Spin Trapping by Use of N-Tert-Butylhydroxylamine. Involvement of Fenton Reactions

Spin trapping of short-lived R. radicals is done by use of N-tert-butylhydroxylamine (1) and H²O². The hydroxylamine is oxidized to the radical t-BuN(O)H (2) which is converted into the spin trap 2-methyl-2-nitrosopropane (3). Simultaneously, hydroxyl radicals. OH are formed from H²O². The latter radical species abstracts hydrogen atoms from suitable molecules HR to give R. radicals, which are trapped with the formation of aminooxyl radicals, i. e., t-BuN(O)R (4) detectable by EPR spectroscopy. The reaction is enhanced by the presence of iron ions. The cleavage of H²O² into. OH radicals is considered to involve both a radical-driven (t-BuN(O)H 2) and an iron-driven Fenton reaction.     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

PKC phosphorylates GluA1-Ser831 to enhance AMPA receptor conductance

AMPA receptors mediate fast excitatory synaptic transmission in the brain, and are dynamically regulated by phosphorylation of multiple residues within the C-terminal domain. CaMKII phosphorylates Ser831 within the AMPA receptor GluA1 subunit to increase single channel conductance, and biochemical studies show that PKC can also phosphorylate this residue. In light of the discovery of additional PKC phosphorylation sites within the GluA1 C-terminus, it remains unclear whether PKC phosphorylation of Ser831 increases GluA1 conductance in intact receptors. Here, we report that the purified, catalytic subunit of PKC significantly increases the conductance of wild-type GluA1 AMPA receptors expressed in the presence of stargazin in HEK293T cells. Furthermore, the mutation GluA1-S831A blocks the functional effect of PKC. These findings suggest that GluA1 AMPA receptor conductance can be increased by activated CaMKII or PKC, and that phosphorylation at this site provides a mechanism for channel modulation via a variety of protein signaling cascades.     

Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.     

Production of Ethanol from Maltose by Zymobacter palmae Fermentation

The production of ethanol from maltose by Zymobacter palmae T109 in monoculture fermentations, and in co-culture fermentations together with Zymomonas mobilis B69 was studies. Zymobacter palmae T109, produced 5.5% (w/v) of ethanol when co-cultured with Zymomonas mobilis B69, but Zymobacter palmae T109 produced only 4.9% (w/v) ethanol from 15% (w/v) maltose medium in monoculture fermentation.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

Analyzing the effect of mutations in SARS-CoV2 papain-like protease from Saudi isolates on protein structure and drug-protein binding: Molecular modelling and dynamics studies

The continuous and rapid development of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus remains a health concern especially with the emergence of numerous variants and mutations worldwide. As with other RNA viruses, SARS-CoV-2 has a genetically high mutation rate. These mutations have an impact on the virus characteristics, including transmissibility, antigenicity and development of drug and vaccine resistance. This work was pursued to identify the differences that exist in the papain-like protease (PL^Pro) from 58 Saudi isolates in comparison to the first reported sequence from Wuhan, China and determine their implications on protein structure and the inhibitor binding. PL^pro is a key protease enzyme for the host cells invasion and viral proteolytic cleavage, hence, it emerges as a valuable antiviral therapeutic target. Two mutations were identified including D108G and A249V and shown to increase the molecular flexibility of PL^Pro protein and alter the protein stability, particularly with D108G mutation. The effect of these mutations on the stability and dynamic behavior of PL^Pro structures as well as their effect on the binding of a known inhibitor; GRL0617 were further investigated by molecular docking and dynamic simulation.