intact human proinsulin  (BioVendor Instruments)

Journal: PLoS ONE

Article Title: Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

doi: 10.1371/journal.pone.0059222

Figure Lengend Snippet: ( a, b ) HEK293 cells were transduced with rAd-proinsulin-WT ( a ) or –B10 ( b ) as indicated and media samples were assayed 24 hr later for both hInsulin and hProinsulin expression. Bars indicate mean+SEM concentration of hInsulin (open bars, left axes) or of hProinsulin (filled bars, right axes) for n = 3 samples per group. ( c,d ) Rat A5 salivary gland cells and mouse NIT1 pancreatic beta-cells were transduced with rAd-proinsulin-WT ( c ) or –B10 ( d ) particles at the doses indicated. Media were collected 24 h later and insulin species were quantified by ELISAs. Bars indicate mean concentration of hInsulin ( c ) or hProinsulin ( d ) in the media of n = 2 samples.

Article Snippet: All measurements of hProinsulin were made in duplicate with the Intact Human Proinsulin ELISA Kit (Millipore, Billerica, MA).

Techniques: Transduction, Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

doi: 10.1371/journal.pone.0059222

Figure Lengend Snippet: Confluent HEK293 cells were switched to serum-free medium with or without rAd-proinsulin-WT or rAd-proinsulin-B10; 24 h later, conditioned media (CM) were collected and hProinsulin concentration was determined by ELISA. Fresh HEK293 cells were seeded, serum starved for 2 h, and treated for 15 min with Control CM (no vector), 10 nM hProinsulin-WT CM (WT proins), 10 nM hProinsulin-B10 CM (B10 proins) or 10 nM recombinant human insulin (rhInsulin). Protein lysates were collected and analyzed by western immunoblot for phospho-Akt(Ser473) and total Akt. Immunoblot band intensities were measured and bars indicate mean+SEM of the P/T Akt ratio for n = 3 samples; *, P<0.0001 vs. control, **, P<0.0001 vs. all (ANOVA).

Article Snippet: All measurements of hProinsulin were made in duplicate with the Intact Human Proinsulin ELISA Kit (Millipore, Billerica, MA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Recombinant, Western Blot

Journal: PLoS ONE

Article Title: Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

doi: 10.1371/journal.pone.0059222

Figure Lengend Snippet: rAd-proinsulin-B10 particles or saline (Control) was delivered by I.V. injection. Serum samples were obtained at 24 h and 48 h post-transduction and saliva, parotid gland (PTG), submandibular gland (SMG), liver and spleen tissues were collected at the conclusion of the experiment (48 h). ( a ) Blood glucose (mg/dl) measurements at 24 h and 48 h post-transduction. No statistical difference in glucose levels was observed between groups (ANOVA) ( b ) Sera, saliva and protein lysates from PTG and SMG were assayed for hProinsulin (pM) by ELISA. ( c ) QPCR analysis of rAd vector genome tissue distribution at the conclusion of the experiment. Data are expressed as rAd genome copies detected in 100 ng of tissue genomic DNA. For all panels, bars indicate mean+SEM of n = 3 mice per group.

Article Snippet: All measurements of hProinsulin were made in duplicate with the Intact Human Proinsulin ELISA Kit (Millipore, Billerica, MA).

Techniques: Saline, Control, Injection, Transduction, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

doi: 10.1371/journal.pone.0059222

Figure Lengend Snippet: rAd-proinsulin-B10 or saline (Control) was delivered to both PTGs of non-diabetic mice by retroductal infusion. Blood samples were obtained at 24 h and 48 h post-transduction and saliva and PTG samples were collected at the conclusion of the experiment (48 h). ( a ) Blood glucose (mg/dl) measurements at 24 h and 48 h post-transduction. No statistical difference in glucose levels was observed between groups (ANOVA) ( b ) Sera, saliva and PTG protein lysates were assayed for hProinsulin (pM) by ELISA. ( c ) QPCR analysis of rAd vector genome distribution in tissues at the conclusion of the experiment. Data are expressed as rAd genome copies detected in 100 ng of genomic DNA. For all panels, ppg = particles per gland and bars indicate mean+SEM of n = 4 mice for 10 9 ppg and Control groups, and n = 5 for all other groups.

Article Snippet: All measurements of hProinsulin were made in duplicate with the Intact Human Proinsulin ELISA Kit (Millipore, Billerica, MA).

Techniques: Saline, Control, Transduction, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

doi: 10.1371/journal.pone.0059222

Figure Lengend Snippet: rAd-proinsulin-B10 or saline (Control) was delivered to both SMG by retroductal infusion. Serum samples were obtained at 24 h and 48 h post-transduction and saliva and SMG samples were collected at the conclusion of the experiment (48 h). ( a ) Blood glucose (mg/dl) measurements at 24 h and 48 h post-transduction. ( b ) Sera, saliva and SMG protein lysates were assayed for hProinsulin (pM) by ELISA. ( c ) QPCR analysis of rAd vector genome tissue distribution at the conclusion of the experiment. Data are expressed as rAd genome copies detected in 100 ng of genomic DNA. For all panels, ppg = particles per gland and bars indicate mean+SEM of n = 4; *, P≤0.021 vs. all at 48 h (ANOVA).

Article Snippet: All measurements of hProinsulin were made in duplicate with the Intact Human Proinsulin ELISA Kit (Millipore, Billerica, MA).

Techniques: Saline, Control, Transduction, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Expression and Secretion of Human Proinsulin-B10 from Mouse Salivary Glands: Implications for the Treatment of Type I Diabetes Mellitus

doi: 10.1371/journal.pone.0059222

Figure Lengend Snippet: Both SMGs of ALX-diabetic mice were transduced with 5×10 9 particles of either rAd-Control (empty vector) or rAd-proinsulin-B10. Sera were collected 24 h later. ( a ) ELISA analysis of hProinsulin (pM) in serum, *P = 0.0003 vs. rAd-Control. ( b ) Serum glucose levels, *P = 0.036 vs. rAd-Control. For all panels, bars indicate mean+SEM for n = 7 animals per treatment group. Statistical significance was determined by unpaired Student t test followed by Mann-Whitney post hoc analysis.

Article Snippet: All measurements of hProinsulin were made in duplicate with the Intact Human Proinsulin ELISA Kit (Millipore, Billerica, MA).

Techniques: Transduction, Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY