Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Translational regulation of somatostatin in cultured sympathetic neurons

Coculture of sympathetic neurons with ganglion nonneuronal cells elevated levels of preprosomatostatin mRNA but did not alter neuronal synthesis, content, or release of somatostatin. Treatment of sympathetic neurons with culture medium conditioned by exposure to ganglion nonneuronal cells similarly elevated preprosomatostatin mRNA. Treatment with conditioned medium elevated somatostatin levels in pure neuronal cultures, but not in neurons cocultured with nonneuronal cells. Conditioned medium also failed to increase peptide levels in neurons cultured on a substratum of killed nonneuronal cells, despite a large increase in preprosomatostatin mRNA. These observations suggest that contact of sympathetic neurons with nonneuronal cell membranes inhibits the increase in peptide synthesis, but not the increase in preprosomatostatin mRNA after treatment with conditioned medium. Thus neuronal interactions with nonneuronal cells regulate somatostatin metabolism at both the mRNA and peptide levels. Regulatory effects on the mRNA and the peptide are separable and do not necessarily occur in parallel, and translational controls may be the rate-limiting factors.     

Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.     

Mechanism of benzo(a)pyrene induction of alpha-human chorionic gonadotropin gene expression in human lung tumor cells

Human lung cells (ChaGo) derived from a bronchogenic carcinoma synthesize and secrete in the culture medium the alpha subunit of the glycoprotein hormone, human chorionic gonadotropin (alpha-hCG). The synthesis of alpha-hCG by ChaGo cells could be further stimulated by treatment with sublethal concentrations of the polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP), or dimethylbenzanthracene. The production of alpha-hCG could be correlated to the levels of alpha-hCG-specific mRNA sequences in control and PAH-treated cells. Further analysis of the RNA species (Northern blot) revealed that the level of the mature (approximately 1.0 kb) and the high molecular weight alpha-hCG specific nuclear RNA sequences (approximately 2.2 and 5 kb) were all greater in PAH-treated cells. Addition of [3H]BaP (0.25 microgram/ml) in the culture medium of ChaGo cells led to immediate uptake of the radioactive compound apparently by simple diffusion. SDS PAGE and subsequent fluorography revealed that the radioactive compound interacted and formed covalent complexes with cytoplasmic and nuclear proteins. This covalent interaction of the [3H]BaP molecule with cellular proteins could be significantly inhibited by either inhibiting the activity of the enzyme aryl hydrocarbon hydroxylase with 7,8-benzoflavone or by reducing the cellular concentration of the enzyme by simultaneous incubation with cycloheximide. These results suggested that in ChaGo cells, the observed covalent complexes were formed by the interaction of the BaP metabolites with cellular proteins. The concentrations at which 7,8-benzoflavone or cycloheximide inhibited formation of metabolites from [3H]BaP and their covalent interaction with cell protein did not affect the BaP-induced stimulation of alpha-hCG gene expression. However, the cytotoxic effects of BaP in ChaGo cells seemed to be exerted by the metabolism of the compounds. Results presented in this report suggest that BaP metabolism and the interaction of the metabolites with cell proteins were not essential for the BaP-induced modulation of alpha-hCG gene expression.     

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.     

The respiratory burst of human neutrophils treated with various stimulators in vitro is dampened by exogenous unsaturated fatty acids

Cis-unsaturated free fatty acids (FFA) at concentrations between 10 and 30 microM suppressed the superoxide respiratory burst induced in human neutrophils by the chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine (FMLP). Corresponding trans-isomers had a reduced efficacy while saturated FFA were inert. The effects of unsaturated FFA were maximally achieved after several min of preincubation with cells and reversed upon washing. Increased concentrations of Ca2+ in the medium also relieved the inhibition. Unsaturated FFA were equally effective in dampening the respiratory burst induced by fluoride ions but less so with bursts elicited by 9 nM phorbol myristate acetate (PMA). Moreover reactions triggered by higher concentrations (e.g., 100 nM) of PMA were resistant to the effects of FFA. Radioimmunoassays showed that unsaturated FFA directly elevated intracellular cyclic adenosine monophosphate (cAMP) by severalfold above basal levels. It is suggested that inhibition is brought about by unsaturated FFA perturbation of the neutrophil membrane structure, perhaps with an independent contribution from a cAMP-dependent mechanism.