The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

An assay for the alpha-tocopherol binding protein mediated transfer of vitamin E between membranes

A model system consisting of donor membrane (egg lecithin liposomes) and acceptor membrane (human erythrocyte ghosts or rat liver mitochondria) were used to investigate the alpha-tocopherol binding protein (alpha TBP) mediated transfer of alpha-tocopherol. Liposomes containing RRR-[alpha-3H]tocopherol ([alpha-3H]T) were incubated with acceptor membrane at 37 degrees C for 0-45 min in the presence or absence of rat liver cytosol or a dialyzed 30-60% saturated ammonium sulfate precipitated fraction of rat liver cytosol (Fraction B). Erythrocyte ghosts and liver mitochondria were compared and found to behave similarly in the presence of Fraction B. alpha-Tocopherol transfer activity (alpha TTA) typically varied 0- to 27-fold greater than buffer blanks, depending upon type and concentration of protein preparation. Gel filtration of Fraction B yielded one alpha TTA peak (liver mitochondria as acceptor) with an estimated Mr of 39,000. [alpha-3H]T recovered from erythrocyte ghosts pellets by HPLC suggest that the [alpha-3H]T was transferred intact. alpha TTA of Fraction B in the presence of varying concentrations of erythrocyte ghosts and liposomal [alpha-3H]T followed saturation kinetics. Optimal concentrations gave alpha TTA responses directly proportional to rat liver cytosol concentration. alpha TTA was inhibited only 5% in the presence of a 32-fold excess of cold liposomal alpha-tocopheryl acetate suggesting that the free hydroxyl group on the chromanol ring of alpha-tocopherol is needed for transfer. Coefficient of variation of repeated measures of alpha TTA in rat liver cytosol was 2.9%. Thus, the intermembrane transfer phenomenon of alpha-tocopherol can be studied quantitatively and can be used to compare liver protein preparations exhibiting transfer activity.     

Encapsulation of eggs of the encyrtid wasp, Metaphycus swirskii, by the hemispherical scale, Saissetia coffeae: Effects of host age and rearing temperature

The combined effects of host age and rearing temperature upon the encapsulation of eggs of Metaphycus swirskii Annecke & Mynhardt (Hymenoptera: Encyrtidae) by the hemispherical scale, Saissetia coffeae (Walker) (Homoptera: Coccidae), were determined. Encapsulation incidence at 16, 20, 24 and 28°C increased with the age of the host, being 5.5–19.6% in 3rd instar nymphs, 45.4–74.2% in young female scales and 88.8–100% in preovipositing female scales. Within each host physiological age range, encapsulation frequency was not materially influenced by most of the temperatures. At 32°C, regardless of host age, S. coffeae encapsulated 99.5 to 100% of parasitoid eggs. Complete immunity to successful parasitism by M. swirskii occurred at 20, 24, 28 and 32°C in preovipositing female scales and at 32°C in 3rd-instar nymphs and young female scales of S. coffeae.

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Résumé Cette étude a déterminé les effets combinés de l'âge de l'hôte et de la température sur l'encapsulation des oeufs de M. swirskii Annecke & Mynhardt (Hym. Encyrtidae) par S. coffeae Walker (Homopt. Coccidae). La fréquence d'encapsulation à 16, 20, 24 et 28°C a augmenté avec l'âge de l'hôte, 5,5–19,6% dans les larves de 3ème stade, 45,4–74,2% chez les jeunes femelles et 88,8–100% chez les femelles avant la ponte. Dans chaque gamme d'âge physiologique de l'hôte, la fréquence d'encapsulation n'est pas influencée par la plupart des températures. A 32°C, quel que soit l'âge de l'hôte, S. coffeae a encapsulé 99,5–100% des oeufs du parasitoïde. Une immunité complète au parasitisme par M. swirskii a été obtenue à 20, 24, 28 et 32°C pour les femelles de cochenilles dans la période précédant la ponte, et à 32°C pour les larves de 3ème stade et les jeunes femelles de S. coffeae.

     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

N-Methylaspartate-Evoked Liberation of Taurine and Phosphoethanolamine In Vivo: Site of Release

The effect of N-methyl-D,L-aspartic acid (NMA) on extracellular amino acids was studied in the rabbit hippocampus with the brain dialysis technique. Administration of 0.5 or 5 mM NMA caused a concentration-dependent liberation of taurine and phosphoethanolamine (PEA). Taurine increased by 1,200% and PEA by 2,400% during perfusion with 5 mM NMA whereas most other amino acids rose by 20-100%. The effect of NMA appeared to be receptor-mediated, as coperfusion with D-2-amino-5-phosphonovaleric acid curtailed the NMA response by some 90%. The NMA-stimulated release of taurine and PEA was suppressed when Ca2+ was omitted and further inhibited when Co2+ was included in the perfusion medium. The effect of NMA was mimicked by the endogenous NMA agonist quinolinic acid and the partial NMA agonist D,L-cis-2,3-piperidine dicarboxylic acid. Although the NMA-evoked release of taurine and PEA was Ca2+-dependent in vivo, NMA had no effect on Ca2+ accumulation in hippocampal synaptosomes. The previously reported NMA-induced activation of dendritic Ca2+ spikes and the lack of effect on synaptosomal Ca2+ uptake suggest that taurine and PEA are released from sites other than nerve terminals, possibly from dendrosomatic sites. This notion was strengthened by the absence of an effect of NMA on the efflux of radiolabelled taurine from hippocampal synaptosomes. In contrast, high K+ stimulated synaptosomal uptake of Ca2+ and release of taurine.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.