IL-7 promotes thymocyte proliferation and maintains immunocompetent thymocytes bearing alpha beta or gamma delta T-cell receptors in vitro: synergism with IL-2

IL-7 induced the proliferation of normal thymocytes and the effect was synergistically potentiated by a small dose of IL-2, which by itself hardly affected thymocyte proliferation. No synergism was observed between IL-7 and any one of the other lymphokines including IL-1, IL-3, and IL-4. The thymocyte culture stimulated with IL-7 and IL-2 consisted of single positive (CD4+CD8- and CD4-CD8+) and double negative (CD4-CD8-) populations, and double positive (CD4+CD8+) cells were completely deleted. Both single positive and double negative thymocytes expressed CD3, but only the former exhibited V beta 8 and V beta 6 in an expected proportion (approximately 30% in BALB/c mice) and the latter none at all. Immunoprecipitation of the cultured thymocytes by anti-TCR gamma antibody, on the other hand, revealed the presence of a TCR gamma chain. Taken together, these results indicated that the thymocyte cultured with IL-7 and IL-2 consisted of mature T cells bearing alpha beta or gamma delta TCR. Experiments using preselected thymocyte subpopulations indicated that double negative cells responded to both IL-7 and IL-2 with positive synergism when combined, while thymocytes enriched for single positive cells preferentially responded to IL-7 with little response to IL-2 and no detectable synergism. Double positive thymocytes showed no proliferation in response to IL-7 and IL-2. In contrast to single positive thymocytes, splenic T cells hardly responded to IL-7, although significant proliferation was induced in the presence of a low dose of IL-2. Thymocytes cultured with IL-7 and IL-2 showed little nonspecific cytotoxic activity, but responded to Con A or alloantigen, whereas those stimulated with a high dose of IL-2 alone exhibited potent cytotoxic activity. These results indicated that IL-7 was involved in the generation of immunocompetent T cells in the thymus in concert with IL-2.     

Autonomous replication of human chromosomal DNA fragments in human cells.

We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.     

Accumulation on the Cytoplasmic Membrane of the Precursor to Dimethyl Sulfoxide Reductase in Molybdenum Cofactor-Deficient Mutants of Rhodobacter sphaeroides f. sp. denitrificans

The role of the molybdenum cofactor (Mo cofactor) in the translocationof dimethyl sulfoxide (DMSO) reductase to the periplasmic spacewas studied in vivo by isolating chlorate-resistant mutantsof Rhodobacter sphaeroides f. sp. denitrificans. More than 50%of the chlorate-resistant mutants isolated were defective inthe biosynthesis of the Mo cofactor and all of these mutantsaccumulated the precursor form of the enzyme. About 45% of themutants contained the same level of Mo cofactor as the parentstrain and exhibited normal levels of DMSO reductase and nitratereductase activities when chlorate was absent from the medium,but the activities of these enzymes were depressed when chloratewas present. Much of the accumulated precursor form of the enzymein a Mo cofactor-deficient mutant was bound to the cytoplasmicmembrane and was sensitive to treatment with proteinase K fromthe periplasmic side of the membrane, an indication that theprecursor was exposed on the periplasmic surface of the membrane.The precursor accumulated on the membrane of the parent strainwhen molybdate was removed from the medium or upon additionof tungstate and this precursor was also sensitive to the treatmentwith proteinase K from the periplasmic side. These results suggestthat the Mo cofactor is necessary for proteolytic processingof the precursor to the mature enzyme on the periplasmic sideof the membrane, whereas binding of the precursor to the membraneand translocation across it can occur in the absence of thecofactor. Almost all of the Mo cofactor available for directreconstitution in vitro of nitrate reductase activity from thenit-l mutant of Neurospora crassa was present in the cytoplasmicfractions. (Received December 11, 1991; Accepted March 25, 1992)     

ANALYSIS OF POLYPEPTIDES ASSOCIATED WITH SHOOT FORMATION IN TOBACCO CALLUS CULTURES

Callus lines of Nicotiana tabacum were selected for competence and lack of competence in shoot formation. Changes in total and chromosomal polypeptides in these shoot-forming and nonshoot-forming tobacco cultures were examined by twodimensional polyacrylamide gel electrophoresis. Qualitative and quantitative differences in total, nonhistone chromosomal, and basic chromosomal polypeptides were evident throughout the 7-d test period. The analysis of total proteins identified polypeptides specific to shoot-forming and nonshoot-forming tissue during the 7-d sampling period. A small number of basic chromosomal proteins were found solely in shoot-forming or nonshoot-forming tissue. One basic chromosomal protein was detected in only nonshoot-forming tissue at all sampling times. Two proteins, although present in shoot-forming tissue, were present at elevated levels in the nonshoot-forming cultures. No temporal changes in basic proteins over the 7-d incubation period were observed. Qualitative differences in total nonhistone chromosomal polypeptides in the shoot-forming and nonshoot-forming tissue were also observed. Differences in chromosomal polypeptides were observed. In contrast to the basic chromosomal proteins, temporal variation in the nonhistone chromosomal polypeptides was demonstrated. Throughout the 7-d sampling period, 29 and 12 nonhistone chromosomal polypeptides varied qualitatively in shoot-forming and nonshoot-forming callus cultures, respectively. In vitro labeling with ³²P-orthophosphate indicated that approximately 1.0% and 0.3% of the nonhistone chromosomal proteins were phosphorylated in the shoot-forming and nonshoot-forming cultures. Of these phosphorylated polypeptides, one was present in nonshoot-forming tissue and three were detected only in the shoot-forming tissue. Phosphorylation occurred at serine or threonine residues.     

Force-dependent changes in movement-related cortical potentials

The purpose of this study was to compare movement-related cortical potentials (MRCPs) associated with different levels of isometric contractions by elbow flexors. Eight healthy, right-handed male subjects participated in this study and performed different levels (10 and 50% of maximal voluntary contraction) of isometric contractions by the right elbow flexors. Electroencephalogram (EEG) signals were recorded from Fz, C3, Cz and C4 of the international 10/20 system. Motor potential (MP) amplitudes (from −200 to approximately −50 ms before force onset) for C3 associated with both force generations was significantly greater (P < 0.01) than those for C4, indicating that contralateral predominance of MRCP was observed in the right arm flexion. In Fz, the potentials of negative slope (NS′) (from −600 to approximately −200 ms) and MPs for 50% MVC were significantly greater than those of 10% MVC. In Cz, the MP associated with 50% MVC revealed a significantly greater (P < 0.05) value than that with 10% MVC. In C3 and C4, the MP associated with 50% MVC tended to be greater than that with 10% MVC, but no statistically significant differences were found. These force-dependent changes in MRCPs imply increased activation of neural circuits involved in motor preparation and initiation. It is therefore suggested that the larger potentials from Fz and Cz for 50% MVC compared with 10% MVC reflect a greater activation of supplementary motor area for the preparation of the larger force generation.     

La procréation médicalement assistée avec spermatozoïdes immatures: Impératifs cliniques et techniques chirurgicales

The authors report their experience with the use of spermatids in TESE programs where mature spermatozoa could not be isolated from testicular biopsies. The details of the indications for spermatid insemination, the technicity of the procedure and the results are exposed.