Oxysterols direct immune cell migration via EBI2

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.     

Spatial distribution of the adaptation field of the surround response mechanism in type X cat retinal ganglion cells

Summary The surround response mechanism in on-center X-cells in cat retina was found to be bimodally distributed and weak or nonexistent in the receptive field middle. An on-inhibition measure was used to assess surround mechanism gain.Acknowledgments. This research is supported by Public Health Service grant No. EY 00701.     

Chemical approaches to intermediates of enzyme catalysis

Zusammenfassung Die Untersuchung von Enzymsubstratkomplexen auf katalyse-induzierte Änderungen der chemischen Reaktivität hat sich als ein aufschlussreicher experimenteller Zugang zum Mechanismus enzymatischer Reaktionen erwiesen. Ternäre Systeme bestehend aus Enzym, Substrat und einem geeigneten Reagens (Tetranitromethan=TNM) sind auf katalyse-abhängige Reaktionen geprüft worden, die nur im vollständigen System, jedoch nicht mit Reagens und Enzym allein oder Reagens und Substrat allein beobachtet werden. Untersuchungen mit Aldolasen und Aspartat-Aminotransferase haben ergeben, dass katalyseinduzierte Reaktivität sowohl auf dem Substrat-als auch auf dem Enzymteil eines Enzym-Substratkomplexes nachgewiesen werden kann.Im Reaktionsmechanismus von Muskelaldolase (eine Lysinaldolase) und von Hefealdolase (eine Metallaldolase) lässt sich mit TNM ein intermediäres Carbanion des Substrats nachweisen. Die TNM-carbanion-Reaktion lässt sich spektralphotometrisch verfolgen und ist benutzt worden, um carboxypeptidase-behandelte Muskelaldolase und den Effekt von Cofaktoren auf die Hefealdolase zu untersuchen.Katalyse-induzierte Reaktivitätsveränderungen einer Enzymseitenkette sind bei der Aspartat-Aminotransferase beobachtet worden. Ein essentieller Tyrosylrest, der in der Abwesenheit von Substrat nicht mit TNM reagiert, wird ungewöhnlich reaktiv im Laufe der Katalyse und ermöglicht dabei seine selektive Nitrierung.Die katalyse-synchrone oder synkatalytische Aktivierung dieses Aminosäurerests scheint ein integraler Bestandteil des katalytischen Mechanismus von Aspartat-Aminotransferase zu sein und wird vermutlich durch katalyse-induzierte Konformationsänderungen des Enzym-Coenzym-Substratkomplexes hervorgebracht. Mögliche funktioneile Zusammenhänge synkatalytischer Reaktivitätsänderungen funktioneller Gruppen mit der Konformationsflexibilität des Enzymproteins und dem Vorkommen metastabiler Zwischenprodukte in der Enzymkatalyse werden diskutiert.     

Effect of adapting target size on the gain of the surround response mechanism in X- and Y-cells in cat retina

Summary The adaptation field of the surround mechanism of X and Y retinal ganglion cells in the cat was assessed with variable size, unmodulated adapting spots. Both an on-inhibition measure and an off-discharge measure of surround gain was used. Results suggest that the surround mechanism in Y-cells is strongest in the receptive field middle but weak or nonexistent in the middle of X-cell receptive fields.Acknowledgment. This research was supported by Public Health Service grant No. EY 00701.     

CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes

A long-standing paradox in cellular immunology concerns the conditional requirement for CD4+ T-helper (T(H)) cells in the priming of cytotoxic CD8+ T lymphocyte (CTL) responses in vivo. Whereas CTL responses against certain viruses can be primed in the absence of CD4+ T cells, others, such as those mediated through 'cross-priming' by host antigen-presenting cells, are dependent on T(H) cells. A clearer understanding of the contribution of T(H) cells to CTL development has been hampered by the fact that most T(H)-independent responses have been demonstrated ex vivo as primary cytotoxic effectors, whereas T(H)-dependent responses generally require secondary in vitro re-stimulation for their detection. Here, we have monitored the primary and secondary responses of T(H)-dependent and T(H)-independent CTLs and find in both cases that CD4+ T cells are dispensable for primary expansion of CD8+ T cells and their differentiation into cytotoxic effectors. However, secondary CTL expansion (that is, a secondary response upon re-encounter with antigen) is wholly dependent on the presence of T(H) cells during, but not after, priming. Our results demonstrate that T-cell help is 'programmed' into CD8+ T cells during priming, conferring on these cells a hallmark of immune response memory: the capacity for functional expansion on re-encounter with antigen.     

Coupling of fructose-1,6-P2 to aminated agarose by Schiff base reduction. Affinity chromatography of yeast aldolase

Summary Fructose-1,6-P₂ was immobilized by sodium borohydride reduction of the Schiff base formed with aminated agarose (AH-Sepharose 4B^®). The coupling occurs with high yield (25 moles immobilized fructose-1,6-P₂ per ml packed gel) at neutral pH and room temperature. Schiff base reduction thus provides a convenient and mild coupling procedure for sugar phosphates preserving their labile phospho ester bonds. As exemplified by a new isolation procedure for fructose-1,6-P₂ aldolase from yeast, sugar phosphates insolubilized in this manner may be used for affinity chromatography of the corresponding enzymes, provided that contaminating unspecific phosphatases are removed in a preceding fractionation step.This work was supported by the Swiss National Science Foundation, grant No. 3.620-0.75. A preliminary account has been presented at the 10th Int. Congress of Biochemistry, Hamburg, 1976, Abstracts. p. 194.Acknowledgment. The excellent technical assistance of Alice Gasser is gratefully acknowledged     

Spatial distribution of the adaptation field of the surround response mechanisms in type X cat retinal ganglion cells.

The surround response mechanism in on-center X-cells in cat retina was found to be bimodally distributed and weak or nonexistent in the receptive field middle. An on-inhibition measure was used to assess surround mechanism gain.     

Effect of adapting target size on the gain of the surround response mechanism in X- and Y-cells in cat retina.

The adaptation field of the surround mechanism of X and Y retinal ganglion cells in the cat was assessed with variable size, unmodulated adapting spots. Both an on-inhibition measure and an off-discharge measure of surround gain was used. Results suggest that the surround mechanism in Y-cells is strongest in the receptive field middle but weak or nonexistent in the middle of X-cell receptive fields.