Identification of a complex of the three forms of the rat liver asialoglycoprotein receptor

We have generated antibodies against synthetic peptides which represent the carboxyl terminus of either the major, or the two minor, forms of the rat hepatic lectin which recognizes galactose-terminated glycoproteins (asialoglycoproteins). The antibodies were shown to be specific for the form of the lectin containing the immunizing peptide sequence by the following: reaction with purified lectin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation of sodium dodecyl sulfate-denatured lectin, immunoprecipitation of lectin synthesized in vitro. These antibodies, however, precipitated all three rat hepatic lectin forms from nonionic detergent extracts of hepatocytes labeled with 125I via the lactoperoxidase catalyzed technique. A similar result was obtained if antibody was bound to intact cells prior to solubilization with detergent and collection of the immune complexes. We conclude that at least the plasma membrane-associated fraction of the rat hepatic lectin forms exists as a heterotypic complex.     

Chromatid damage induced by fluorescent light during G2 phase in normal and Gardner syndrome fibroblasts. Interpretation in terms of deficient DNA repair

Skin fibroblasts from Gardner syndrome (GS) compared with those from normal donors showed a significantly higher incidence of chromatid gaps and breaks following exposure to low-intensity, cool-white fluorescent light during G2 phase of the cell cycle. Considerable evidence supports the concept that chromatid gaps and breaks seen directly after exposure to DNA-damaging agents represent unrepaired DNA single- and double-strand breaks respectively. The changes in incidence of chromatid aberrations with time after light exposure are consistent with the sequence of events known to follow DNA damage and repair. Initially, the incidence of light-induced chromatid gaps was equivalent in GS and normal fibroblasts. In the normal cells, the chromatid gaps disappeared by 1 h post-exposure, presumably as a result of efficient repair of DNA single-strand breaks. In contrast, the incidence of gaps increased in GS cells by 0.5 h followed by a decrease at 1 h and concomitant increase in chromatid breaks. It appears from these findings that the increased incidence of chromatid damage in GS fibroblasts results from deficient repair of DNA single-strand breaks which arise from incomplete nucleotide excision of DNA damage during G2 phase.     

Stable transformation of papaya via microprojectile bombardment

Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1^–1 2,4-D, 400 mg 1^–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1^–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - X-gluc 5-Br-4-Cl-3-indolyl--D-glucuronic acid - CaMV cauliflower mosaic virus - NOS nopaline synthase - NPTII neomycin phosphotransferase II Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Stimulation of vascular cell proliferation by beta-galactoside specific lectins

An investigation was conducted to assess the effects of various beta-galactoside specific lectins on the growth of vascular cells in vitro. The plant lectins from peanut (Arachis hypogaea), mushroom (Agaricus bisporus), and coral tree (Erythrina corallodendron) were used in these studies with the ultimate purpose of comparing those findings with data derived with the lectin isolated from rat lung. Peanut lectin was added to confluent and subconfluent cultures of smooth muscle cells (SMC), pulmonary arterial (PEC), and aortic endothelial cells (BAEC) at concentrations of 2, 3.5, and 7.0 micrograms/ml. There was a dose-dependent increase in cell proliferation for both confluent and subconfluent SMC, with maximal stimulation noted between 3.5 and 7 micrograms/ml of peanut lectin. A dose-dependent stimulation of PEC proliferation was also found with maximal stimulation between 3.5 and 7.0 micrograms/ml. Peanut lectin did not stimulate BAEC to multiply. The stimulation of PEC and SMC by peanut lectin could be prevented by the addition of 50 mM lactose. Peanut and mushroom lectin stimulated the proliferation of sparse cultures of SMC in a dose-dependent fashion in both standard (10% fetal bovine serum, or FBS) or low (0.5% FBS) serum to about the same degree. Coral tree lectin did not have a significant stimulation of proliferation under either serum conditions. The incorporation of [3H]thymidine into the DNA of PEC was increased 30 and 150% by peanut lectin and lung galaptin, respectively, under standard serum conditions. However, under low serum conditions, both lectins increased incorporation by about the same extent (93 and 78% for peanut lectin and galaptin, respectively). Both lectins produced a 30% increase in DNA synthesis by SMC under standard serum conditions, and about a 200% increase under low serum conditions. These studies indicate that beta-galactoside specific lectins such as lung galaptin have mitogenic activity toward vascular cells.     

Changes in nuclear proteins of astrocytes as a result of acute ammonia or ethanol exposure

We have used an in vitro system to monitor the effects of high levels of ammonia and ethanol on glial cells. Nuclei were isolated and the protein profiles examined by two-dimensional gel electrophoresis. Acute exposure of rat astrocyte cell cultures to ammonia or ethanol resulted in changes in cellular morphology and the level of some nuclear proteins. Glial fibrillary acidic protein (GFAP) levels remained constant under both treatments. Several nuclear proteins were increased specifically. Only one protein was visually detected which was unique to treatment with ammonia or ethanol. This protein (p2a) appeared only in the presence of ammonia. There were no changes in previously observed astrocyte-associated proteins (Silverman et al. Neurochem Int. 12, 513–518, 1988). Two proteins appeared de novo upon either treatment with either ammonia or ethanol. These latter proteins had a molecular weight and pI profile similar to the major class of nuclear stress proteins (hsp70). However, results from immunoblot experiments clearly demonstrated that hsp70 was not induced in astroycte cultures following exposure to ammonia or ethanol.     

Systemic Lipopolysaccharide and Pokeweed Mitogen Increase Quinolinic Acid Content of Mouse Cerebral Cortex

The effects of lipopolysaccharide and pokeweed mitogen on brain L-tryptophan and quinolinic acid (QUIN) concentrations were investigated in C57BL/6NCR mice. Twenty-four hours after an intraperitoneal injection of lipopolysaccharide (5 micrograms from Salmonella abortus equii) or pokeweed mitogen (500 micrograms), cortical QUIN concentrations were increased by 81 +/- 6% and 182 +/- 15%, respectively. Plasma QUIN was increased 175 +/- 7% of control in pokeweed-mitogen treated mice only. Brain L-tryptophan concentrations were increased, whereas plasma L-tryptophan concentrations were decreased. The consequences of increased QUIN concentrations during endotoxin and mitogen exposure remain to be determined.     

Applying the PDR principle to AIDS

The principle of pathogen-derived resistance (the PDR principle) has been put forward as a broadly-applicable conceptual tool for use in designing genes which will confer resistance to pathogens. This paper reveals an example of how the PDR principle may be applied in the field of human medicine. Specifically it is shown how the PDR principle can be employed in designing a series of genes which should be capable of protecting human blood cells from the retrovirus causing the AIDS disease. Prospects are discussed for using such genes in gene therapy treatment of people infected with this virus.     

Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol

Allopurinol (1H-pyrazolo-[3,4-d]pyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide-forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (1H-pyrazolo-[3,4-d]pyrimidine-4,6-diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the O₂ concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.