Erratum

Neurotransmitter receptor trafficking and the regulation of synaptic strength. Traffic 2001:2(7):437–448.     

N-Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells

Abstract: In previous studies, we demonstrated that the neuropeptide, N -acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1–6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans -1-aminocyclopentane-1,3-dicarboxylate ( trans -ACPD) and l -2-amino-4-phosphonobutyrate ( l -AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans -ACPD, activated each of the transfected receptors, whereas l -AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans -ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.     

Potential biomarkers of an exaggerated response to endotoxemia

Serial plasma protein analysis was used to study the acute plasma proteome response to endotoxemia (presence of toxic bacterial products called endotoxins in the blood stream). Plasma samples from healthy volunteers before and multiple time points up to 24 h following administration of low-dose endotoxin were evaluated. Plasma protein profiles were obtained by rapid extraction of whole plasma followed by analysis with matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The profiles were unique to each individual and stable over the time of the experiment. Administration of low-dose endotoxin caused profound change in six of 18 individuals. At 8 h many proteins showed quantitative oxidation, in addition to the appearance of new components and disappearance of common baseline components. An exceptionally intense new component at 4154 mass units was identified as the activation peptide of C1 esterase inhibitor. While recovery of baseline protein structure was nearly complete by 24 h, serum amyloid A, an acute-phase reactant, was still increasing and minor profile changes persisted. Clinical features did not distinguish these extreme responders from others, suggesting that plasma proteome changes offered unique insights into and potential biomarkers of subclinical events following endotoxin exposure.     

Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.     

Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity

Glutamate is an important excitatory amino acid at many central nervous system synapses. After its release from presynaptic nerve terminals, glutamate transiently binds to specific neuronal membrane receptors, which transduce its signal by the generation of intracellular second messengers before being rapidly cleared from the synapse. However, during ischemia, the glutamate concentration at synapses surrounding the focal lesion can be increased for sustained periods of time, resulting in abusive stimulation of glutamate receptors that can eventually be neurotoxic. To develop drugs capable of selectively blocking the pathological effects of glutamate in neurons surrounding ischemic lesions while leaving the physiological actions of glutamate in nonlesioned areas of the brain unaffected, it is essential to delineate glutamate-induced intracellular events that are specific to receptor abuse. This article describes the intracellular sequelae of physiological and pathological glutamate receptor activation and suggests potential targets for such receptor abuse-dependent antagonists (RADAs).     

N-Methyl-d-Aspartate-Sensitive Glutamate Receptors Induce Calcium-Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N -methyl-d-aspartate (NMDA) induced a dose-dependent release of [³H]arachidonic acid ([³H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [³H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A₂ (PLA₂) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA₂ rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [³H]AA release was dependent on the presence of extracellular Ca²⁺ and was mimicked by the Ca²⁺ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [³H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA₂ that is mediated by Ca²⁺ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.     

The effect of oligonucleotide microarray data pre-processing on the analysis of patient-cohort studies

Background  

Intensity values measured by Affymetrix microarrays have to be both normalized, to be able to compare different microarrays by removing non-biological variation, and summarized, generating the final probe set expression values. Various pre-processing techniques, such as dChip, GCRMA, RMA and MAS have been developed for this purpose. This study assesses the effect of applying different pre-processing methods on the results of analyses of large Affymetrix datasets. By focusing on practical applications of microarray-based research, this study provides insight into the relevance of pre-processing procedures to biology-oriented researchers.     

OBATOCLAX and ABT-737 Induce ER Stress Responses in Human Melanoma Cells that Limit Induction of Apoptosis

Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.     

Nascent HDL formation by hepatocytes is reduced by the concerted action of serum amyloid A and endothelial lipase

Inflammation is associated with significant decreases in plasma HDL-cholesterol (HDL-C) and apoA-I levels. Endothelial lipase (EL) is known to be an important determinant of HDL-C in mice and in humans and is upregulated during inflammation. In this study, we investigated whether serum amyloid A (SAA), an HDL apolipoprotein highly induced during inflammation, alters the ability of EL to metabolize HDL. We determined that EL hydrolyzes SAA-enriched HDL in vitro without liberating lipid-free apoA-I. Coexpression of SAA and EL in mice by adenoviral vector produced a significantly greater reduction in HDL-C and apoA-I than a corresponding level of expression of either SAA or EL alone. The loss of HDL occurred without any evidence of HDL remodeling to smaller particles that would be expected to have more rapid turnover. Studies with primary hepatocytes demonstrated that coexpression of SAA and EL markedly impeded ABCA1-mediated lipidation of apoA-I to form nascent HDL. Our findings suggest that a reduction in nascent HDL formation may be partly responsible for reduced HDL-C during inflammation when both EL and SAA are known to be upregulated.