Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Partial Purification and Characterization of a Glycoprotein Cell Fusion Hormone from Griffithsia pacifica, a Red Alga

In filaments of the red alga Griffithsia, dead intercalary cells are replaced by the process of cell repair by cell fusion. This process is coordinated by a morphogenetic cell fusion hormone, rhodomorphin, which accelerates cell division and induces the production of a specialized repair cell. We have isolated rhodomorphin from Griffithsia pacifica Kylin and have purified it by concanavalin A affinity chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. This molecule binds specifically to concanavalin A, is proteinase sensitive, and is inactivated by short treatments at temperatures of 50°C or above. It therefore appears that rhodomorphin from G. pacifica is a glycoprotein; its molecular weight, as estimated by gel filtration, is approximately 14,000.     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

In vitro growth of B lymphocytes infiltrating human melanoma tissue by transformation with EBV: evidence for secretion of anti-melanoma antibodies by some transformed cells

Epstein Barr virus was used to transform the B lymphocytes infiltrating metastatic tumor tissue from seven patients with melanoma. In this way it was possible to establish continuously growing B lymphoblastoid cell lines (LCL) derived from the tumor-infiltrating B lymphocytes from each of the seven patients. Antibody production of up to 50 micrograms/ml could be achieved by such cultures, and the lymphoblastoid cells could be cloned readily by limit dilution on a feeder layer of irradiated fetal fibroblasts. Preliminary analysis of the antibodies produced by lymphoblastoid cell lines established from tumors from two of the patients indicated that most were of IgM type and bound to a panel of melanoma cell target cells, as well as to some nonmelanoma tumor cell lines. Cloned LCL were produced from the tumor-infiltrating B cells from one of the patients, and of 100 such clones tested, 9% secreted antibody that bound to autologous tumor cells, and one of these clones produced antibody that appeared to be melanoma specific.     

Immunoregulation in experimental disseminated histoplasmosis: flow microfluorometry (FMF) studies of the Thy and Lyt phenotypes of T lymphocytes from infected mice

Previous studies have shown that mice infected i.v. with 6 X 10(5) yeast phase Histoplasma capsulatum (Hc) develop suppressed immune responses during weeks 1 to 4 of infection but that by weeks 8 to 12 of infection these responses return to normal. In this study total and differential cell counts showed that as early as the third day of infection there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymus of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 28 both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric (FMF) studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (slg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 5 and 7 the thymocytes from infected mice displayed a higher relative fluorescence intensity (RFI) of the Thy-1.2 marker than normal thymocytes, whereas at day 10, the RFI was less than that of normal thymic lymphocytes. Between days 7 and 10 of infection the RFI of the Lyt-2 marker was less on thymocytes from Hc-infected mice; however, there was no change in the Lyt-1 marker. Examination of these lymphocyte markers in blood, spleen, and mesenteric lymph nodes showed that there were decreases in the RFI of both the Thy-1.2 and Lyt-2 between days 5 and 10 of infection. No changes were observed in the Lyt-1 or slg markers. By day 28 there were no differences between the normal and infected mice with respect to any surface marker in any of the organs studied. In other experiments, the effect of adrenalectomy before infection on these surface markers was studied. Absolute numbers of Thy-1.2+, Lyt-1+, and Lyt-2+ cells were significantly increased in the spleen and significantly decreased in the thymus and peripheral blood of infected mice relative to normal controls. These studies suggest that there is a migration of cells from the thymus, blood, and bone marrow to the spleens of mice with disseminated Hc infection.     

The action of nitrous acid on C-teichoic acid (C-substance) from the walls of Diplococcus pneumoniae

1. C-teichoic acid (C-substance) from the walls of Diplococcus pneumoniae contained free amino groups accessible to attack by nitrous acid. Treatment with nitrous acid, followed by reduction with borohydride and hydrolysis with acid, gave ribitol, glucitol and their respective phosphates. 2. Hydrolysis of the polymer with alkali followed by treatment of products with nitrous acid yielded glucose. 3. When alkali hydrolysis was followed by treatment with a phosphomonoesterase, nitrous acid degradation of C-substance yielded glucose and a disaccharide identified as 2-O-(N-acetylgalactosaminyl)-d-ribitol. 4. A partial structure for C-teichoic acid was deduced in which the order of the constituent residues and the position of phosphodiester linkages were established.     

Ribonuclease H activity present in purified DNA polymerase from avian myeloblastosis virus

The presence of ribonuclease H activity in the purified complex of DNA polymerase from avian myeloblastosis virus is described. Evidence includes co-chromatography of the two activities during all purification steps; the presence of ribonuclease H activity in the purified two-polypeptide complex of DNA polymerase; ion requirements for optimal activity of purified ribonuclease H are identical to those for the purified DNA polymerase; and monospecific antiserum against purified DNA polymerase neutralizes the ribonuclease H activity.     

Replication of nucleolus-associated DNA during "G2 phase" in Physarum polycephalum

In the myxomycete, Physarum polycephalum, the bulk of nuclear DNA replication occurs during a period of a few hours immediately following upon mitosis. During the remainder of the intermitotic period, incorporation of thymidine-³H continues at a low rate in the region of the nucleolus (radioautographs). A few nuclei incorporated thymidine-³H into the extranucleolar chromatin at a high rate at all times of the intermitotic period. These nuclei were exceptionally large and they frequently contained several small nucleoli of different sizes rather than the one, central nucleolus which is characteristic of a normal interphase nucleus.