Joint report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus x Bos indicus, and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

Mutational evidence for competition between the P1 and the P10 helices of a mitochondrial group I intron.

A guanosine to cytosine transversion at position 2 of the fifth intron of the mitochondrial gene COB blocks the ligation step of splicing. This mutation prevents the formation of a base pair within the P1 helix of this group I intron--the RNA duplex formed between the 3' end of the upstream exon and the internal guide sequence. The mutation also reduces the rate of the first step of splicing (guanosine addition at the 5' splice junction) while stimulating hydrolysis at the 3' intron-exon boundary. Consequently, the ligation of exons is blocked because the 3' exon is removed prior to cleavage at the 5' splice junction. The lesion can be suppressed by second-site mutations that preserve the potential for base-pairing at this position. Because the P1 duplex and the P10 duplex (between the guide sequence and the 3' exon) overlap at the affected pairings represent alternative structures that do not, indeed cannot, form simultaneously.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence

A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.