is not stabilized by inhibition of proteasomal degradation . Cells were treated with proteasomal inhibitors (MG132 or Lactacystin) 1 h prior to Dox addition and incubated for 48 h. Thereafter, cells were treated with Chx for indicated time points and western blots were performed. (A) MG132 treatment in HTet23p53 and HTet26p53 cells and p53 protein expression. (B) Lactacystin treatment in HTet23p53 and HTet26p53 cells and p53 protein expression.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Incubation, Western Blot, Expressing

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is functionally impaired by HPV E6 . (A) HTet23p53, HTet26p53 or HTet43GFP cells were transfected with vector or HPV18 E6 plasmid and 18 h post transfection cells were treated with OA 1 h prior to Dox addition. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) HTet26p53 cells were transfected with HPV18 E6 plasmid and treated as mentioned in A and immunoprecipitation was performed first with E6 antibody (first IP) and secondly by p53 antibody. p53 or E6 was detected in immunoprecipitated complex. (C) p53 post-IP (second IP) following E6 IP was performed and p53 or phospho-p53detection by western blotting was performed. (D) Cells were transfected with p21luciferase construct with or without HPV18 E6 plasmid and treated as mentioned in A. Luciferase assay was performed and luciferase/GFP reading was plotted. Bar represents results from an experiment done in triplicate. (± SE). * Indicates P < 0.05.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, MTT Assay, Immunoprecipitation, Western Blot, Construct, Luciferase

Journal: Cell & Bioscience

Article Title: Human papillomavirus 18 E6 inhibits phosphorylation of p53 expressed in HeLa cells

doi: 10.1186/2045-3701-2-2

Figure Lengend Snippet: Overexpressed p53 is active and is made non-functional by HPV 18 E6 in H1299, a p53 and E6 null cell line . (A) H1299 cells were transfected with pC53-SN3 and/or HPV18 E6 plasmids and 18 h post transfection cells were treated with OA. MTT assay was performed after 48 h. Bar represents variations among the wells of an experiment done twice in triplicate. * Indicates P < 0.01 (B) H1299 cells were transfected with pC53-SN3 and/or HPV18E6 plasmids and 18 h post transfection cells were treated with OA for 48 h and western blot was performed for pSer46p53 and p53.(C) Densitometric analysis was performed by normalization with p53 and ratio of pSer53 and p53 was plotted.

Article Snippet: Antibodies against p53 (FL-393 goat polyclonal; DO1-HRP conjugated mouse monoclonal), E6 (goat polyclonal and mouse monoclonal), GAPDH (goat polyclonal), β-Tubulin (rabbit polyclonal) and β-Actin (goat polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Functional Assay, Transfection, MTT Assay, Western Blot

Journal: International journal of cancer

Article Title: The screening of the second-site suppressor mutations of the common p53 mutants.

doi: 10.1002/ijc.22724

Figure Lengend Snippet: FIGURE 1 – Transactivity of SSS mutations for 7 distinct p53-binding sequences in yeast. The transactivity was shown as a relative value to the activity of wild-type p53. Transactivity of mutant p53 (G245S) was rescued by SS mutations, H178Y and S183P. Columns, means of 3 inde- pendent experiments; bars, SD.

Article Snippet: The expressed p53 was detected by immunoblot analysis using an HRP-conjugated antihuman p53 antibody, p53 (FL-393) HRP, (Santa Cruz Biotechnology), a HRP-conjugated goat polyclonal anti-HA antibody (Bethyl Laboratories) or an anti-HRP-conjugated goat polyclonal antiMyc antibody (Bethyl Laboratories).

Techniques: Binding Assay, Activity Assay, Mutagenesis

Journal: International journal of cancer

Article Title: The screening of the second-site suppressor mutations of the common p53 mutants.

doi: 10.1002/ijc.22724

Figure Lengend Snippet: FIGURE 2 – Transactivity of SSS mutations for 5 distinct p53-bind- ing sequences in Saos-2 cells. The transactivity was shown as a rela- tive value to the activity of wild-type p53. Transactivity of mutant p53 (G245S) was rescued by SS mutations, H178Y, C182F, S183P, D184A, D184G and D186N for 5 p53-binding sequences. Columns, values of 2 independent experiments.

Article Snippet: The expressed p53 was detected by immunoblot analysis using an HRP-conjugated antihuman p53 antibody, p53 (FL-393) HRP, (Santa Cruz Biotechnology), a HRP-conjugated goat polyclonal anti-HA antibody (Bethyl Laboratories) or an anti-HRP-conjugated goat polyclonal antiMyc antibody (Bethyl Laboratories).

Techniques: Activity Assay, Mutagenesis, Binding Assay

Journal: International journal of cancer

Article Title: The screening of the second-site suppressor mutations of the common p53 mutants.

doi: 10.1002/ijc.22724

Figure Lengend Snippet: FIGURE 3 – Intertermolecular rescue of mutant p53 (G245S) function by another coexpressed mutant p53 (H178Y). The transactivity was shown as a relative value to the activity of wild-type p53. (a) p53 expression with wild type (WT), G245S, H178Y, H178Y-G245S and the coex- pression of H178Y and G245S (H178Y 1 G245S) in Saos-2 cells. The total amount of plasmid DNA used for transfection was the same in each lane. The net intensities of each lanes were calculated by the 1D Image Analysis Software (Kodak Digital Science) and indicated as a relative value of wild-type p53. (b) Rescue of G245S transactivity by coexpressed H178Y was shown in all but the p53R2 promoter. We adjusted the transactibvation levels by the protein expression levels indicated by the net intensities. Columns, means of 3 independent experiments; bars, SD.

Article Snippet: The expressed p53 was detected by immunoblot analysis using an HRP-conjugated antihuman p53 antibody, p53 (FL-393) HRP, (Santa Cruz Biotechnology), a HRP-conjugated goat polyclonal anti-HA antibody (Bethyl Laboratories) or an anti-HRP-conjugated goat polyclonal antiMyc antibody (Bethyl Laboratories).

Techniques: Mutagenesis, Activity Assay, Expressing, Plasmid Preparation, Transfection, Software

Journal: International journal of cancer

Article Title: The screening of the second-site suppressor mutations of the common p53 mutants.

doi: 10.1002/ijc.22724

Figure Lengend Snippet: FIGURE 4 – Rescue of apoptotic function of mutant p53 (G245S) by coexpressed mutant p53 (H178Y). (a–e) Representative DNA histo- gram of the Saos-2 cells expressing null, wild-type, mutant p53 (G245S), mutant p53 (H178Y) or both mutant p53 (G245S) and mutant p53 (H178Y). (f) Apoptotic fraction of the transfection experi- ments was estimated as DsubG1 fraction (see Materials and methods section) by FACS analysis. Columns, values of 2 independent experi- ments.

Article Snippet: The expressed p53 was detected by immunoblot analysis using an HRP-conjugated antihuman p53 antibody, p53 (FL-393) HRP, (Santa Cruz Biotechnology), a HRP-conjugated goat polyclonal anti-HA antibody (Bethyl Laboratories) or an anti-HRP-conjugated goat polyclonal antiMyc antibody (Bethyl Laboratories).

Techniques: Mutagenesis, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

doi: 10.1074/jbc.ra118.004301

Figure Lengend Snippet: Figure 1. P472L mutation increases p97 ATPase activity and desensitizes the enzyme to ATP-competitive and allosteric inhibitors. A, p97 mutations found in NMS-873- or CB-5083–resistant HCT116 cells are localized to the D1–D2 linker and D2 and are distinct from disease-causing MSP-1 mutations. N615V and N616F are mutations shown to disrupt NMS-873 analog binding from cross-linking studies (37). Residues implicated in NMS-873 (pink) and CB-5083 (cyan) resistance are shown (PDB code 5FTL) with inhibitor-binding sites indicated. Additional mutations found in HCT116 cells resistant to CB-5083 and NMS-873 during this study are in bold. B, ATP titration experiments were performed with p97 and the P472L mutant to obtain steady-state kinetic parameters. Resulting Michaelis-Menten constants (KM), turnover numbers (kcat), and catalytic efficiencies (kcat/KM) are shown in Table 1. C, sensitivities of the ATPase activities of p97 and the P472L mutant to CB-5083 or NMS-873 were evaluated in inhibitor titration experiments. IC50 values obtained from dose-response curves are shown in Table 1. Data points represent the mean (n 4) with standard deviation (S.D.) error.

Article Snippet: To generate engineered P472L cell lines, HCT116 cells were co-transfected with a Cas9-expressing plasmid (PX458, Addgene plasmid no. 48138) containing an sgRNA corresponding to bases 35,060,894 to 35,060,875 of the p97 gene exon 12 on chromosome 9 and a linear repair template corresponding to 35,060,974 to 35,060,787 with mutations at nucleotide 35,060,873 (G to A; disrupts PAM) and 35,060,868 (C to T; mutates Pro-472 codon to Leu).

Techniques: Mutagenesis, Activity Assay, Binding Assay, Titration, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

doi: 10.1074/jbc.ra118.004301

Figure Lengend Snippet: Figure 3. P472L mutation does not disrupt the binding sites of p97 inhibitors. A, ITC was used to measure the binding of CB-5083 to p97 and the P472L mutant. Top panels are raw data, and lower panels show isotherms with fitted curves and representative KD values. B, effect of increasing NMS-873 concentra- tions on TR-FRET generated by p97 and the P472L mutant in the presence of BODIPY-FL–ATP, and a terbium-labeled anti-His tag antibody was measured (n 4, S.D.). Apparent KD values extrapolated from NMS-873–dependent increases in TR-FRET are shown. C, fluorescence polarization experiments were performed (n 2, S.D.) to measure the enhanced binding of EDA–ADP–ATTO-495 to p97 and the P472L mutant with increasing NMS-873 concentrations. Apparent KD values with S.E. are shown.

Article Snippet: To generate engineered P472L cell lines, HCT116 cells were co-transfected with a Cas9-expressing plasmid (PX458, Addgene plasmid no. 48138) containing an sgRNA corresponding to bases 35,060,894 to 35,060,875 of the p97 gene exon 12 on chromosome 9 and a linear repair template corresponding to 35,060,974 to 35,060,787 with mutations at nucleotide 35,060,873 (G to A; disrupts PAM) and 35,060,868 (C to T; mutates Pro-472 codon to Leu).

Techniques: Mutagenesis, Binding Assay, Generated, Labeling, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

doi: 10.1074/jbc.ra118.004301

Figure Lengend Snippet: Figure 4. P472L mutation increases D2 ATPase activity and alters its intra-subunit control by D1. A, schematic shows the location of the P472L mutation relative to Walker A and Walker B mutations in D1 and D2. B, ATP titration experiments (n 4, S.D. error) were performed to obtain steady-state kinetic parameters of p97 and the P472L mutant proteins containing the indicated Walker motif mutations. The sensitivities of the ATPase activities of p97 and the P472L mutant with Walker motif mutations in D1 or D2 were evaluated in the presence of increasing concentrations of CB-5083 (C) and NMS-873 (D) (n 4, S.D. error). Resulting kinetic properties and IC50 values are summarized in Table 2.

Article Snippet: To generate engineered P472L cell lines, HCT116 cells were co-transfected with a Cas9-expressing plasmid (PX458, Addgene plasmid no. 48138) containing an sgRNA corresponding to bases 35,060,894 to 35,060,875 of the p97 gene exon 12 on chromosome 9 and a linear repair template corresponding to 35,060,974 to 35,060,787 with mutations at nucleotide 35,060,873 (G to A; disrupts PAM) and 35,060,868 (C to T; mutates Pro-472 codon to Leu).

Techniques: Mutagenesis, Activity Assay, Control, Titration

Journal: Journal of Biological Chemistry

Article Title: Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

doi: 10.1074/jbc.ra118.004301

Figure Lengend Snippet: Figure 5. P472L mutation enhances cooperative D2 ATP binding and hydrolysis. A, p97 schematic (top) and D2 catalytic pocket model (bottom, PDB code FTM)showargininefingerresiduesArg-635andArg-638fromanadjacentD2domaininvolvedincooperativeD2ATPhydrolysis.TheaffinitiesofADP(B)orATP (C) to the D2 domains of the indicated p97 or P472L mutant proteins were measured by fluorescence polarization (n 2, S.D. error). D, kinetic properties of p97 and the P472L mutant proteins in the presence or absence of R635A or R638A were obtained from ATP titration experiments (n 4, S.D. error). E, sensitivities of p97 and the P472L mutant proteins to CB-5083 and NMS-873 were tested in inhibitor titration experiments (n 4, S.D. error). Dissociation constants for ATP and ADP binding to D2, enzyme kinetic properties, and IC50 values are summarized in Table 3.

Article Snippet: To generate engineered P472L cell lines, HCT116 cells were co-transfected with a Cas9-expressing plasmid (PX458, Addgene plasmid no. 48138) containing an sgRNA corresponding to bases 35,060,894 to 35,060,875 of the p97 gene exon 12 on chromosome 9 and a linear repair template corresponding to 35,060,974 to 35,060,787 with mutations at nucleotide 35,060,873 (G to A; disrupts PAM) and 35,060,868 (C to T; mutates Pro-472 codon to Leu).

Techniques: Mutagenesis, Binding Assay, Fluorescence, Titration

Journal: Journal of Biological Chemistry

Article Title: Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

doi: 10.1074/jbc.ra118.004301

Figure Lengend Snippet: Figure6.Proposeddesensitizationmechanismofp97toitsATP-compet- itive and allosteric inhibitors. NMS-873 allosterically inhibits p97 activity by blocking cooperative D2 ATP hydrolysis. CB-5083 functions as an ATP-com- petitive and D2-selective inhibitor. Our data suggest that intra-subunit con- trol of D2 activity through ATP-bound D1 is critical for p97 sensitivity to this molecule (orange arrow). The P472L mutant, identified from CB-5083– resistant cells, is desensitized to both inhibitors. This mechanism involves enhanced cooperative ATP binding and hydrolysis by D2 that alter its intra- subunit control by ATP-bound D1.

Article Snippet: To generate engineered P472L cell lines, HCT116 cells were co-transfected with a Cas9-expressing plasmid (PX458, Addgene plasmid no. 48138) containing an sgRNA corresponding to bases 35,060,894 to 35,060,875 of the p97 gene exon 12 on chromosome 9 and a linear repair template corresponding to 35,060,974 to 35,060,787 with mutations at nucleotide 35,060,873 (G to A; disrupts PAM) and 35,060,868 (C to T; mutates Pro-472 codon to Leu).

Techniques: Activity Assay, Blocking Assay, Mutagenesis, Binding Assay, Control

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: Schematic presentation of the 3,139-bp-long cloned and sequenced chromosomal L. casei DNA fragment containing the hprK gene. Indicated are the five ORFs (yvlB, yvlC, yvlD, hprK, and lgt) detected in this fragment and several restriction sites. The DNA sequence shown above this scheme represents the region preceding hprK and includes a putative promoter (−10 and −35), a presumed ribosome binding site (SD), and the ATG start codon. The L. casei DNA fragments present in the plasmids pHKLc1, pHKLc2, and pHKLc3 are aligned underneath the schematic presentation of the cloned L. casei DNA. The asterisk in pHKLc3 indicates the position of the hprK208(Am) mutation.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Clone Assay, Sequencing, Binding Assay, Mutagenesis

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: [γ-32P]ATP-dependent phosphorylation of B. subtilis and L. casei HPr(His)6 with crude extracts prepared from either the L. casei wild-type strain (BL23) or the hprK208(Am) mutant (LcG102). Phosphorylation experiments were carried out with 1.5 μg of HPr as described in Materials and Methods. After electrophoresis, the 15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate was treated with 16% boiling trichloroacetic acid, dried, and exposed to autoradiography. Lane 1, HPr from B. subtilis and crude extract from BL23; lane 2, HPr from B. subtilis and crude extract from LcG102; lane 3, HPr from L. casei and crude extract from BL23; lane 4, HPr from L. casei and crude extract from LcG102.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Phospho-proteomics, Mutagenesis, Electrophoresis, Autoradiography

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: The effect of FBP and Pi on ATP-dependent phosphorylation of B. subtilis HPr. HPr and P-Ser-HPr were separated on nondenaturing 12.5% polyacrylamide gels. (A) Effect of FBP on the L. casei HPr kinase activity. HPr phosphorylation was carried out with 20 ng of HprK/P and the indicated concentrations of FBP for 3 min at 37°C as described in Materials and Methods. HPr standard (2.5 μg) was loaded on lane 6. (B) Effect of Pi on L. casei P-Ser-HPr phosphatase activity. HprK/P-catalyzed dephosphorylation of P-Ser-HPr was performed with 50 ng of HprK/P and the indicated Pi concentrations by incubating the assay mixture for 5 min at 37°C as described in Materials and Methods. P-Ser-HPr standard (2.5 μg) was loaded on lane 1. (C) Effect of FBP on ATP-dependent HPr phosphorylation in the presence of Pi. ATP-dependent HPr phosphorylation was carried out for 5 min at 37°C with 20 ng of HprK/P and the indicated amounts of Pi in the presence (+) or absence (−) of 20 mM FBP. Lanes 10 and 11 contain 2.5 μg of HPr and P-Ser-HPr standards, respectively. After electrophoresis, gels were stained with Coomassie blue.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Phospho-proteomics, Activity Assay, De-Phosphorylation Assay, Electrophoresis, Staining

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: P-Ser-HPr phosphatase activity in crude extracts of L. casei wild-type or hprK208(Am) mutant. P-Ser-HPr phosphatase assays were carried out with 10 μl of crude extracts in the absence (−) or presence (+) of 2.5 μg of P-Ser-HPr as described in Materials and Methods. P-Ser-HPr and HPr standards (2.5 μg each) were loaded on lanes 5 and 6, respectively. Samples were separated on a nondenaturing 12.5% polyacrylamide gel which was stained with Coomassie blue.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Activity Assay, Mutagenesis, Staining

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: Catabolite repression of N -acetylglucosaminidase in L. casei

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Activity Assay

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: [14C]Maltose uptake in the presence (diamonds) and absence (squares) of glucose by L. casei wild-type (A) and hprK208(Am) mutant (B) cells. Transport studies were carried out by using the rapid filtration method. Glucose at a final concentration of 1 mM was added at the time indicated by the arrow.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Mutagenesis, Filtration, Concentration Assay

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: Consumption of maltose (0.025%) and glucose (0.15%) by resting L. casei wild-type (A) or hprK208(Am) mutant (B) cells. The cell suspension containing 18 mg of cells (dry weight) in 5 ml of 50 mM sodium phosphate buffer, pH 7, was incubated at 37°C. Samples of 300 μl were withdrawn at the indicated time intervals and were centrifuged. The maltose concentration in experiments carried out in the presence (diamonds) or absence (circles) of glucose and the glucose concentration (squares) were determined in the supernatant.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Mutagenesis, Suspension, Incubation, Concentration Assay

Journal:

Article Title: Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

doi:

Figure Lengend Snippet: Expulsion of preaccumulated [14C]TMG-6-P in different L. casei strains. Cells preloaded with [14C]TMG-6-P were washed and resuspended in 1 ml of transport buffer. At time 0, glucose was added at a final concentration of 5 mM, and 100-μl aliquots were withdrawn at the indicated time intervals. The radioactivity remaining inside the cells was measured with the wild-type strain BL23 (squares) and the ptsH1 (triangles), the ptsH2 (cross within squares), and the hprK208(Am) (diamonds) mutant strains. Two control samples were taken before glucose was added. One was immediately filtered and provided time point 0, the other was incubated at 37°C and was filtered at the end of the experiment (crosses within circles). This end point is shown only for the experiment with the ptsH1 mutant. A similar leakage (less than 5%) of [14C]TMG-6-P in the absence of glucose was observed with all other strains.

Article Snippet: L. casei BL23 (ATCC 393 cured of plasmid pLZ15) was used in this study.

Techniques: Concentration Assay, Radioactivity, Mutagenesis, Control, Incubation