In vivo priming of helper and suppressor T cells by alloantigens. Frequency analysis with the use of an in vitro limiting dilution assay

Earlier studies have demonstrated that T cells activated in mixed lymphocyte reactions can exert positive as well as negative allogeneic effects on B cells expressing the appropriate alloantigens on their surface. We investigated the effect of in vivo priming of T cells with alloantigens on their capacity to help or suppress allogeneic B cell cultures against sheep erythrocytes. We used immunization protocols that have been shown to be optimal for induction of alloantigen-specific delayed-type hypersensitivity (DTH) and alloantigen-specific suppressor T (Ts) cells for DTH. The results show that in vivo stimulation with alloantigens, depending on the immunization route and the lymphoid organ studied, can be as effective as in vitro stimulation in increasing the frequency of alloantigen-specific helper T (Th) cells and Ts cells. Subcutaneous immunization induced a 10-fold frequency raise of Th cells as well as of Ts cells in the lymph nodes. In the spleen the Th cell population was hardly affected by s.c. immunization, whereas the Ts cell population increased by at least a factor 20. Intravenous immunization, on the other hand, selectively expanded the Th cell population in the spleen, whereas the splenic Ts cell population and the Th and Ts cells in the lymph nodes were not affected. Comparison of these results with our previous data concerning characteristics and the requirements of in vivo activation of alloantigen-specific DTH reactive T cells and of alloantigen-specific Ts cells suggest that different Ts cell populations are involved in suppression of alloantigen-specific DTH in vivo and of allogeneic suppression of in vitro induced sheep erythrocytes specific antibody formation.     

Inactivation of the proteolytic activity of mouse nerve growth factor by human C1(activated)-inhibitor

The interaction between the serine protease gamma subunit of NGF (gamma-NGF) and human C1(activated)-inhibitor (C1-Inh) has been studied. C1-Inh inactivates the protease activity of gamma-NGF as measured by its ability to cleave the synthetic substrate benzoyl-arginine-p-nitroanilide (L-BAPNA). Experiments in which gamma-NGF and C1-Inh were mixed at differing molar ratios indicated that inhibition was due to the formation of a 1:1 stoichiometric complex. Analysis of the interaction of 125I-labeled gamma-NGF with C1-Inh by SDS-PAGE and autoradiography indicated that a covalent bond was formed between gamma-NGF and C1-Inh. The covalent bond was hydrolyzed by hydroxylamine, which suggested that the two proteins were linked via an acyl linkage. The formation of this complex was time dependent and required the proteolytic activity of the gamma-NGF.     

Repeated sequence elements in the high molecular weight basic nuclear proteins from the winter flounder

In maturing sperm of the winter flounder, histones are not replaced by protamines but instead joined by a group of high molecular weight basic nuclear proteins. Despite their large size and number of components, these proteins were reduced to a relatively simple set of peptides by a "limit" digestion with endoprotease Lys-C. Nine of these peptides, that together account for half of the mass of the digest, were purified by two rounds of chromatography on a C18 reverse-phase high pressure liquid chromatographic column and analysed by sequential Edman degradation. Their sequences can be divided into two homology groups. Seven of the peptides contain all or part of a dodecapeptide consensus sequence, NH2-Ser-Pro-Met-Arg-Ser-Arg-Ser-Pro-Ser-Arg-Ser-Lys-COOH, which appears to be tandemly repeated. This dodecapeptide contains a previously recognized consensus phosphorylation sequence, NH2-Arg-Ser-Arg-Ser-Pro-COOH, in which both serines are phosphorylated during the early stages of spermiogenesis. The other homology group has the sequence NH2-Arg-Arg-Val-X-X-Pro-Lys-COOH, where X-X is either Gln-Thr or Pro-Ser. The dodecapeptide and heptapeptide sequences form at least 35 and 11%, respectively, of the high molecular weight basic nuclear proteins and are, therefore, repeated many times over in these proteins. A search for identical or homologous sequences within the Protein Sequence Database indicated that they are unique. The closest matches were to protamines and some viral DNA-binding proteins.     

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.     

Are isolated maternity units run by general practitioners dangerous?

A retrospective survey was carried out of women admitted in labour to an isolated maternity unit run by general practitioners in Penrith. In the five years 1980-4, 1267 women began labour in Penrith, of whom 1153 (91%) never required help from a consultant unit. Ninety required transfer during labour. Ten mothers and four neonates required transfer during the early puerperium, all to one receiving unit in Carlisle. There were six perinatal deaths during the five years; five occurred in babies delivered after transfer. The perinatal mortality was 4.7/1000. The low mortality, the low level of intervention, and the preference of women all support the retention of isolated units.     

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm-2a region of chromosome 9 in tomato

Summary A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number of single copy clones and the suitability of these enzymes for the mapping of large DNA fragments was evaluated. Furthermore, five genetically tightly linked single copy clones have been used to begin the construction of a physical map in a region of the genome containing the Tm-2a gene which confers resistance to tobacco mosaic virus. Two of the five clones were found to be on the same 560 kb SalI fragment and therefore are no further apart than that distance. The remaining three markers are distributed over at least 3 million bp, so that the total minimum physical distance of that cluster is at least 4 million bp. The results are discussed with respect to correlations between recombination frequencies and physical distance as well as physical mapping large regions of a complex plant genome like tomato.