Control of outer vestibule dynamics and current magnitude in the Kv2.1 potassium channel

In Kv2.1 potassium channels, changes in external [K+] modulate current magnitude as a result of a K+-dependent interconversion between two outer vestibule conformations. Previous evidence indicated that outer vestibule conformation (and thus current magnitude) is regulated by the occupancy of a selectivity filter binding site by K+. In this paper, we used the change in current magnitude as an assay to study how the interconversion between outer vestibule conformations is controlled. With 100 mM internal K+, rapid elevation of external [K+] from 0 to 10 mM while channels were activated produced no change in current magnitude (outer vestibule conformation did not change). When channels were subsequently closed and reopened in the presence of elevated [K+], current magnitude was increased (outer vestibule conformation had changed). When channels were activated in the presence of low internal [K+], or when K+ flow into conducting channels was transiently interrupted by an internal channel blocker, increasing external [K+] during activation did increase current magnitude (channel conformation did change). These data indicate that, when channels are in the activated state under physiological conditions, the outer vestibule conformation remains fixed despite changes in external [K+]. In contrast, when channel occupancy is lowered, (by channel closing, an internal blocker or low internal [K+]), the outer vestibule can interconvert between the two conformations. We discuss evidence that the ability of the outer vestibule conformation to change is regulated by the occupancy of a nonselectivity filter site by K+. Independent of the outer vestibule-based potentiation mechanism, Kv2.1 was remarkably insensitive to K+-dependent processes that influence current magnitude (current magnitude changed by <7% at membrane potentials between -20 and 30 mV). Replacement of two outer vestibule lysines in Kv2.1 by smaller neutral amino acids made current magnitude dramatically more sensitive to the reduction in K+ driving force (current magnitude changed by as much as 40%). When combined, these outer vestibule properties (fixed conformation during activation and the presence of lysines) all but prevent variation in Kv2.1 current magnitude when [K+] changes during activation. Moreover, the insensitivity of Kv2.1 current magnitude to changes in K+ driving force promotes a more uniform modulation of current over a wide range of membrane potentials by the K+-dependent regulation of outer vestibule conformation.     

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C₃ plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C₄ plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C₄ plants, 0.5% in the CAM plant, and less than 0.01% in C₃ plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C₃ and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C₃ and CAM plants than to those of C₄ plants.     

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote™ as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote™, as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the silicon-coating of glass petri dishes by Sigmacote™ is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.     

A Mutation Causes MuSK Reduced Sensitivity to Agrin and Congenital Myasthenia

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A>G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient’s biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation.     

Exploration and local utilization of medicinal vegetation naturally grown in the Deusai plateau of Gilgit,Pakistan

In the study area, the traditional knowledge regarding the uses of local wild medicinal plants for the health care of human and domestic animals is totally in hold of old people. The young ones are unaware about such an indispensable practices. The objective of the current study was to transfer this sort of precious knowledge from old members of the community to young generations in documented form.     

Cytokines as potential combination agents with PD-1/PD-L1 blockade for cancer treatment

Immunotherapy has caused a paradigm shift in the treatment of several malignancies, particularly the blockade of programmed death-1 (PD-1) and its specific receptor/ligand PD-L1 that have revolutionized the treatment of a variety of malignancies, but significant durable responses only occur in a small percentage of patients, and other patients failed to respond to the treatment. Even those who initially respond can ultimately relapse despite maintenance treatment, there is considerable potential for synergistic combinations of immunotherapy and chemotherapy agents with immune checkpoint inhibitors into conventional cancer treatments. The clinical experience in the use of cytokines in the clinical setting indicated the efficiency of cytokine therapy in cancer immunotherapy. Combinational approaches to enhancing PD-L1/PD-1 pathways blockade efficacy with several cytokines such as interleukin (IL)-2, IL-15, IL-21, IL-12, IL-10, and interferon-α (IFN-α) may result in additional benefits. In this review, the current state of knowledge about PD-1/PD-L1 inhibitors, the date in the literature to ascertain the combination of anti-PD-1/PD-L1 antibodies with cytokines is discussed. Finally, it is noteworthy that novel therapeutic approaches based on the efficient combination of recombinant cytokines with the PD-L1/PD-1 blockade therapy can enhance antitumor immune responses against various malignancies.     

Endothelial cells' biophysical,biochemical, and chromosomal aberrancies in high‐glucose condition within the diabetic range

To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia‐induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells—especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high‐glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high‐glucose condition, the dynamic of the fatty acid profile—including polyunsaturated, monounsaturated, and saturated fatty acids—was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31⁺/CD34⁺) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell‐cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)‐1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold‐coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.     

Radiometrically determined dates and sedimentation rates for recent sediments in nine North African wetland lakes (the CASSARINA Project)

Sediment cores were collected from nine wetland lakes in Morocco, Tunisia and Egypt for the CASSARINA project investigating environmental change in Northern African wetlands. The cores were dated radiometrically by using natural (²¹⁰Pb) and artificial (¹³⁷Cs and ²⁴¹Am) radionuclides. At sites in Morocco and Tunisia with mean annual rainfall totals ranging from 500–1000 mm yr^–1, fallout records were generally satisfactory and it was possible to develop independent sediment chronologies based on the radiometric data alone. At the Egyptian sites, rainfall was less than 200 mm yr^–1 and fallout records were much less distinct. At these sites the radiometric data could only be used to give an indication of mean sedimentation rates during the past 30–40 years. By using a combination of fallout radionuclide, pollen, and macrofossil stratigraphic records it was however possible to determine a credible sediment chronology spanning the major part of the 20th century. Applying this chronology to records of spheroidal carbonaceous particles (SCP) from the same sediment cores, the onset of significant levels of atmospheric pollution in the Nile Delta is dated in all three cores to the mid 1950s. Results from a number of lakes (Sidi Bou Rhaba, Ichkeul and Korba) revealed high and accelerating siltation rates, threatening their continued existence beyond the next few decades. In contrast, sedimentation rates at all three Nile Delta sites appear to have declined in recent decades, most probably due to the impact of the Nile barrages.