alpha-D-mannosidase forms in chicken liver

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.     

Centromeric index versus DNA content flow karyotypes of human chromosomes measured by means of slit-scan flow cytometry

We have applied slit-scan flow cytometry (SSFCM) to classify human chromosomes according to their centromeric index (CI) and relative DNA content. The resulting bivariate--CI vs. DNA content--distributions shows 14 peaks for normal human chromosomes. Distinct peaks are produced by chromosomes 1, 2, 3, 4 + 5, 6 + 7 + X, 8, 13 + 14 + 15, 16, 17 + 18, 19 + 20, and 21 + 22 + Y. In addition, chromosomes 9 through 12 are resolved into three peaks. The identity of the chromosomes comprising each peak was determined by comparing CI vs. DNA content distributions measured for normal human chromosomes by means of SSFCM with CI and DNA content values measured for human chromosomes with image analysis. The accuracy of CI measurement by SSFCM was verified by measuring CIs for human chromosomes isolated from human/rodent hybrid cell lines containing only a few known human chromosomes. These studies showed CIs measured for human chromosomes 1-19 and 21 to be in close agreement with the CIs calculated by means of image analysis. We further confirmed the chromosome assignments for each peak by showing that the relative volumes of the peaks in the CI vs. DNA content distributions for chromosomes from normal cells are similar to the relative frequencies of chromosomes expected for these peaks based on the peak assignments.     

Human T-cell tumours containing chromosome 14 inversion or translocation with breakpoints proximal to immunoglobulin joining regions at 14q32.

T-cell tumours are frequently found to carry an inversion of chromosome 14 (inv(14)) (q11;q32) or more rarely a chromosome 14 translocation t(14;14) with the same cytogenetic breakpoints (q11;q32). We have examined the molecular junctions of an inv(14) and a translocation t(14;14) using T-cell receptor (TCR) alpha joining (J) region probes. Both of these chromosomal abnormalities have breakpoints within the TCR J alpha locus at 14q11 and both have breakpoints which are proximal (i.e. on the centromeric side) to the immunoglobulin heavy chain JH region at 14q32. The cloned segments corresponding to the junctions at 14q32 are not associated with obvious immunoglobulin-like sequences. This contrasts to the previously described inv(14) in the cell line SUP-T1 and places a potential cluster of chromosome 14 breakpoints downstream of the Ig JH locus. The possible role of the varying breakpoints in the development of these tumours is discussed.     

Production and Regeneration of Protoplasts from Pseudocercosporella herpotrichoides (Fron) Deighton

This paper describes the development of a system for protoplast isolation from this pathogen using the commercially available, hydrolytic enzyme preparations Rhozyme HP150, Driselase and Cellulase CP. An analysis of the active components of these enzymes believed to be involved in protoplast release has been made. Factors affecting protoplast release including mycelial age, enzyme concentration and choice of osmotic stabilizer have been optimised, and conditions for high frequency protoplast regener, ation have been determined. The possible significance of the interaction of the several components of each enzyme is discussed. The developments described in this paper can now be exploited in genetic studies in this imperfect fungus.     

Biochemical and molecular characterization of the glucocorticoid receptor of lymphosarcoma P1798 variants

Three phenotypically distinct isolates from lymphosarcoma P1798 have been compared with respect to properties of the glucocorticoid receptor. Wild type P1798 cells express functional receptors and glucocorticoid treatment of such cells causes cytolysis in vivo. Wild type cells do not undergo cytolysis in culture. Rather, such cells exhibit reversible inhibition of proliferation in the presence of dexamethasone. Two variant populations were selected from this background. One was selected for the ability to form tumors in mice receiving pharmacological doses of glucocorticoids. Cells from such tumors are resistant to the cytolytic effects of glucocorticoids in vivo, but are sensitive to the antiproliferative effects of the hormone in culture. Variants were also selected based upon their ability to proliferate in the presence of dexamethasone in culture. These variants were resistant to glucocorticoid-mediated cytolysis in vivo. Wild type P1798 cells express approximately 20,000 high affinity dexamethasone-binding sites per cell. Dexamethasone-mesylate labeling and immunoblotting experiments indicate that hormone binding is due to a polypeptide of Mr 90-100 K. This polypeptide is encoded in an mRNA species that resolved as a single entity of approximately 7000 nucleotides. Variants selected for resistance to cytolysis in vivo are indistinguishable in any of these respects from wild type cells. The receptors are fully functional, as evidenced by their ability to precipitate growth arrest of dexamethasone-treated cultures. Variants selected for resistance in culture harbor a receptor mutation. They express fewer than 500 dexamethasone-binding sites per cell. Such variants contain neither detectable dexamethasone-mesylate-binding protein nor any protein that is recognized by a receptor antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Escherichia coli STa enterotoxin and other secretagogues on mucosal surface pH of rat small intestine in vivo

The mucosal surface pH of rat small intestine was measured in vivo. The surface pH in the normal jejunum was 6.20 +/- 0.02 (67) and 7.00 +/- 0.05 (5) in the ileum. Escherichia coli STa toxin induced a rapid and reversible alkalinization of both jejunal and ileal mucosae to a pH of 6.91 +/- 0.08 (10) and 7.67 +/- 0.06 (5) respectively. The synthetic ST analogue, STh-(6-19), had an effect identical to native STa toxin on jejunal surface pH. Theophylline (20 mM) maintained the STa-elevated jejunal surface pH after toxin removal but had no effect on untreated tissue. 8-Bromo cyclic GMP resembled STa by causing similar mucosal alkalinization in the jejunum; 8-bromo cyclic AMP, forskolin and cholera toxin individually had considerably smaller effects on surface pH, although combining forskolin or cholera toxin with theophylline resulted in alkalinization of the jejunal mucosa to a pH of 6.92 +/- 0.03 (5) and 6.76 +/- 0.04 (4). These results indicate that cyclic-GMP-dependent secretory processes are more capable of inducing surface pH changes than those dependent on cyclic AMP. The ability of STa to alter mucosal surface pH makes it a useful tool to investigate the microclimate hypothesis for weak electrolyte absorption.     

Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6 to 1.0 ng/ml of PMA and 125 micrograms/ml of IgG isolated from a goat anti-5'-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2R expression and IL-2 secretion were also induced by anti-5'-NT antibodies and PMA. Anti-5'-NT-induced proliferation was inhibited greater than 95% by a murine anti-IL-2 receptor mAb and required less than 0.3% monocytes. Similar results have been obtained with a murine mAb specific for 5'-NT. As expected, anti-5'-NT antibodies and PMA did not induce the proliferation of ecto-5'-NT-T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol-specific phospholipase C removed an average of 89% of the 5'-NT activity from the cell surface and also inhibited by 83% the ability of the cells to proliferate in response to anti-5'-NT antibodies and PMA. Thus, the activation signal provided by anti-5'-NT antibodies is apparently transduced, in large part, by a form of the enzyme that is attached to the membrane via glycosyl-phosphatidylinositol linkage. These data suggest that 5'-NT may play a role in lymphocyte activation as has been proposed for other glycosyl-phosphatidylinositol-anchored lymphocyte surface proteins.     

Immunomodulation by low-dose methotrexate. I. Methotrexate selectively inhibits Lyt-2+ cells in murine acute graft-versus-host reactions

We have studied the effect of methotrexate in murine acute graft vs host (GvH) disease at concentrations analogous to those used in human rheumatoid arthritis. The GvH reaction was induced by i.v. injection of parental spleen cells into a normal F1 recipient. The acute suppression of T cell function in GvH mice was prevented by methotrexate given orally for 10 days at 1.0 or 0.5 mg/kg but not at 0.25 mg/kg. T cell mitogen response and IL-2 secretion that were inhibited in GvH mice were restored by methotrexate. Protection from immunosuppression in drug-treated GvH mice lasted at least 3 wk after drug dosing was stopped. The mechanism of the protective effect appears to be a preferential inhibition of donor and host Lyt-2+ Ts cell proliferation. In mixing experiments we found that methotrexate inhibited Ts function in GvH mice. By dual fluorescence labeling we showed that the engraftment of donor Lyt-2+ cells was prevented by drug treatment. This was not true of donor L3T4+ cells which were clearly present in the spleens of GvH mice after methotrexate treatment. These donor L3T4 cells were functional in that they induced the production of anti-DNA autoantibodies in the methotrexate-treated GvH mice.