Showing papers by "Howard Hughes Medical Institute published in 1979"

Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding: application of a computerized model fitting technique.

Abstract: Frog myocardium appears to possess both beta1 and beta2 receptors, based on the potency order of several adrenergic agonists to compete for [3H]dihydroalprenolo1 binding. Selective beta blocking agents are able to distinguish two receptor subtypes in frog myocardium, but only one site in rat ventricle. Computer modeling using a PDP 11/45 indicates that all rat beta receptors are beta1, whereas only 15%-25% of frog ventricular beta receptors are of the beta1 subtype. Computerized curve fitting can provide a more accurate estimate of receptor parameters than currently available graphical methods of analysis.

α -Globin gene organisation in blacks precludes the severe form of α -thalassaemia

Abstract: IN most human populations, the α-globin structural gene loci are duplicated so that each diploid cell contains four copies of α-globin genes1–3. In α-thalassaemia, a hereditary disorder of α-globin chain synthesis, the most common molecular lesion is due to the deletion of the α-globin genes4–7. In the Asian population, four main α-thalassaemia syndromes of increasing clinical severity are recognised: (1) the silent carrier state (α-thalassaemia-2) with no clinical manifestation; (2) α-thalassaemia trait (α-thalassaemia-1), characterised by microcytic red blood cells but little or no anaemia; (3) haemoglobin-H disease, which manifests as haemolytic anaemia; and (4) homozygous α-thalassaemia, in which the afflicted fetus dies at or around term from hydrops fetalis. These four syndromes are due to the deletion of from one to all four copies of the α-globin genes. In this study, we have characterised α-thalassaemia in people of African origin. Haemoglobin screening programmes have shown that α-thalassaemia occurs in the black population8–10. Recently we have demonstrated by complementary DNA–DNA hybridisation that in black individuals with clinically well defined α-thalassaemia trait, two of the four normal α-globin genes were deleted11. However, in this population, haemoglobin-H disease is rare and homozygous α-thalassaemia has never been found12,13. For this study we have used the restriction endonuclease mapping technique of Southern14 to delineate the nature of the deletion of the α-globin genes.

Agonist-specific effects of guanine nucleotides on alpha-adrenergic receptors in human platelets.

Abstract: The effect of GTP on the binding affinity of alpha-adrenergic receptors for alpha-adrenergic agents was studied in human platelet lysates using direct ligand binding methods with [3H]dihydroergocryptine. GTP at a concentration of 0.1 mM markedly decreased the binding affinity of the agonist (-)epinephrine for the receptors (more than 10-fold) but had no effect on the binding of antagonists. The half maximal effect of GTP on epinephrine binding occurred at a concentration of 4 µM. Gpp(NH)p was as effective as GTP at the same concentration, whereas GDP was only 80% as effective. Other nucleotides such as ATP and ITP were less effective. The extent of the GTP-induced reduction in the affinity of alpha-adrenergic agents for the receptors was directly related to the intrinsic activity of these agents for inhibition of PGE1-stimulated adenylate cyclse. The effect of GTP appears to depend on the concurrent presence of Mg++. In the absence of Mg++, GTP caused only a slight decrease in the agonist binding affinity. When Mg++ was present without GTP, the binding affinity of the agonist (-)epinephrine was increased by 5-fold. GTP in the presence of Mg++ induces a state of diminished affinity of the receptor for the agonist which is lower than that induced by the nucleotide in the absence of MgCl2. The relationship between GTP and MgCl2 in the regulation of platelet alpha-adrenergic receptors which are inhibitory to adenylate cyclase activity appears to be analogous to their role in regulating beta-adrenergic receptors which are stimulatory for the enzyme in other tissues.

Allograft tolerance after total lymphoid irradiation (TLI).

Abstract: The ability to induce permanent and specific transplantation tolerance in neonatal mice was first reported by Medawar and his colleagues {Medawar 1953, Billingham & Brent 1956) more than 25 years ago. Since then, several models of tolerance induction in adult laboratory animals have been developed (Binz & Wigzell 1976, Muller-Rucholtz et al. 1976, Von Boehmer et al. 1976, Yunis et ai. 1976), but as yet none have had successful clinical application. During the past several years, we developed a regimen for the induction of transplantation tolerance which allows for the permanent survival of BM, skin and heart allografts in both inbred and outbred adult animals which differ at the major histocompatibility genetic region. The tolerization regimen is based upon the use of total lymphoid irradiation (TLI), a radiotherapy technique which has been used to treat lymphoid malignancies in humans (Kaplan 1972). TLI has few severe side effects, and the mortality rate associated with this procedure is negligible (Kaplan 1972). We review here the essential features of our experimental findings, and suggest that this regimen merits further investigation with regard to applicability to clinical bone marrow and organ transplantation.

Corticotropin and beta-endorphin: construction and analysis of recombinant DNA complementary to mRNA for the common precursor

Abstract: A cDNA fragment synthesized from mouse mRNA (ACTH/LPH mRNA) that codes for the precursor polypeptide containing corticotropin (ACTH), beta-lipotropin (LPH), and several other peptides has been cloned in bacteria. The mRNA was enriched for ACTH/LPH mRNA translational activity (to about 75%) prior to cDNA synthesis. It appears to contain about 1200 bases, of which approximately 450 bases are not translated. The cloned DNA fragment is complementary to the region of the mRNA coding for the protein fragment beta-LPH-(44--90); this contains all of the amino acids of [Met]-enkephalin (residues 61--65 of beta-LPH), most of the amino acids of beta-melanocyte-stimulating hormone, and all but the carboxy-terminal amino acid of beta-endorphin. Based on assignment of the amino acid sequence of mouse beta-LPH from the nucelic acid sequence, it appears that there is extensive homology of mouse beta-LPH with human and porcine beta-LPH. The data also establish the linkage between beta-melanocyte-stimulating hormone and beta-endorphin as a Lys-Arg sequence. It is hoped that this cloned DNA can be used as a probe to study the expression and structure of the ACTH/LPH gene.

The regulatory and effector roles of eosinophils.

Abstract: Publisher Summary This chapter discusses the regulatory and effector role of eosinophils. The eosinophilic polymorphonuclear (PMN) leukocyte or eosinophil was initially recognized and defined by the characteristic staining of its cytoplasmic granules with aniline dyes, such as eosin. The passive transfer of eosinophilia by T cells from animals with peripheral blood eosinophilia and the special role of the stimulated mast cell in local eosinophilia are produced. Factors that are preferentially chemotactic for eosinophils as compared to other types of leukocytes are obtained. The long-noted association of eosinophilia with infection by helminths is given special significance by increasing evidence for the concept of a protective effector function of these cells in parasitic infections. Specific immunological principles capable of augmenting the production of eosinophils have been recognized recently by the application of in vitro bone marrow culture techniques. Contemporary investigations of the functions of eosinophils have been facilitated by the development of improved approaches for obtaining purified populations of eosinophils and of more sensitive biochemical and immunochemical methods to characterize cellular constituents and receptors. Eosinophils differ from other leukocytes in their genesis and maturation, their morphology and biochemistry, and their functional abilities. Eosinophils possess apparently unique capabilities to modulate immediate-type hypersensitivity reactions by virtue of specific biochemical constituents that degrade mast cell-derived mediators.

Pitfalls in the Interpretation of Leukocyte Counts of Newborn Infants

Abstract: Some pitfalls in the interpretation of neonatal leukocyte counts are identified. The well-known variability in neonatal leukocyte counts was investigated by simultaneously sampling arterial, venous and capillary blood, and during periods of rest and mild and violent exercise. Venous blood leukocyte counts were 82% +/- 3.5 (mean +/- SE, P = less than .001) of counts in simultaneously drawn capillary blood from heel punctures; arterial blood counts were 77% +/- 5.3 (P less than .001) of capillary blood values. Following violent crying, capillary blood leukocyte counts increased to 146% +/- 6.1 (P less than .001) of baseline values, and a shift to the left occurred. Milder exercise induced an increase to 113% +/- 5.2 (P less than .05), without a leftward shift. Thus, counts from different vascular sources cannot be considered equivalent. Also, counts from vigorously crying babies may show leukocytosis and a leftward shift, and erroneously suggest bacterial infection. It is recommended that serial counts be obtained from a consistent vascular source in resting babies.

Multiple reactive sulfhydryl groups modulate the function of adenylate cyclase coupled beta-adrenergic receptors.

Abstract: The beta -adrenergic receptor adenylate cyclase complex in the frog erythrocyte contains at least two reactive sulfhydryl groups, which have been identified by their different sensitivities to N-ethylmaleimide (NEM). Adenylate cyclase catalytic activity is completely inhibited by [NEM] = 1 mM. In contrast, ability of beta -adrenergic agonists to form a high affinity guanine nucleotide sensitive state is reduced by NEM only at concentrations ≥ 1 mM. The inhibition of cyclase activity did not affect high affinity agonist binding nor perturb the ability of guanine nucleotides to reduce agonist affinity as determined in direct radioligand binding assays utilizing the beta -adrenergic agonist [ 3 H]hydroxybenzylisoproterenol and (-)isoproterenol competition for antagonist [ 3 H]dihydroalprenolol binding. Preincubation of frog erythrocyte membranes with antagonists or guanine nucleotide did not alter the effect of NEM on agonist affinity. However, once formed by preincubation of membranes with agonist, the high affinity state is resistant to the effects of NEM. The data suggest that the sulfhydryl group modulating agonist affinity 1) is located on the receptor complex but distal to the hormonal binding site and 2) may be associated with guanine nucleotide regulation of agonist affinity. The widely different sensitivities of adenylate cyclase activity and agonist binding affinity toward NEM have been exploited to investigate the mechanism of desensitization in frog erythrocytes. Incubation of intact erythrocytes with NEM under appropriate conditions inactivates the cyclase enzyme without altering the ability of membranes prepared from these cells to bind agonist with high affinity. This NEM treatment prior to prolonged exposure to (-)isoproterenol prevents receptor desensitization in frog erythrocytes, suggesting that agonist occupancy of a nucleotide-sensitive receptor is insufficient, by itself, to initiate the receptor regulation process.

DNA restriction endonuclease analysis for localization of human beta- and delta-globin genes on chromosome 11.

Abstract: DNA from a clone of a mouse-human hybrid that retained a human chromosome consisting of the major part of chromosome 11 and region q25-26-qter of the X chromosome was digested with various restriction endonucleases, subjected to electrophoresis in agarose gels, and transferred to nitrocellulose filters. The restriction digest pattern of the clone, when hybridized with a 32P-labeled plasmid fragment containing human beta-globin gene sequences, was a composite of the normal human and mouse (A9) patterns. When back-selected in 6-thioguanine to eliminate the 11 translocation chromosome, the hybrid cells showed only the A9 restriction pattern. These results substantiate the localization of beta- and delta-globin genes to human chromosome 11 and exclude the region 11q23-qter as the site.

Molecular cloning and sequence analysis of adult chicken β globin cDNA

Abstract: The molecular cloning and nucleotide sequence analysis of adult chicken beta globin mRNA is reported. DNA sequences derived from in vitro transcrption of globin mRNA were purified and amplified as recombinant DNA using the plasmid pBR322. Sequence analysis of several clones coding for beta globin strongly suggests that transcription errors may be generated near the 5' end of transcripts in vitro by reverse transcription. The complete sequence of the longest beta globin insert containing 51 bases of the 5' untranslated region as well as the complete coding and 3' untranslated regions has been determined.

Comparison of thyroxine and 3,3′,5′-triiodothyronine metabolism in rat kidney and liver homogenates☆☆☆

Abstract: The effects of agents added in vitro, in vivo PTU treatment, and fasting for 72 hr on T 4 -T 3 conversion rates and rT 3 degradation rates in rat kidney and liver homogenates were compared. In kidney homogenates, 5 m M DTT stimulated both reactions, whereas 0.3 m M diamide, 0.1 μ M iopanoic acid, 17 μ M PTU and 1 m M 2,4-dinitrophenol inhibited both reactions; 25 μ M methimazole had no effect. DTT also stimulated both of these reactions in liver homogenates. Diamide was a less potent inhibitor in liver than in kidney homogenates. Kinetic analysis showed that the k m for T 4 in kidney and liver homogenates were similar, but not identical, and that the k m for rT 3 in kidney and liver homogenates were again similar, but not precisely the same. When a particulate fraction of the homogenates was employed, the k m for T 4 in two kidney preparations was 0.8 and 1.0 μ M , and in two liver preparations it was 2.9 and 5.5 μ M . PTU administered in vivo reduced the T 4 -T 3 conversion rates and rT 3 degradation rates in kidney and liver homogenates to 3 concentration to 3 concentration to nine times control, but did not alter the mean serum T 4 concentration or the hepatic glutathione content. A 72-hr fast had no effect on T 4 -T 3 conversion or rT 3 degradation rates in kidney homogenates and had no effect on renal glutathione content, but fasting had the expected inhibitory effect on T 4 -T 3 conversion in liver homogenates and lowered the hepatic glutathione content to 79% of control. These results, along with previous findings from this and other laboratories, strongly suggest that there is a single iodothyronine 5′-monodeio-dinase in rat kidney that metabolizes both T 4 and rT 3 . The results are also compatible with the hypothesis that the iodothyronine 5′-monodeiodinases in rat kidney and liver are the same enzyme.

Is UAA or UGA part of the recognition signal for ribosomal initiation

Abstract: In none of the 92 published prokaryotic sequences is a translation codon preceeded by UAG as the first "termination codon". In most cases the UAA or UGA is close to the initiation codon and may be part of the ribosome recognition signal.