Hands-on experiments on glycemia regulation and type 1 diabetes

2015 ◽  
Vol 39 (3) ◽  
pp. 232-239 ◽  
Author(s):  
M. Mingueneau ◽  
A. Chaix ◽  
N. Scotti ◽  
J. Chaix ◽  
A. Reynders ◽  
...  

In the present article, we describe a 3-day experimental workshop on glycemia regulation and type 1 diabetes that engages students in open-ended investigations and guided experiments leading to results that are not already known to them. After an initial questioning phase during which students observe PowerPoint slides depicting the glycemia (blood glucose levels) of individuals in various situations, students design, execute, and interpret experiments to address one of the following questions: 1) Which criteria must an animal model of diabetes fulfill? 2) How do pancreatic cells maintain glycemia constant? and 3) Is there a way to produce an insulin protein similar to the one released by human pancreatic cells? Students then 1) measure glycemia and glycosuria in control mice and in a mouse model of type 1 diabetes (Alloxan-treated mice), 2) measure the release of insulin by pancreatic β-cells (INS-1 cell line) in response to different concentrations of glucose in the extracellular medium, and 3) transfect Chinese hamster ovary cells with a plasmid coding for green fluorescent protein, observe green fluorescent protein fluorescence of some of the transfected Chinese hamster ovary cells under the microscope, and observe the characteristics of human insulin protein and its three-dimensional conformation using RASMOL software. At the end of the experimental session, students make posters and present their work to researchers. Back at school, they may also present their work to their colleagues.

1988 ◽  
Vol 8 (5) ◽  
pp. 2229-2232 ◽  
Author(s):  
A M Brunner ◽  
L E Gentry ◽  
J A Cooper ◽  
A F Purchio

Analyses of cDNA clones coding for simian type 1 transforming growth factor beta (TGF-beta 1) suggest that there are three potential sites for N-linked glycosylation located in the amino terminus of the precursor region. Analysis of [3H]glucosamine-labeled serum-free supernatants from a line of Chinese hamster ovary cells which secrete high levels of recombinant TGF-beta 1 indicate that the TGF-beta 1 precursor, but not the mature form, is glycosylated. Digestion with neuraminidase resulted in a shift in migration of the two TGF-beta 1 precursor bands, which suggests that they contain sialic acid residues. Endoglycosidase H had no noticeable effect. Treatment with N-glycanase produced two faster-migrating sharp bands, the largest of which had a molecular weight of 39 kilodaltons. TGF-beta 1-specific transcripts produced by SP6 polymerase programmed the synthesis of a 42-kilodalton polypeptide which, we suggest, is the unmodified protein backbone of the precursor. Labeling with 32Pi showed that the TGF-beta 1 precursor was phosphorylated in the amino portion of the molecule.


1988 ◽  
Vol 8 (5) ◽  
pp. 2229-2232
Author(s):  
A M Brunner ◽  
L E Gentry ◽  
J A Cooper ◽  
A F Purchio

Analyses of cDNA clones coding for simian type 1 transforming growth factor beta (TGF-beta 1) suggest that there are three potential sites for N-linked glycosylation located in the amino terminus of the precursor region. Analysis of [3H]glucosamine-labeled serum-free supernatants from a line of Chinese hamster ovary cells which secrete high levels of recombinant TGF-beta 1 indicate that the TGF-beta 1 precursor, but not the mature form, is glycosylated. Digestion with neuraminidase resulted in a shift in migration of the two TGF-beta 1 precursor bands, which suggests that they contain sialic acid residues. Endoglycosidase H had no noticeable effect. Treatment with N-glycanase produced two faster-migrating sharp bands, the largest of which had a molecular weight of 39 kilodaltons. TGF-beta 1-specific transcripts produced by SP6 polymerase programmed the synthesis of a 42-kilodalton polypeptide which, we suggest, is the unmodified protein backbone of the precursor. Labeling with 32Pi showed that the TGF-beta 1 precursor was phosphorylated in the amino portion of the molecule.


1997 ◽  
Vol 138 (6) ◽  
pp. 1333-1341 ◽  
Author(s):  
Christof Zitt ◽  
Alexander G. Obukhov ◽  
Carsten Strübing ◽  
Andrea Zobel ◽  
Frank Kalkbrenner ◽  
...  

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.


BioTechniques ◽  
2021 ◽  
Author(s):  
Elizabeth R Denn ◽  
Joseph M Schober

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.


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