Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.     

Characterization of the putative operon containing arylamine N-acetyltransferase (nat) in Mycobacterium bovis BCG

Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N-acetyltransferase whose gene is predicted to occur within a six-gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC5) and a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3-dihydroxybiphenyl (2,3-DHB) to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3-DHB or an inhibitor of BphC, 3-chlorocatechol (3-CC). In addition, incubation with 2,3-DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Deltanat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis.     

Developmental Changes in τ Phosphorylation: Fetal τ Is Transiently Phosphorylated in a Manner Similar to Paired Helical Filament-τ Characteristic of Alzheimer's Disease

Rat and human fetal brain τ were probed with a panel of monoclonal antibodies (tau-1, AT8, 8D8, RT97, SMI31, SMI34) that distinguish between paired helical filament (PHF)-τ of Alzheimer's disease and normal adult brain τ. These antibodies discriminate between normal and PHF-τ because their epitopes are phosphorylated in PHF-τ. Although only one molecular isoform of τ was shown to be expressed in fetal brain, two fetal τ species could be distinguished on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the slower migrating species was recognized by all of the PHF-τ-specific antibodies. Moreover, this immunoreactivity was shown to be phosphorylation dependent. Our observations suggest that the abnormal phosphorylation of τ in Alzheimer's disease may be the result of reactivation of pathways governing the phosphorylation of τ in the developing brain.     

Aggregation of Neurofilaments in NF-L Transfected Neuronal Cells: Regeneration of the Filamentous Network by a Protein Kinase C Inhibitor

Abstract: Cytoplasmic inclusion bodies that are accumulations of neurofilaments are the pathological hallmark of many neurodegenerative diseases and have been produced in transgenic mice by overexpression of mouse (NF-L and NF-M; light and medium chains, respectively) and human (NF-M and NF-H; medium and heavy chains, respectively) neurofilament subunits. This report describes a neuronal culture model in which human NF-L was overexpressed to produce cytoplasmic accumulations of neurofilaments within cell bodies concomitant with the collapse of the endogenous neurofilament network. Electron microscopy showed that, within accumulations, neurofilaments retained a filamentous structure. The culture model thus provides a novel system in which the effect on neurofilament accumulations of manipulating protein phosphorylation can be studied. Treatment of cells containing neurofilament accumulations with bisindolylmaleimide, a specific protein kinase C inhibitor, resulted in regeneration of the filamentous network; this effect was not due to a change in the level of transfected NF-L expression. These findings lend support to the suggestion that an impairment in the regulation of protein phosphorylation may lead to the accumulation of neurofilaments seen in neurodegenerative disease.     

A Kainate Receptor Linked to Nitric Oxide Synthesis from Arginine

In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate. When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate. Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory. The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine. It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.     

Modification of the fatty acid composition of phospholipid in measles virus-persistently infected cells

The composition of phospholipid was studied in BGM cells uninfected, persistently infected or lytically infected with measles virus, strain Hallé. In persistently infected cells, phosphatidylcholine palmitic acid content, and phosphatidylethanolamine palmitic acid and arachidonic acid contents were significantly increased. Lytically infected cells had a similar phospholipid fatty acid composition to the uninfected. Phosphatide composition showed minor modifications, but the content of total choline-derivative phospholipids was unchanged in either type of infection.     

Monoclonal antibodies against plant proteins recognise animal intermediate filaments

Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.     

Separation of membrane proteins in an undenatured form by isoelectric focusing

Conditions are described which allow good resolution of membrane proteins in an undenatured form by isoelectric focusing in thin polyacrylamide gels in Triton X-100. High voltages and deionization of the acrylamide are essential. Streaking is grossly reduced by sample application in Bio-Gel P-60, by deionization of the Triton, and dialysis of membrane samples against low ionic strength buffer at slightly alkaline pH. The latter step also greatly improves solubilization of the membrane components. Reproducible isoelectric focusing patterns of proteins from red cell, thyroid, and lymphocyte membrane are presented.     

Direct analysis of tau from PSP brain identifies new phosphorylation sites and a major fragment of N-terminally cleaved tau containing four microtubule-binding repeats

Tangles containing hyperphosphorylated aggregates of insoluble tau are a pathological hallmark of progressive supranuclear palsy (PSP). Several phosphorylation sites on tau in PSP have been identified using phospho-specific antibodies, but no sites have been determined by direct sequencing due to the difficulty in enriching insoluble tau from PSP brain. We describe a new method to enrich insoluble PSP-tau and report eight phosphorylation sites [Ser46, Thr181, Ser202, Thr217, Thr231, Ser235, Ser396/Ser400 (one site) and Thr403/Ser404 (one site)] identified by mass spectrometry. We also describe a 35 kDa C-terminal tau fragment (tau35), lacking the N-terminus of tau but containing four microtubule-binding repeats (4R), that is present only in neurodegenerative disorders in which 4R tau is over-represented. Tau35 was readily detectable in PSP, corticobasal degeneration and 4R forms of fronto-temporal dementia with parkinsonism linked to chromosome 17, but was absent from control, Alzheimer's disease and Pick's disease brain. Our findings suggest the aggregatory characteristics of PSP-tau differ from those of insoluble tau in Alzheimer's disease brain and this might be related to the presence of a C-terminal cleavage product of tau.