Comparing Methods for Analysis of Pupillary Response

Changes in eye-pupil size index a range of cognitive processes. However, variations in the protocols used to analyze such data exist in the psychological literature. This raises the question of whether different approaches to pupillary response data influence the outcome of the analysis. To address this question, four methods of analysis were compared, using pupillary responses to sexually appetitive visual content as example data. These methods comprised analysis of the unadjusted (raw) pupillary response data,z-scored data, percentage-change data, and data transformed by a prestimulus baseline correction. Across two experiments, these methods yielded near-identical outcomes, leading to similar conclusions. This suggests that the range of approaches that are employed in the psychological literature to analyze pupillary response data do not fundamentally influence the outcome of the analysis. However, some systematic carryover effects were observed when a prestimulus baseline correction was applied, whereby dilation effects from an arousing target on one trial still influenced pupil size on the next trial. This indicates that the appropriate application of this analysis might require additional information, such as prior knowledge of the duration of carryover effects

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Automated large-scale isolation, in vitro function and xenotransplantation of porcine islets of Langerhans

Abstract: To evaluate the potential of utilizing porcine islet tissue as an alternative to human islet tissue for transplantation, we developed a method for the isolation of large amounts of highly purified porcine islets, and assessed the in vitro and in vivo function of the isolated islets after 1, 4, and 7 days of culture. The pancreatic duct of the splenic lobe was cannulated and distended by, injection of Hanks' balanced salt solution containing 1.5 mg/ml collagenase. The pancreas was then processed by a modification of the automated digestion-filtration method developed in this laboratory, and with purification accomplished by Euro-Ficoll gradients (dialyzed Ficoll in Eurocollins solution), consisting of two layers of 1.108 and 1.091 g/cm3 density, topped with a layer of HBSS. The postpurification yield was 5203 +/- 645 (mean +/- SEM) islets per gram of pancreas with a number of islet equivalents (IE) per gram pancreas (islet equivalence: 150-mu-m-sized islets) of 3551 +/- 305, and a volume of 6.27 +/- 1.7 mm3 islet tissue per gram of pancreas. The islet purity exceeded 90%. Overnight-cultured, perifused porcine islets released 53.1 +/- 8.2 pM insulin/200 IE at 3.3 mM glucose, and 114.9 +/- 25.4 p.M insulin/200 IE at 16.7 mM glucose (P < 0.001 vs. basal output). When theophylline was added, insulin secretion increased to 264.2 +/- 63.2 pM/200 IE (P < 0.001 vs. basal secretion and P < 0.005 vs. secretion at 16.7 mM glucose). After 4 days of culture, the islets still responded to secretagogues. The functional integrity of the isolated islets was confirmed by reversal of diabetes in aL3T4 antibody-treated C57B/B6 diabetic mice: normoglycemia was promptly restored by transplanting 1000 over-night- or 7-day-cultured (24-degrees-C) islets under the kidney capsule. These results suggest that continued improvements of porcine islet isolation and culture could permit the use of porcine islets in immunoalteration and immunoisolation studies that may lead to eventual human transplantation

Factors affecting in vitro and in vivo function of isolated porcine islets

[abstract non disponibile

Prevention of contamination of isolated porcine islets of Langerhans

[abstract non disponibile

Insulin inhibits its own secretion from isolated, perifused human pancreatic islets

Abstract: It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification, Aliquots of 200 islet equivalents (IE, 150-mu m sized-islets) were sequentially perifused at 37 degrees C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37 degrees C culture in CMRL 1066 medium with or without the addition of either 200 or 400 mu U/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perifusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) mu U/ml added insulin, basal Cp release was 0.12 +/- 0.03, 0.14 +/- 0.02 and 0.14 +/- 0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47 +/- 0.13 ng/ml, P < 0.02) and Ins400 (0.68 +/- 0.15 ng/ml, P < 0.05) than C (0.83 +/- 0.15 ng/ml). The second-phase secretion peak was also significantly (P < 0.05) reduced with added insulin (Ins200: 0.47 +/- 0.08 ng/ml; Ins400: 0.45 +/- 0.07 ng/ml) than in its absence (C: 0.65 +/- 0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6 +/- 2.3 ng/ml, P = 0.03) and Ins400 (11.8 +/- 2.3 ng/ml) than with C (16.0 +/- 2.2 ng/ml). Thus, the addition for 24 h of either 200 or 400 mu U/ml insulin in the culture medium caused a significant decrease of insulin (as assessed by Cp measurement) secretion from perifused human islets, suggesting that feedback suppression of insulin release is at least in part due to a direct action of insulin on the islets

Cryogenic storage of isolated, purified porcine pancreatic islets

Abstract: Highly purified islets of Langerhans were prepared in the present study from adult pigs by collagenase digestion and density gradient purification. After overnight culture, the tissue was equilibrated with DMSO at 25 degrees C, supercooled to -7.5 degrees C, nucleated, slowly cooled at 0.25 degrees C/min to -40 degrees C, and stored at -130 degrees C. Then, after variable periods of storage, the islets were rapidly thawed at 37 degrees C. Postthaw actual islet and islet equivalent (150-mu m sized islets) recovery were 75+/-7% and 66+/-4%, respectively. The frozen-thawed porcine islets maintained good morphology on histological staining by hematoxylin-eosin and aldehyde-fuchsin. Upon perifusion, basal insulin secretion was 43+/-10 and 67+/-18 pmol/L from noncryopreserved, control islets, and cryopreserved islets, respectively (P=0.2). Peak insulin release at 16.7 mmol/L glucose was 85+/-28 pmol/L from noncryopreserved islets and 157+/-48 pmol/L from the frozen-thawed islets (P=0.1). When 10 mmol/L theophylline was added to 16.7 mmol/L glucose, the secretion of the hormone peaked to 221+/-83 (control islets) and 479+/-140 pmol/L (cryopreserved islets, P=0.1). Total insulin secretion differed significantly for the noncryopreserved and the cryopreserved islets at both 16.7 mmol/L (1412+/-306 vs. 3756+/-764 pmol/L, respectively, P=0.007) and 16.7 mmol/L glucose plus 10 mmol/L theophylline (2161+/-371 vs. 7505+/-2075 pmol/L, respectively, P=0.011). Normoglycemia was restored within 7 days from implantation in temporarily immunosuppressed (aL3T4 antibody) mice with streptozotocin-induced diabetes by transplanting 1500-2000 cryopreserved porcine islets under the kidney capsule. Mean survival time of frozen-thawed islet xenografts (39+/-3 days) was similar to that of noncryopreserved islet xenografts (43+/-6 days). This study demonstrates that cryogenic storage is feasible of isolated porcine islets, with the frozen-thawed pancreatic endocrine tissue maintaining morphological integrity and both in vitro and in vivo viability. Further studies are needed to define the effect of cryopreservation on the immunogenic properties of porcine islets