Acylation of endogenous myelin proteolipid protein with different acyl-CoAs

Fatty acyltransferase activity that catalyzes the transfer of palmitic acid from palmitoyl-CoA to the endogenous myelin proteolipid protein has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the acylation of proteolipid protein was obtained in 0.1% Triton X-100, 2 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.5 and at 37 degrees C. Other detergents had little or no effect on the reaction whereas acylation was completely abolished by sodium dodecyl sulphate (0.1%). Pulse-chase experiments indicated that the reaction involves the net addition of fatty acid to the protein and not a rapid fatty acid exchange. The rate of acylation was linear up to 30 min, indicating that the concentration of endogenous protein acceptor was constant. Under these conditions and at short time periods, the enzyme activity versus acyl-CoA concentration showed a hyperbolic curve. The apparent Km and Vmax for palmitoyl-CoA was 41 microM and 115 pmol/mg protein/min. Similar values were obtained for stearoyl and oleoyl-CoA, whereas myristoyl-CoA showed a lower specificity for the enzyme. The acyl-CoA specificity was also studied in competition experiments using several saturated and unsaturated fatty acid-CoAs. The product of the reaction was identified as myelin proteolipid protein and the fatty acid was shown to be attached to the protein via an ester linkage. Limited proteolysis and peptide mapping showed that the same sites on the proteolipid protein were acylated when the reaction was carried out in isolated myelin preparations or in brain tissue slices, suggesting physiological importance for the in vitro acylation of endogenous myelin proteolipid protein.     

Haemopoiesis in the beagle foetus after in utero irradiation

On day 33 of gestation, foetal beagles were irradiated in utero (0.9 Gy of 60Co gamma-irradiation, 0.4 Gy/min). Foetal haematocytopoiesis was studied during the third trimester of gestation (days 42-55). Peripheral blood nucleated cell counts were 33 per cent lower than normal on day 44 and continued to be lower until day 49, when values became higher than normal. Splenic cellularities of irradiated pups on day 44 were more than 3 times those of the nonirradiated, but thereafter they were similar to normal. Differences in haemopoietic progenitor cell activity between irradiated and normal foetuses were observed. In comparison with the other foetal tissues, the foetal liver appeared to experience greater radiation injury. For example, on day 44, the irradiated liver BFU-E, CFU-E, and GM-CFC per 10(5) cells were almost fivefold lower than normal values. Spleens of irradiated foetal beagles contained a marked increase in all haemopoietic progenitor cells (BFU-E, CFU-E, and GM-CFC) and recognizable proliferative granulocytic cells and nucleated erythroid cells. The haemopoietic activity of the irradiated bone marrow during days 42-44 was similar to that of the irradiated spleen, and compensated for the damaged liver. However, unlike the irradiated spleen, the irradiated bone marrow had decreased BFU-E activity compared with the values for the nonirradiated bone marrow during days 48-55. Until day 50, the irradiated marrow contained fewer recognizable proliferative granulocytic cells but more nucleated erythroid cells.     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase

This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme. Specifically, we propose that SiR performs its 6-electron reduction via intramolecular or intermolecular electron transfer. Our model explains both the significance of the stoichiometric mismatch between reductase and oxidase subunits in the holoenzyme and how SiR can handle such a large volume electron reduction reaction that is at the heart of the sulfur bio-geo cycle.     

Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.     

Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound

Feces from 15 dogs at 2 different foxhound kennels in the U.K. were examined microscopically for the presence of oocysts of Neospora caninum. One sample containing approximately 400 candidate oocysts per gram was positive in a polymerase chain reaction (PCR) using N. caninum-specific primers. In a sample taken 4 mo later from the same hound. N. caninum oocysts were again detected visually and by PCR. This is the third reported case of a dog naturally excreting oocysts of N. caninum and suggests that oocyst excretion can occur over a relatively long period of time in some circumstances or that reshedding may occur.