ALBUMIN CAN REVERSE THE RELEASE OF POTASSIUM FROM HUMAN ERYTHROCYTES TREATED WITH THE NON-IONIC DETERGENT,BRIJ 58

Exchange of erythrocyte intracellular (i/c) K⁺for extracellular (e/c) Na⁺in human erythrocytes treated with sub-CMC concentrations of the non-ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K⁺contents of detergent-treated human erythrocytes with the incubation medium was reversed by an albumin-mediated withdrawal of detergent molecules from the cell. Re-establishment of near normal [K⁺] in terms of K⁺/kg water proceeds in two ways: (i) a metabolism-dependent net accumulation of K⁺ions; and (ii) a metabolism-independent shrinkage of erythrocytes, this being the more significant factor.     

Serum Level of Soluble Receptor for Advanced Glycation End Products Is Associated with A Disintegrin And Metalloproteinase 10 in Type 1 Diabetes

Background

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of diabetic complications, and soluble forms of the receptor (sRAGE) can counteract the detrimental action of the full-length receptor by acting as decoy. Soluble RAGE is produced by alternative splicing [endogenous secretory RAGE (esRAGE)] and/or by proteolytic cleavage of the membrane-bound receptor. We have investigated the role of A Disintegrin And Metalloproteinase 10 (ADAM10) in the ectodomain shedding of RAGE.

Methods

Constitutive and insulin-induced shedding of RAGE in THP-1 macrophages by ADAM10 was evaluated using an ADAM10-specific metalloproteinase inhibitor. Serum ADAM10 level was measured in type 1 diabetes and control subjects, and the association with serum soluble RAGE was determined. Serum total sRAGE and esRAGE were assayed by ELISA and the difference between total sRAGE and esRAGE gave an estimated measure of soluble RAGE formed by cleavage (cRAGE).

Results

RAGE shedding (constitutive and insulin-induced) was significantly reduced after inhibition of ADAM10 in macrophages, and insulin stimulated ADAM10 expression and activity. Diabetic subjects have higher serum total sRAGE and esRAGE (p<0.01) than controls, and serum ADAM10 was also increased (p<0.01). Serum ADAM10 correlated with serum cRAGE in type 1 diabetes (r = 0.40, p<0.01) and in controls (r = 0.31. p<0.01) but no correlations were seen with esRAGE. The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.

Conclusion

Our data suggested that ADAM10 contributed to the shedding of RAGE. Serum ADAM10 level was increased in type 1 diabetes and was a significant determinant of circulating cRAGE.     

The interplay of cyclic stretch and vascular endothelial growth factor in regulating the initial steps for angiogenesis

Angiogenesis is regulated by chemical and mechanical factors in vivo. The regulatory role of mechanical factors and how chemical and mechanical angiogenic regulators work in concert remains to be explored. We investigated the effect of cyclic uniaxial stretch (20%, 1 Hz), with and without the stimulation of vascular endothelial growth factor (VEGF), on sprouting angiogenesis by employing a stretchable three‐dimensional cell culture model. When compared to static controls, stretch alone significantly increased the density of endothelial sprouts, and these sprouts aligned perpendicular to the direction of stretch. The Rho‐associated kinase (ROCK) inhibitor Y27632 suppressed stretch‐induced sprouting angiogenesis and associated sprout alignment. While VEGF is a potent angiogenic stimulus through ROCK‐dependent pathways, the combination of VEGF and stretch did not have an additive effect on angiogenesis. In the presence of VEGF stimulation, the ROCK inhibitor suppressed stretch‐induced sprout alignment but did not affect stretch‐induced sprout density; in contrast, the receptor tyrosine kinase (RTK) inhibitor sunitinib had no effect on stretch‐induced alignment but trended toward suppressed stretch‐induced sprout density. Our results suggest that the formation of sprouts and their directionality do not have completely identical regulatory pathways, and thus it is possible to separately manipulate the number and pattern of new sprouts. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:248–257, 2015     

Asymmetric boundary shifts of tropical montane Lepidoptera over four decades of climate warming

Aim To estimate whether species have shifted at equal rates at their leading edges (cool boundaries) and trailing edges (warm boundaries) in response to climate change. We provide the first such evidence for tropical insects, here examining elevation shifts for the upper and lower boundaries shifts of montane moths. Threats to species on tropical mountains are considered. Location Mount Kinabalu, Sabah, Malaysia. Methods We surveyed Lepidoptera (Geometridae) on Mount Kinabalu in 2007, 42 years after the previous surveys in 1965. Changes in species upper and lower boundaries, elevational extents and range areas were assessed. We randomly subsampled the data to ensure comparable datasets between years. Estimated shifts were compared for endemic versus more widespread species, and for species that reached their range limits at different elevations. Results Species that reached their upper limits at 2500–2700 m (n= 28 species, 20% of those considered) retreated at both their lower and upper boundaries, and hence showed substantial average range contractions (?300 m in elevational extent and ?45 km² in estimated range area). These declines may be associated with changes in cloud cover and the presence of ecological barriers (geological and vegetation transitions) which impede uphill movement. Other than this group, most species (n= 109, 80% of the species considered) expanded their upper boundaries upwards (by an average of 152 m) more than they retreated at their lower boundaries (77 m). Main conclusions Without constraints, leading margins shifted uphill faster than trailing margins retreated, such that many species increased their elevational extents. However, this did not result in increases in range area because the area of land available declines with increasing elevation. Species close to a major ecological/geological transition zone on the mountain flank declined in their range areas. Extinction risk may increase long before species reach the summit, even when undisturbed habitats are available.     

Parallel gene cloning and protein production in multiple expression systems

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5′‐EcoRI/3′‐XhoI identical in these vectors for ligating with the sticky‐end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.     

Bax Activates Endophilin B1 Oligomerization and Lipid Membrane Vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.