Clonal specificity of human gamma delta T cells: V gamma 9+ T-cell clones frequently recognize Plasmodium falciparum merozoites, Mycobacterium tuberculosis, and group-A streptococci.

Peripheral blood gamma delta T cells expressing a V gamma 9/V delta 2 T-cell receptor are stimulated by killed bacteria including Mycobacterium tuberculosis (m.tb.) and group-A streptococci (strep A). In addition, recent data indicate that V gamma 9/V delta 2 T cells from unexposed individuals also respond to Plasmodium falciparum (P. falcip.) merozoites. Here we analyzed the reactivity to these ligands of 23 V gamma 9/V delta 2, 3 V gamma 9/V delta 1, and 4 V gamma 9-/V delta 1 clones derived from 8 healthy individuals after phytohemagglutinin stimulation of cell sorter-selected gamma delta T cells. Upon restimulation in the presence of irradiated antigen-presenting cells, the majority of V gamma 9/V delta 2 clones recognized m.tb. and strep A (but not strep D), and about one third of the clones also recognized P. falcip. Some clones, however, recognized only one or two of the tested ligands, and 4 V gamma 9/V delta 2 clones did not react at all. Interestingly, 2 of 3 V gamma 9/V delta 1 clones proliferated in response to m.tb., P. falcip., strep A and strep D, while V gamma 9-/V delta 1 clones were not activated by any of the tested ligands. Nucleotide sequence analysis indicated a broad diversity of V gamma 9 N regions in V gamma 9/V delta 2 clones. At the clonal level, our results demonstrate that individual V gamma 9/V delta 2 T cells can recognize m.tb., strep A, as well as P. falcip.-infected erythrocytes, with no influence of the expressed V gamma 9 N region.

Identification and sequence analysis of the ribosomal DNA promoter region of Crithidia fasciculata.

We have identified the promoter region of the large ribosomal DNA repeat unit of Crithidia fasciculata by northern blotting and nuclear run-on analyses. These data show that transcription starts approximately 1 kb upstream of the 18S rRNA gene. S1 protection experiments and sequence analysis of this area resulted in a precise localization of the start site. We have been unable to identify conserved sequence element(s) by a direct comparison of the crithidial RNA polymerase I promoter region and similar promoter regions of other eukaryotes; not even to the promoter region of the more closely related kinetoplastid species, Trypanosoma brucei. The absence of homology within the primary sequence of the promoter region, which is also found in other eukaryotes, might explain the observed species specificity of in vivo and in vitro rDNA transcription, since this resides in the interaction of initiation factor(s) and the core promoter domain.

Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III.

To analyze transcription in Typanosoma brucei, we have characterized the trypanosomal RNA polymerases. Here we present our results, which allow a discrimination between the different classes of RNA polymerases in nuclear run-on experiments by polymerase inhibitors and Mn2+ dependence. We also describe the separation of trypanosomal RNA polymerases by chromatography, demonstrating that T. brucei contains RNA polymerases I-III. The outcome of our experiments suggests that the VSG genes of T. brucei are not transcribed by RNA polymerase I, as previously reported, but by RNA polymerase II. We propose that an additional factor modifies RNA polymerase II, resulting in the alpha-amanitin-resistant transcription of VSG genes. Our data also suggest that the mini-exon genes, which encode the 5' end of each trypanosomal mRNA, are probably transcribed by RNA polymerase III.

Monospecific antiserum against 5'-nucleotidase from Torpedo electric organ: immunocytochemical distribution of the enzyme and its association with Schwann cell membranes.

The cellular and subcellular distribution of 5'-nucleotidase in tissues of the electric ray Torpedo marmorata has been investigated by means of an antiserum raised against the native enzyme purified from the electric organ. As revealed by immunohistochemistry the enzyme is associated with the surface of the axons of the electric nerves and of spinal nerves. Using the post-embedding colloidal gold technique at the electron-microscopical level 5'-nucleotidase could be located at the plasma membrane of the Schwann cells including the myelin and the fine processes covering the terminal axon ramifications. Also the perineurial sheath of the axons inside the electric organ is 5'-nucleotidase positive. The plasma membrane of the axon and the terminal axon region or the postsynaptic membrane do not contain 5'-nucleotidase. Immunoprecipitation studies using polyacrylamide beads suggest that the ecto-Ca2+- or -Mg2+-adenosine 5'-triphosphatase previously ascribed to synaptosomes of the Torpedo electric organ is not associated with the same membranes as 5'-nucleotidase. Within the electric organ the dorsal plasma membrane of the electroplaque cell, blood capillaries and the connective tissue layer surrounding the columns of electroplaque cells also bind the antibodies. In central nervous tissue solely blood vessels show immunofluorescence. Within the electric lobe both the surface of the electromotor neurons as well as the myelinated axons giving rise to the electric nerve are negative. This also applies to the axons of the optic nerve suggesting that the antiserum is Schwann cell specific, and does not bind to a potential oligodendroglial 5'-nucleotidase. In peripheral tissue the surface of skeletal muscle fibres as well as that of individual myofibrils bind the anti-5'-nucleotidase antibodies. Our results demonstrate that the Schwann cell plasma membrane, including myelin, contains 5'-nucleotidase and that one can distinguish by means of a specific antiserum between Schwann cell and oligodendroglia plasma membranes. The functional significance of the association of 5'-nucleotidase with Schwann cells along the entire surface of axons including the synaptic region as well as with other parts of the electric tissue is discussed regarding its catalytic activity and also the possibility that this surface glycoprotein may be involved in mediating cellular interactions.

Purification, characterization and cellular localization of 5'-nucleotidase from Torpedo electric organ.

5'-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5'-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.

Ca(2+)-dependency and substrate specificity of cholinergic synaptic vesicle ATPase.

The Mg(2+)-ATPase associated with highly purified cholinergic synaptic vesicles isolated from the Torpedo electric organ can be activated by millimolar concentrations of Ca(2+) only to a maximum of 46%. ATP is hydrolyzed in preference to other tri-nucleotides on Mg(2+)-activation but not on Ca(2+)-activation of the enzyme. Analysis of the Ca(2+)-dependency of vesicle associated ATPase-activities under controlled free Ca(2+)-concentrations (10(?7) to 10(?3)M) shows that vesicles do not contain a Mg(2+)-dependent, Ca(2+)-stimulated (Ca(2) + Mg(2+)) ATPase. Our results suggest that cholinergic synaptic vesicles contain a single ATPase preferentially stimulated by Mg(2+)-ions and that ATP-stimulated vesicular uptake of Ca(2+) is not due to the presence of a Mg(2+)-ATPase stimulated by micromolar Ca(2+)-concentrations.

Ectonucleotidase activities associated with cholinergic synaptosomes isolated from Torpedo electric organ.

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.