Amphiphysin I phosphorylation on residue threonine 260 in a pentylenetetrazole-induced seizure model

A method to evaluate kinase inhibitor action was reported [L. Morgan, S.J. Neame, H. Child, R. Chung, B. Shah, L. Barden, J.M. Staddon, T.R. Patel, Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system, Neurosci. Lett. 395 (2006) 143–148]. In this, acute administration of the GABA antagonist pentylenetetrazole triggers seizures through glutamate-dependent pathways. Under such conditions, activation of the c-Jun N-terminal kinase (JNK) pathway was detected in hippocampal extracts. Phosphorylation of the upstream JNK kinase MKK4 was also revealed through use of a phospho-MKK4-specific antibody. Here, this antibody is shown to also react with a protein of ∼125 kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the ∼125 kDa protein as it may provide novel insight into signalling, neuronal activity and seizures. Using chromatographic methods and mass spectrometry, the protein was identified as amphiphysin I. This was confirmed by 2D gel analysis and immunoblot with amphiphysin I-specific antibodies. Although the phospho-MKK4 antibody was raised against an MKK4-specific peptide, partial sequence homology between this sequence and a region of amphiphysin was discerned. New antibodies raised against the phospho-threonine 260-amphiphysin-specific sequence detected increased phosphorylation in response to pentylenetetrazole treatment. This particular phosphorylation site does not seem to have been described before, possibly reflecting a novel regulatory aspect of amphiphysin biology. As amphiphysin is involved in the regulation of endocytosis, phosphorylation at this site may play a role in the regulated re-uptake of synaptic vesicles after neurotransmitter release.     

Neurological Manifestations of COVID-19 Feature T Cell Exhaustion and Dedifferentiated Monocytes in Cerebrospinal Fluid

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Procedure for concentration and conditioning of hybridoma cell culture medium for monoclonal antibody purification

Summary Concentration and conditioning the hybridoma cell culture medium is an important step in the monoclonal antibody purification procedure. This report describes a frequent first-step concentration of the hybridoma cell culture medium and the conditioning of the concentrated medium for the affinity purification of the monoclonal antibodies.     

The enzymatic degradation of polymers in vitro

Specimens of 14C-labeled poly(ethylene terephthalate), nylon 66, and poly(methyl methacrylate) have been synthesized and exposed, in vitro, to a number of enzyme solutions. Poly(ethylene terephthalate) was found to be affected by esterase and papain, although in different ways, but not by trypsin or chymotrypsin. Nylon 66 was unaffected by esterase but degraded by the other three. Poly(methyl methacrylate) was not affected by any of these enzymes. This indicates that some nominally stable polymers are susceptible to degradation by enzymes under some circumstances. The amount of degradation is small, but could have significant sequelae should it be reproduced in vivo.     

Crossing the barrier: Evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis

A paradigm for Streptococcus interspecies gene transfer is represented by the mosaic pbp genes encoding the target enzymes for beta-lactam antibiotics, the penicillin-binding proteins, in Streptococcus pneumoniae. We investigated a collection of oral streptococci from three continents by comprehensive multi-locus sequence typing analysis in order to trace the origin of a mosaic block belonging to a dominant family of mosaic pbp2x implicated in penicillin resistance of S. pneumoniae. One widespread family of mosaic pbp2x occurred in all three distinct clusters of S. pneumoniae, Streptococcus mitis and Streptococcus oralis, documenting independent inter- and intraspecies recombination events. Moreover, potential ancestor genes of this mosaic block could be identified in two penicillin-susceptible S. mitis strains from South Africa and Spain, facilitating the identification of pbp2x mutations relevant for resistance development.     

Protection of granulocytes by vitamin E in glutathione synthetase deficiency.

The administration of vitamin E (alpha-tocopherol) was found to improve polymorphonuclear leukocyte function in an infant with congenital deficiency of glutathione synthetase activity. Before therapy with vitamin E the abnormal leukocytes exposed to phagocytic challenge showed oxidant damage. They released 60 per cent more hydrogen peroxide than did normal leukocytes, iodinated only 20 to 25 per cent of the normal number of particles, and were unable to kill bacteria as effectively as normal leukocytes although the rates of phagocytosis were normal. These functional abnormalities disappeared when the patient was placed on 400 IU of alpha-tocopherol daily for three months. Associated with the functional improvement was a normalization of microtubule assembly during phagocytic challenge.     

ES cell-derived glial precursors contribute to remyelination in acutely demyelinated spinal cord lesions

Pluripotent embryonic stem (ES) cells have emerged as a powerful tool for disease modeling and neural regeneration. Transplantation studies in rodents indicate that ES cell-derived glial precursors (ESGPs) efficiently restore myelin in dysmyelinating mutants and chemically induced foci of myelin loss. Here we explore the myelination potential of ESGPs in an antibody/complement-induced demyelination model. Microinjection of an antibody to myelin oligodendrocyte glycoprotein (MOG) and complement was employed to generate circumscribed areas of demyelination in the adult rat spinal cord. ESGPs transplanted into 2-day-old lesions were found to survive and differentiate into both oligodendrocytes and astrocytes. The engrafted cells remained largely confined to the lesion site and showed no evidence of tumor formation up until 4 weeks after transplantation. Within areas of pronounced microglial activation and macrophage extravasation, engrafted ES cell-derived oligodendrocytes contacted and enwrapped host axons and alongside endogenous glia, contributed to the formation of new myelin sheaths. These findings demonstrate that ESGPs transplanted into acutely demyelinated lesions can contribute to myelin repair.