Joint Report of the Second International Workshop on Lymphocyte Alloantigens of the Horse, held 3–8 October 1982

The Second International Workshop on Lymphocyte Alloantigens of the Horse was held 3–8 October 1982. At this workshop, the 6 specificities identified at the first workshop were confirmed and an additional 5 new specificities were identified and given workshop nomenclature. Four of the new specificities, products of the ELA locus, were named ELA-W7, W8, W9, and W10. An additional specificity, designated ELY-2.1, is the product of a locus independent of the ELA locus.
Cell isolation methods were compared at this workshop, Technical variation in methods clearly affected reactivity of many reagents. However, when highly selected reagents were used, antigen assignment did not differ regardless of the cell isolation method. Based on the comparison of methods, isolation procedures in which thrombin was used were more effective than those relying on carbonyl iron or slow centrifugation.     

Measles virus-induced suppression of lymphocyte proliferation

The mechanism by which measles virus induces immunosuppression was investigated using an in vitro system employing phytohemagglutinin (PHA)-induced human peripheral mononuclear cell (PBMC) proliferation. At a multiplicity of infection of 1.0 or greater measles virus significantly inhibited (45%) the proliferation of PBMC. This inhibition was not due to an alteration in the kinetics of proliferation. PHA-stimulated PBMC were then infected with measles virus for 72 hr and irradiated (3200 rad) to prevent further proliferation. These infected, irradiated PBMC when added to fresh autologous PBMC caused significant inhibition of lymphoproliferation over a wide range of infected:fresh cell ratios (maximum inhibition seen at a 1:1 ratio, 85% inhibition). Virus recovered from the irradiated, infected cells was 100-fold lower than the virus titer needed to cause inhibition by direct addition of measles virus. However, antibody to measles virus reversed the inhibition. Virus-free supernatant fluids from the infected irradiated cells caused immunosuppression of the PHA response. This immunosuppressive material induced by the measles virus was maximally produced after 72 hr and did not appear to require viral replication. This factor was not prostaglandin E or interferon-alpha or -gamma. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be detected.     

Modification of flow-force relationships by external ATP in yeast mitochondria

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP. ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP. At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase, and support the view the condition mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system.     

Influence of coenzyme structure on the transient chemical intermediate formed during horse-liver alcohol-dehydrogenase-catalyzed reduction of aromatic aldehydes

The influence of coenzyme structure on the transient chemical intermediate formed in the reaction between the horse-liver alcohol dehydrogenase-NADH complex and an aromatic aldehyde such as 4-trans-(N,N-dimethylamino)cinnamaldehyde or 4-(N,N-dimethylamino)benzaldehyde was investigated by substituting various adenylic dinucleotides for NADH. Two classes of dinucleotide were studied. (a) Dinucleotides which, in the presence of horse-liver alcohol dehydrogenase and either 4-(N,N-dimethylamino)benzaldehyde or 4-trans-(N,N-dimethylamino)cinnamaldehyde, lead to a chromophore structurally analogous to the transient chemical intermediate formed with NADH under the same experimental conditions. This includes dinucleotides with a neutral 1,4-dihydropyridine ring, analogues of NADH and adducts of NAD+ (or analogues) with enolizable carbonyl compounds. (b) Dinucleotides which, under the same experimental conditions, do not form any new chromophores when mixed with horse-liver alcohol dehydrogenase and either 4-trans-(N,N-dimethylamino)cinnamaldehyde or 4-trans-(N,N-dimethylamino)benzaldehyde. This includes oxidized coenzyme analogues, NADPH and NADP+ adducts. Our data suggest that a neutral 1,4-dihydropyridine ring is crucial for the formation of the transient chemical intermediate. When the NAD+-sulphite complex, which has a 1,4-dihydronicotinamide structure and a positive charge at position 4 neutralized by sulphite ions, was substituted for NADH, the transient chemical intermediate chromophore was observed. The implications of this phenomenon are examined by assuming the existence of intermediate-activated forms of substrates and coenzymes during the horse-liver alcohol dehydrogenase catalytic reduction of aldehydes.     

Thermodynamic and kinetic control of ATP synthesis in yeast mitochondria: role of delta pH

ATP synthesis rate, measured as the variation in external Pi concentration, varied as a linear function of either delta mu H+ or delta Gpin, in such a manner that the delta Gpin/delta mu H+ ratio increased while VATP increased. We also observed a linear dependence of the flux control coefficient of the Pi carrier on delta pH. All the results presented can be explained by a relatively large delta pH drop when VATP increases.     

Preparation of an inner membrane-matrix fraction from yeast yeast mitochondria.

Treatment of yeast mitochondria with digitonin was used in order to prepare an inner membrane-matrix fraction preserving its permeability properties. The incubation time of mitochondria with digitonin was an essential parameter for the selective solubilization of the outer membrane. The incubation of mitochondria for l min at different concentrations of digitonin led to a three-step release of mitochondrial enzymes: (a) at low concentrations of digitonin, adenylate kinase was released; (b) higher concentrations were required to solubilize kynurenine hydroxylase, an outer membrane marker; (c) inner membrane markers (succinate dehydrogenase and oligomycin-sensitive adenosine triphosphatase) and matrix markers (fumarase and isocitrate dehydrogenase) were significantly released at concentrations of digitonin higher than 0.4 mg/mg of protein. The electron microscopic aspects of yeast mitoplasts (inner membrane-matrix fraction obtained by treatment with 0.4 mg of digitonin) showed an orthodox and a twisted configuration. These new organelles retained respiratory control when assayed with ethanol as the substrate. Their selective permeability properties were preserved as shown by isoosmotic swelling in potassium or ammonium salt solutions.     

Differences in specificity and catalytic efficiency between allozymes of esterase-4 from Drosophila mojavensis

A more than 10-fold difference in the specificity and catalytic efficiency for 1-naphthyl esters was measured between two allozymes of esterase-4 from Drosophila mojavensis. This difference is mainly caused by a difference in the affinity for the 1-naphthyl esters. The amino acid compositions of the allozymes are not significantly different, which means that the difference in primary structure is small. Small differences in primary structure generally do not result in such a large increase in catalytic efficiency and such a large shift in substrate specificity as was found in the present study.