Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Characterization of Haemophilus parainfluenzae strains with low-Mr or ladder-like lipopolysaccharides

Twenty-five Haemophilus parainfluenzae strains were characterized for lipopolysaccharide (LPS) profiles, outer membrane protein profiles, serum sensitivity, plasmid profiles and DNA homology. Seventeen strains produced low-Mr LPS that did not contain O-sidechains, while the remaining eight strains contained ladder-like LPS suggestive of O-repeated units. This is the first time in the genus Haemophilus that LPS with O-repeated groups has been described. The strains producing the different types of LPS could not be distinguished from each other in outer membrane protein profiles or the other characteristics examined.     

A followup study of the academic attainment and classroom behavior of children with elevated dentine lead levels

To investigate the impact of low-level lead exposure on children's behavior within a natural setting, we assessed the school performance of a sample of 141 elementary school children classified according to dentine lead level. Twenty-two children had “elevated” levels (≥20 parts per million), 71 had “midrange” levels (10.0–19.9 parts per million), and 48 had “low” levels (<10 parts per million). Four types of outcome data were collected: (1) scores on a standardized group intelligence test, (2) teachers' ratings, (3) incidence of academic failure (assignment to remedial aid, grade retention), and (4) observations of classroom behavior. In general, higher dentine lead levels were associated with less favorable school performance, with most covariance-adjustedp-values in the range of 0.05–0.15. The incidence of grade retention was the outcome most strongly related to lead level. The pattern of results suggests a consistent, though weak relationship between children's dentine lead levels and elementary school performance.     

Totipotent hematopoietic stem cells: normal self-renewal and differentiation after transplantation between mouse fetuses

Successful engraftment of mouse fetal liver cells in early fetal recipients, after microinjection via the placental circulation, is attributable to seeding of the recipient's liver by a cell type that is ancestral to both the myeloid and lymphoid definitive lineages and is capable of sustained self-renewal and differentiation for more than 2 years. This primitive cell is therefore the normal totipotent hematopoietic stem cell (THSC). The use of a large series of mutant anemic recipients with decreasing severity of an endogenous stem-cell defect (W/W, Wv/Wv, Wf/Wf, Wv/+), and therefore of graded selective advantage to normal donor cells, has revealed that engraftment entails marginal numbers of cells--probably individual ones--in the least afflicted hosts. Thus the observed progressive and coordinate shift toward donor-strain erythrocytes, granulocytes and B and T lymphocytes, over time, indicates THSC expansion to form a larger stem-cell pool and normally regulated differentiation of cells from the pool. This transplant system allows allogeneic combinations with impunity and therefore provides many novel experimental possibilities for investigating THSC normal development, genetic abnormalities or neoplastic potential in relation to the intact developmental succession of hematopoietic tissue environments in vivo.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Establishment of rat pancreatic endocrine cell lines by infection with simian virus 40.

The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Association of CAT polymorphisms with catalase activity and exposure to environmental oxidative stimuli

We tested the hypotheses that catalase activity is modified by CAT single nucleotide polymorphisms (SNPs) (-262;-844), and by their interactions with oxidant exposures (coal dusts, smoking), lymphotoxin alpha (LTA, NcoI) and tumor necrosis factor (TNF, -308) in 196 miners. Erythrocyte catalase, superoxide dismutase, and glutathione peroxidase activities were measured. The CAT -262 SNP was related to lower catalase activity (104, 87 and 72 k/g hemoglobin for CC, CT and TT, respectively, p < 0.0001). Regardless of CAT SNPs, the LTA NcoI but not the TNF-308 SNP was associated with catalase activity (p = 0.04 and p = 0.8). CAT -262 T carriers were less frequent in highly exposed miners (OR = 0.39 [0.20–0.78], p = 0.007). In CAT -262 T carriers only, catalase activity decreased with high dust exposure (p = 0.01). Haplotype analyses (combined CAT SNPs) confirm these results. Results show that CAT -262 and LTA NcoI SNPs, and interaction with coal dust exposure, influenced catalase activity.