Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of synthetic peptide octarphin (TPLVTLFK) with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of non‐opioid β‐endorphin receptor, was labeled with tritium to specific activity of 29 Ci/mmol. The analysis of [³H]octarphin binding to human T and B lymphocytes separated from normal human blood revealed the existence of one type of high‐affinity binding sites (receptors): K_d 3.0 and 3.2 nM, respectively. Besides unlabeled octarphin, unlabeled β‐endorphin possessed the ability to inhibit the specific binding of [³H]octarphin to Т and B lymphocytes (K_i 1.9 and 2.2 nМ, respectively). Tests of the specificity of the receptors revealed that they are not sensitive to naloxone, α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin. Thus, both T and B lymphocytes from normal human blood express non‐opioid receptor for β‐endorphin. Binding of the hormone to the receptor provides a fragment 12–19. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Wide-Area Estimates of Stand Structure and Water Use of Tamarix spp. on the Lower Colorado River: Implications for Restoration and Water Management Projects

Tamarix spp. removal has been proposed to salvage water and allow native vegetation to recolonize western U.S. riparian corridors. We conducted wide‐area studies on the Lower Colorado River to answer some of the scientific questions about Tamarix water use and the consequences of removal, combining ground surveys with remote sensing methods. Tamarix stands had moderate rates of evapotranspiration (ET), based on remote sensing estimates, averaging 1.1 m/yr, similar to rates determined for other locations on the river and other rivers. Leaf area index values were also moderate, and stands were relatively open, with areas of bare soil interspersed within stands. At three Tamarix sites in the Cibola National Wildlife Refuge, groundwater salinity at the site nearest to the river (200 m) was relatively low (circa 2,250 mg/L) and was within 3 m of the surface. However, 750 and 1,500 m from the river, the groundwater salinity was 5,000–10,000 mg/L due to removal of water by the Tamarix stands. Despite the high groundwater salinity, the sites away from the river did not have saline surface soils. Only 1% of the mean annual river flow is lost to Tamarix ET on the Lower Colorado River in the United States, and the opportunities for water salvage through Tamarix removal are constrained by its modest ET rates. A possible alternative to Tamarix removal is to intersperse native plants among the stands to improve the habitat value of the riparian zone.     

KIF13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

Membrane blebbing‐dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D‐environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D‐collagen, while RhoB overexpression enhanced blebbing and 3D‐collagen migration in a manner dependent on its plasma membrane localization and down‐stream effectors ROCK and Myosin II. RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11‐positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D‐migration of ALL cells. In conclusion, KIF13A‐mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.