Journal: Sensors (Basel, Switzerland)

Article Title: Development of Optical Sensors Based on Neutral Red Absorbance for Real-Time pH Measurements

doi: 10.3390/s24175610

Figure Lengend Snippet: Optimized mirror geometry in 316 L stainless steel, using Zemax/optic studio software (version 20.1).

Article Snippet: The mirror’s geometry was optimized using Zemax/optic studio software (version 20.1).

Techniques: Software

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of the N-terminal preproTRH(31–52) and C-terminal preproTRH(241–255) sequence deletion on precursor protein levels in AtT20 cells under steady state conditions. A, Western blot analysis of extracted peptides from AtT20 cells transfected with wild type (WT) proTRH, Δ31–52, or Δ241–255 mutants. After 48 h of transfection, levels of wild type (26 kDa) and mutated (∼23–24 kDa) prohormone levels were analyzed by densitometric analysis of the Western blot signals (n = 4, ±S.E.). Student's t test was done to compare each with wild type (*, p < 0.05). B, proTRH gene expression analysis. Transcript levels of wild type and Δ31–52 under steady state conditions were assayed by reverse transcription-PCR (n = 3; ± S.E.). Δ31–52 mRNA was transcript as much efficiently as than wild type. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, proTRH gene translation analysis. Protein translation for wild type and mutant Δ31–52 was performed using an in vitro cell free translation system (see “Experimental Procedures”). After protein immunoprecipitation,3H-labeled translation products were separated onto 12% SDS-PAGE polyacrylamide gels. The gels were sliced, and the cpm for each slice was counted. The peaks depicted in the graph represent the amount of prohormone (26 kDa) translated. Equal amount of protein was observed in both wild type and Δ31–52 (n = 4, ±S.E.). NC represents none DNA on the reaction.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Sequencing, Western Blot, Transfection, Gene Expression, Reverse Transcription, Mutagenesis, In Vitro, Immunoprecipitation, Labeling, SDS Page

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Analysis of Δ31–52 prohormone degradation by proteasome and lysosome systems. AtT20 cells transfected with wild type proTRH or Δ31–52 were treated with vehicle solution or proteasome inhibitor lactacystin (5 μm) for 16 h (A) or BFA (5 μg/ml) for 4 h (B). Equal amounts of protein from each sample were immunoblotted with anti-pCC10 antibody that recognizes the entire precursor protein (26 kDa for wild type (WT) and 23 kDa for Δ31–52 mutant) or anti-β-tubulin. NC represents AtT20 cells transfected with pEGFP-N1 plasmid as negative control. Protein levels were quantitated by AlphaEraseFC software. Graphics show the ratio between cells treated/untreated in wild type and mutant Δ31–52. Prohormone levels of Δ31–52 are recovered ∼2-fold by proteasomal inhibitor and BFA treatment (n = 4; ±S.E.). A representative blot from a single experiment is shown.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Negative Control, Software

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of specific mutations within the N-terminal proregion on protein abundance under steady state conditions in ATt20 and GH4C1. Wild type (WT) and PGL mutant DNAs were transfected into AtT20 (A) and GH4C1 (C) cells. NC indicates cells transfected with pEGFP-N1 plasmid. 48 h after transfection cells were lysed for immunoblotting with anti-pCC10. This antibody recognizes the precursor (∼26 kDa for ΔPGL, PGL/EKA, PGL/GGG, and PGL/AAA ∼23 kDa for Δ31–52), but it also recognizes N-terminal intermediate forms of the precursor (15 and 9.5 kDa). Precursor protein levels (26 kDa) are expressed as percentages of the levels in wild type cells. Values represent the mean ± S.E., n = 3. *, p < 0.05 relative to wild type by Student's t test. B, wild type, AVT, and R51G/R52G mutant DNAs were transfected into AtT20 cells, and the precursor protein levels were analyzed as described above (n = 3; ± S.E.). *, p < 0.05 relative to wild type by Student's t test. NC, negative control.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Quantitative Proteomics, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Negative Control

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Analysis of proTRH-derived peptides secretion in wild type and N-terminal mutants. N-terminal (pEH24) (A), C-terminal (5.4 kDa) (B), and TRH (C) proTRH derived peptides from 1-h basal release (BR), 1-h stimulated release (SR) with 1 mm BaCl2, and cell content (CC) were measured by radioimmunoassay in AtT20 cell expressing wild type (WT) and mutants Δ31–52, PGL/EKA, ΔPGL, PGL/GGG, PGL/AAA, R51G/R52G, and AVT/GGG. Peptides levels in each fraction are expressed as percentage of the levels in wild type cells (100%) (n = 3; ± S.E.). *, p < 0.05 relative to wild type by Student's t test. See Table 1 for a description of mutants.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Derivative Assay, RIA Assay, Expressing

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of proteasome inhibition in wild type (WT), ΔPGL, and PGL/GGG on precursor protein levels. AtT20 cells expressing ΔPGL (A) or PGL/GGG (B) mutants and AtT20 cells expressing wild type preproTRH were exposed to vehicle solution (–) or 5 μm lactacystin (+) for 16 h. NC represents AtT20 cells transfected with pEGFP-N1 plasmid. Cells extracts were prepared, and equal amounts of protein (30 μg) were analyzed by Western blotting with anti pCC10. Protein recovered is expressed as the ratio lactacystin+/lactacystin–. Inhibition of the proteasome induced 2-fold recovered ΔPGL precursor compared with wild type (A) but was 1.3-fold lower for PGL/GGG precursor (n = 4, ±S.E.). NC, negative control.

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot, Negative Control

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Effect of ER transport with Brefeldin A in wild type, ΔPGL, and PGL/GGG on precursor protein levels. AtT20 cells expressing ΔPGL (A) or PGL/GGG (B) mutants and AtT20 cells expressing wild type (WT) preproTRH were exposed to vehicle solution (–) or 5 μg/ml BFA (+) for 4 h. Cells extracts were prepared, and equal amounts of protein (30 μg) were analyzed by Western blotting with anti pCC10. Protein recovered was expressed as the ratio BFA+/BFA–. For 4 h of incubation, BFA proportionally induces an equal accumulation in wild type and both ΔPGL and PGL/GGG precursors (n = 4, ±S.E.).

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Expressing, Western Blot, Incubation

Journal:

Article Title: Role of a Pro-sequence in the Secretory Pathway of Prothyrotropin-releasing Hormone *

doi: 10.1074/jbc.M803413200

Figure Lengend Snippet: Determination of wild type proTRH, ΔPGL, and PGL/GGG precursor half-lives by pulse-chase experiments. 48 h after AtT20 cells transfection with wild type (WT), ΔPGL, and PGL/GGG DNA constructs, protein precursors were radiolabeled for 2 h with [3H]leucine, and protein levels (cpm) were followed at 0-, 45-, and 90-min chase times. Graphics show the evolution of proTRH precursor (26 kDa) through 90 min. The results are the means ± S.E. (n = 3).

Article Snippet: All mutants generated were confirmed by DNA sequencing. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Nomenclature Point mutation Description proTRH Wild type Wild type preproTRH 1-255 Δ31-52 preproTRH 31-52 Deletion of N-terminal 22 amino acids leaving intact the first 6 amino acids preceded by the signal sequence Δ241-255 preproTRH 241-255 Deletion of C-terminal 15 amino acids PGL/EKA preproTRH 40-42 Pro-Gly-Leu replaced by Glu-Lys-Ala ΔPGL preproTRH 40-42 Deletion of Pro-Gly-Leu PGL/GGG preproTRH 40-42 Pro-Gly-Leu replaced by Gly-Gly-Gly AVT/GGG preproTRH 34-36 Ala-Val-Thr replaced by Gly-Gly-Gly ΔAVT preproTRH 34-36 Deletion of Ala-Val-Thr R51G/R52G preproTRH 51,52 Arg-Arg replaced by Gly-Gly PGL/AAA preproTRH 40-42 Pro-Gly-Leu replaced by Ala-Ala-Ala Open in a separate window List of the mutant constructs used in this study Cell Culture and Transient Transfection —AtT20 mouse pituitary cells (D16v-F2 subclone; ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with high glucose, 30 m m sodium bicarbonate, 1 m m sodium pyruvate, 10% fetal bovine serum, and 0.1% penicillin/streptomycin (Invitrogen) at 37 °C and 5% CO 2 as previously described ( 20 ).

Techniques: Pulse Chase, Transfection, Construct