Immunoregulatory influence of thymic epithelium and thymopoietin on human B-lymphocyte differentiation

Thymic influence upon immunoregulation of B-lymphocyte differentiation in human bone marrow was investigated. Mononuclear cells isolated from marrow of normal adult volunteers were incubated with thymic epithelial monolayers or with the polypeptide thymopoietin. Generation of pokeweed mitogen-stimulated anti-sheep red blood cell antibody-secreting direct plaque-forming cells (PFC) was found to be inhibited following incubation of marrow mononuclear cells with thymic epithelial monolayers. Addition of 50 ng/ml thymopoietin to pokeweed mitogen-stimulated cultures resulted in enhanced marrow PFC responses, whereas higher doses of thymopoietin were inhibitory for the generation of PFC in this assay system. The data suggest that both helper and suppressor T cells are recruited from their precursors in human bone marrow by thymic influences. Generation of helper or suppressor cells may be dependent upon (a) the stage of differentation of precursor T cells and (b) upon the specific action and intensity of the thymic influence.     

Functional effects of thymopoietin32-36 (TP5) on cytotoxic lymphocyte precursor units (CLP-U). I. Enhancement of splenic CLP-U in vitro and in vivo after suboptimal antigenic stimulation

Cytotoxic lymphocyte precursor units (CLP-U) were enumerated in the spleens of C57BL/6 mice 3 days after i.p. injections of synthetic thymopoietin32-36 (TP5). One hundred to 1000 ng TP5/mouse potentiated splenic CLP-U, this effect being detectable only after suboptimal allogeneic sensitization (with 1.2 x 10(5) mitomycin-C treated DBA cells). This elevation of CLP-U persisted in the injected mice for at least 14 days. Control peptide did not affect CLP-U. In vitro incubation of 0.01 to 0.1 ng/ml of TP5 with normal C57BL/6 spleen cells also enhanced CLP-U after suboptimal allogeneic stimulation; high concentrations of TP5 caused suppression of CLP-U and this was detectable with optimal sensitization conditions. Thus TP5, in vitro and in vivo, appears to regulate immune responsiveness and this regulation varies with TP5 dosage and with the immune stimulus.     

Effect of temperature on morphology and DNA-content of polytene chromosomes in Drosophila

Feulgen-DNA contents and chromosome lengths and projected areas were measured in salivary gland nuclei from Drosophila prepupae which had developed at 25° or 15° C. Nuclei from a given prepupa fell into 3 to 5 DNA classes corresponding to different levels of polyteny. The 15° nuclei tended to fall into higher classes than those from 25°-reared animals, and their chromosomes were, on average, about 50% wider. Chromosomes within a given DNA class did not differ significantly in mean area, length or width between the temperature groups, and slight apparent differences in mean DNA content were attributable to systematic microdensitometric errors associated with differences in the spreading behaviour of the nuclei. On cytological examination, chromosomes from the two temperature groups differed mainly in width and stain intensity, but some other differences in appearance could not be accounted for by levels of polyteny. The mean length of the chromosome complement was about 400 m. From one polytenic level to the next the chromosomes increased by about 10% in length, 40% in width and 17% in mean absorbance. The DNA content approximately doubled; small apparent deviations from the 12 ratio could have been due to microdensitometric error or to underreplication of heterochromatin.     

Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Karyotype analysis of Meloidogyne hapla by 3-D reconstruction of synaptonemal complexes from electron microscopy of serial sections

Meloidogyne hapla Race A (meiotic, n=17) females have 17 synaptonemal complexes (SC). The karyotype length is constant throughout pachytene, although nuclear volume increases as pachytene progresses. Each SC has at least one region in which two pairs of lateral elements run parallel to each other for a distance of 1–2 m, thus forming a double SC (dSC). Decondensed chromatin regions (DCR) occur along some SCs and represent 5% of the length of the karyotype. The DCRs may be the location of the sex-determining chromatin. Spermatocytes from males of the same meiotic parthenogenetic race (A) have SCs and cylindrical granular complexes (CGC) while males and females from a mitotic (3n=45) parthenogenetic race (B) lack such structures. The CGCs may contribute precursors necessary for the formation of SCs.     

Molecular characterization of Ascaris suum DNA and of chromatin diminution

A technique for the extraction of pure somatic (post-diminution) and germ line (pre-diminution) DNA from the parasitic nematode Ascaris is described. Uncontaminated post- and pre-diminution DNAs were sheared and reassociated to different C₀t values. Computer analysis of the complete reassociation kinetics determined that 33% of the germ line genome is eliminated during the process of chromatin diminution. The eliminated DNA is comprised of repetitive and unique sequences in an approx. 1:1 ratio.     

Genetic and Segregation Analysis of Escherichia coli Strains Containing a Tandem Duplication of the trpD-purB Region of the Chromosome

Genetic and segregation analysis of Escherichia coli strains containing a partial duplication of the trp operon reveal that the 2.5-min-long region trpD-purB is duplicated in tandem in the chromosome. The adjacent loci cysB and fabD are not duplicated. Although one copy of the duplicated region is longer than the maximum size of bacteriophage P1kc transducing fragments, the frequency at which the duplicated segment trpDCBA is transferred by transduction to tonB-trp deletion strains is equal to that observed for transfer of the normal trp operon. This suggests that three-point recombination events believed to account for transduction of long duplications occur as frequently as two-point recombination events believed to account for normal transduction. Cotransduction frequencies of trpDCBA with the duplicated loci tonB, galU, tyrT, and hemA are very similar to those for the trp operon with the same loci. This indicates that normal genetic linkage is maintained during the three-point recombination event. However, purB, which is normally unlinked to trp by transduction, is closely linked to trpDCBA and thus must be near the repeat point of the duplication. Transduction tests with point mutations in the trp operon indicated that the repeat point occurs near the normal boundary between trpE and trpD. Segregation analysis of heterogenotes constructed from tonB-trp deletion strains shows that the frequency at which a marker is lost is approximately proportional to its distance from the repeat point. This finding is consistent with a random, singlesite crossover event during segregation. Several observations indicate that non-reciprocal genetic exchange also occurs between copies of the duplication. Analysis of heterogenotes containing dadR1 and dadR(+) demonstrate that the mutant allele is transdominant.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.