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.     

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.     

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.     

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.     

Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

N-acetoxy-2-acetylaminofluorene modification of a deoxyoligonucleotide duplex.

The carcinogen N-acetoxy-2-acetylaminofluorene was reacted with d (CCACGCACC) to form a covalent adduct with attachment at the single guanine. The sample was purified, mixed 1:1 with d (GGTGCGTGG) and studied by thermal denaturation experiments. The Tm for the mixture was 35 +/- 3 degrees C, consistent with duplex formation. The method of continuous variation shows that the modified oligomer, d (CCACGAAFCACC), forms a 1:1 duplex with d (GGTGCGTGG). Circular dichroism spectra also indicate the formation of a duplex and suggest that the modified duplex has a left-handed conformation. Addition of the intercalating drug ethidium alters the CD spectrum of the modified duplex, resulting in a CD spectrum similar to that of ethidium bound to right-handed DNA.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

Oxidation and chemical modification of lung beta-galactoside-specific lectin.

Galaptins are small, soluble, lectins with a specificity for beta-galactose residues. Many galaptins are inactivated by atmospheric oxygen and are protected by disulphide-reducing reagents. We find that each subunit of rat lung galaptin contains one residue of tryptophan and six of cysteine. Oxygen inactivates rat lung galaptin by oxidation of the cysteine residues. During oxidation, the normal dimeric structure is maintained and all disulphide bonds are formed within individual subunits. Exogenous thiols protect against inactivation, but oxidized thiols accelerate inactivation. Human lung fibroblast galaptin is almost completely inactivated within 1 h in tissue culture medium at 37 degrees C. Alkylation of native rat lung galaptin with iodoacetate or ethyleneimine causes substantial loss of activity. The dimeric galaptin structure is maintained. In contrast, alkylation with iodoacetamide yields carboxamidomethyl-galaptin, which is fully active and stable to atmospheric oxygen in the absence of disulphide-reducing reagents. This derivative is very useful for studies of galaptin properties and function.