Showing papers by "Howard Hughes Medical Institute published in 1984"

A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase.

Abstract: Arachidonate and other unsaturated long-chain fatty acids were found to activate protein kinase C from human neutrophils. Kinase activation by arachidonate required calcium and was enhanced by diolein but did not require exogenous phosphatidylserine. Submaximal levels of arachidonate also enhanced the affinity of the kinase for calcium during activation by phosphatidylserine. Thus the release of arachidonate, which is triggered in many cell types by ligand-receptor interactions, could play a second messenger role in the regulation of cellular function by activation of protein kinase C.

Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene

Abstract: In this communication, we describe several features of the D. melanogaster gene which codes for ribosomal protein 49 (rp49). Nucleotide sequence analysis in conjunction with primer extension and S1 nuclease protection experiments show that the structure of the rp49 gene consists of a 102 bp 5' exon, a single 59 bp intron, and a 420 bp 3' exon, encoding a total of 132 amino acids. The rp49 gene shares many features with other abundantly expressed Drosophila genes, including codon preference, which are discussed.

Leukotriene B4 produces hyperalgesia that is dependent on polymorphonuclear leukocytes.

Abstract: Leukotriene B4, at the same intracutaneous doses as bradykinin, reduced the nociceptive threshold in the rat paw. The mechanism of leukotriene B4-induced hyperalgesia was distinguished from that of the hyperalgesia elicited by prostaglandin E2 and bradykinin by its dependence on polymorphonuclear leukocytes and independence of the cyclooxygenation of arachidonic acid.

Pre-proglucagon messenger ribonucleic acid: nucleotide and encoded amino acid sequences of the rat pancreatic complementary deoxyribonucleic acid.

Abstract: Glucagon, a pancreatic peptide hormone of 29 amino acids that regulates carbohydrate, fat, and protein metabolism, is one of a family of structurally similar regulatory peptides which include GH-releasing hormone, vasoactive intestinal peptide, secretin, and gastric inhibitory peptide. The synthesis of glucagon involves its specific proteolytic cleavage from preproglucagon, a large polyprotein precursor. To facilitate analyses of the cellular processing of pre-proglucagon and to begin studies of the regulation of glucagon gene expression in the rat, we have cloned and sequenced two cDNAs derived from rat neonatal pancreas. The cDNAs represent close to the entire transcriptional sequence of the glucagon gene and encode a pre-proglucagon of 180 amino acids. The coding region of the pre-proglucagon contains, in addition to the sequence of glucagon, the sequences of two peptide domains that are related in their structures to glucagon. Glucagon and the two glucagon-like peptides are flanked in the precursor by...

Molecular evidence for new mutation at the hprt locus in Lesch-Nyhan patients.

Abstract: Hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC2.4.2.8), which functions in the metabolic salvage of purines, is encoded by an X-linked gene in man. Partial HPRT deficiencies are associated with gouty arthritis, while absence of activity results in Lesch-Nyhan syndrome (L-N). L-N patients fail to reproduce and the heterozygous state appears to confer no selective advantage. Thus, Haldane's principle predicts that new mutations at the hprt locus must occur frequently in order for L-N syndrome to be maintained in the population. This constant introduction of new mutations would be expected to result in a heterogeneous collection of genetic lesions, some of which may be novel. As we report here, the mutations in the hprt gene of seven L-N patients, selected from an initial survey of 28 patients, have been characterized and all were found to be distinctly different, as predicted. The origin of one unusual mutation has been identified by analysis of DNA from four generations of family members. Further molecular analysis of the origin of new mutations at the hprt locus should aid in resolving the issue of an apparent difference in the frequency of hprt mutations in males and females.

Elevated concentrations of leukotriene D4 in pulmonary edema fluid of patients with the adult respiratory distress syndrome

Abstract: The possible contribution of metabolites of arachidonic acid to the increased permeability of the alveolar-capillary barrier in the adult respiratory distress syndrome was examined by quantifying the pulmonary edema fluid concentrations of lipoxygenase and cyclooxygenase products. The concentration of leukotriene D4 in pulmonary edema fluid of 10 patients with the adult respiratory distress syndrome (18.5±6.8 pmol/ml; mean±SD), assessed by specific radioimmunoassay after isolation of the mediator, was significantly higher (P<0.001) than that of five patients with cardiogenic pulmonary edema (4.4±1.1 pmol/ml). The concentrations of leukotrienes B4 and C4, prostaglandin E2, and thromboxane B2 in edema fluid were not significantly different in the adult respiratory distress syndrome patients than in the other subjects with pulmonary edema. The edema fluid concentration of leukotriene D4 correlated with the ratio of edema fluid to plasma concentrations of albumin (r=0.64). Leukotriene D4 thus may contribute to the permeability defect which allows an accumulation of proteinrich alveolar fluid in the adult respiratory distress syndrome.

Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum.

Abstract: Calmodulin was purified from the eukaryotic microorganism Dictyostelium discoideum and characterized in terms of its nearly complete primary structure and quantitative activator activity. The strategy for amino acid sequence analysis took advantage of the highly conserved structure of calmodulin and employed a new procedure for limited cleavage of calmodulin that uses a protease from mouse submaxillary gland. Fourteen amino acid sequence differences between Dictyostelium and bovine calmodulin were identified unequivocally, as well as an unmethylated lysine at residue 115 instead of N epsilon, N epsilon, N epsilon-trimethyllysine. Seven of the amino acid substitutions in Dictyostelium calmodulin are novel in that the residues at these positions are invariant in all calmodulin sequences previously examined, most notably an additional residue at the carboxy terminus. Comparison of the Dictyostelium calmodulin sequence with other calmodulin sequences shows that the region with the greatest extended sequence identity includes parts of the first and second structural domains and the interdomain region between domains 1 and 2. Dictyostelium calmodulin activated bovine brain cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of bovine brain calmodulin. However, Dictyostelium calmodulin activated pea NAD kinase to a maximal level 4.6-fold greater than that produced by bovine brain calmodulin. This functional difference demonstrates the potential biological importance of the limited number of amino acid sequence differences between Dictyostelium calmodulin and other calmodulins and provides further insight into the structure, function, and evolution of the calmodulin family of proteins.

Structural characterization of a higher plant calmodulin : spinacia oleracea.

Abstract: Calmodulin is a eukaryotic calcium binding protein which has several calcium-dependent in vitro activities. Presented in this report is a structural characterization of calmodulin from spinach leaves (Spinacia oleracea). Spinach calmodulin may be representative of higher plant calmodulins in general since calmodulin from the monocotyledon barley (Hordeum vulgare) is indistinguishable by a variety of physical, chemical, and functional criteria (Schleicher, Lukas, Watterson 1983 Plant Physiol 73: 666-670). Spinach calmodulin is homologous to bovine brain calmodulin with only 13 identified amino acid sequence differences, excluding a blocked NH2-terminal tripeptide whose sequence has not been elucidated. Two extended regions of sequence identity are in the NH2-terminal half of the molecule, while nine of the 13 identified differences are in the COOH-terminal half of the molecule. Two of the changes, a cysteine at residue 26 and a glutamine at residue 96, require a minimum of two base changes in the nucleotide codons. Both of these changes occur in the proposed calcium binding loops of the molecule. Five additional amino acid differences found in spinach calmodulin had not been observed previously in a calmodulin. As described in an accompanying report (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798), these limited number of amino acid sequence variations appear to result in differential effects on the activation of calmodulin-dependent enzymes by plant and vertebrate calmodulins.

Repetitive episodes of brief ischaemia (12 min) do not produce a cumulative depletion of high energy phosphate compounds.

Abstract: During myocardial ischaemia the purine (ATP, GTP) and pyrimidine (CTP, UTP) nucleotide content of the myocyte falls. When the ischaemic episode resolves, many hours or even days are required for restoration of nucleotide pools. These obervations suggest that repetitive episodes of ischaemia might produce progressive depletion of nucleotide pools. In order to determine the effect of repetitive episodes of brief ischaemia on nucleotide pools, open-chest dogs underwent three 12 min periods of occlusion of the left anterior descending coronary artery, with each occlusion followed by 10 min of reperfusion. During the first occlusion nucleotide pools decreased by 30% (ATP); 36% (GTP), 52% (CTP), and 48% (UTP). The subsequent two occlusions produced no further decrease in nucleotide pools. The myocardial content of adenine nucleotide catabolites (adenosine + inosine + hypoxanthine) tended to be greater during the first occlusion than during the subsequent occlusions, and substrate delivery (ie regional myocardial blood flow) was similar during each of the periods of ischaemia. These results indicate that a decrease in the rate of nucleotide degradation, rather than an increase in nucleotide synthesis, accounts for the maintenance of nucleotide content during subsequent ischaemic episodes after the initial ischaemic period. Thus repetitive episodes of regional ischaemia do not produce a cumulative decrease in the high energy phosphate content of the myocardium.

Characteristics of insulin and epidermal growth factor stimulation of receptor autophosphorylation in detergent extracts of rat liver and transplantable rat hepatomas.

Abstract: Insulin and epidermal growth factor (EGF) share a number of metabolic actions, including stimulation of protein synthesis and growth in certain tissues, and activation of apparent receptor tyrosine kinase activities. We have shown that insulin and EGF promote the phosphorylation of a number of intracellular proteins in common, suggesting that some of the shared metabolic actions of these hormones might be due to shared effects on protein kinases or phosphatases. We, therefore, compared the effects of these hormones on their respective membrane receptor autophosphorylation reactions in detergent extracts prepared from rat liver microsomes. Under appropriate conditions, ligand-promoted receptor autophosphorylation could be observed without further receptor purification. Insulin- and EGF-stimulated receptor autophosphorylation exhibited differing requirements for divalent cations and optimum ATP concentrations, and were enhanced by detergent extraction of the membranes. Insulin-promoted receptor autophosphorylation occurred on a tyrosine residue. In liver microsomal extracts prepared from rats exposed to various dietary conditions, insulin-stimulated receptor autophosphorylation was enhanced in extracts prepared from starved or diabetic animals when compared to those prepared from fed or fasted-refed animals. Experiments with extracts from several transplantable rat hepatomas of varying degrees of differentiation indicated that both insulin binding and insulin-stimulated receptor autophosphorylation were remarkably preserved in all tumors; in contrast, EGF binding and EGF-induced receptor autophosphorylation were diminished or absent in all tumors studied. These studies suggest that the relationship between insulin-mediated receptor phosphorylation and subsequent metabolic events might be profitably studied in these tissues.

The enzymatic synthesis of 5-amino-4-imidazolecarboxamide riboside triphosphate (ZTP)

Abstract: 5-Amino-4-imidazolecarboxamide riboside triphosphate (ZTP) is thought to play a regulatory role in cellular metabolism Unlike other nucleoside triphosphates, ZTP is synthesized in a one-step reaction in which the pyrophosphate group of 5-phosphoribosyl-l-pyrophosphate is transferred to the riboside monophosphate (ZMP) in a reaction catalyzed by 5-phosphoribosyl-l-pyrophosphate synthetase; reversal of this reaction leads to dephosphorylation of ZTP to ZMP This unusual route of synthesis (and catabolism) of ZTP may be important in defining its metabolic effects in the cell

Translocation and Uncoupling of the β-Adrenergic Receptor in Rat Lung after Catecholamine Promoted Desensitization in Vivo

Abstract: Beta-adrenergic agonists and antagonists are widely used in many clinical situations to regulate beta-adrenergic stimulation. However, responsiveness to beta-stimulation may be reduced by the process of desensitization. We have established an in vivo mammalian model system, the rat lung, to study the cellular and biochemical basis for beta-agonist induced desensitization. After in vivo administration of a beta-agonist [(-)isoproterenol] the adenylate cyclase becomes rapidly insensitive to further stimulation by beta-agonists with no change in basal or NaF-stimulated activity. The in vivo desensitization can be blocked by the simultaneous administration of a beta-antagonist [(+/-)propranolol] and the process displays the pharmacological characteristics typifying the beta 2 receptor of rat lung. This indicates that the in vivo desensitization is itself a receptor-mediated event. The processes of de- and resensitization are very rapid with onset within 5 min, maximal effect at 10 min, and complete reversal by 2-3 h. The change of adenylate cyclase sensitivity is paralleled by a translocation of approximately 40% of the beta-receptors from the plasma membrane fraction to a light membrane fraction, which has very low activities of plasma membrane marker enzymes. The receptors translocated to the light membrane fraction as well as those remaining in the plasma membranes are uncoupled with loss of their ability to form the high affinity, nucleotide sensitive, physiologically active state of the receptor. During resensitization the receptors in the plasma membrane fraction are recoupled before all the translocated receptors have returned. This suggests that translocation and uncoupling of the receptors are two distinct, probably independent processes. During the entire process of de- and resensitization no structural change of the receptor protein residing in the plasma membranes or light membrane fraction can be demonstrated as visualized by photoaffinity labeling.

Direct demonstration of impaired functionality of a purified desensitized beta-adrenergic receptor in a reconstituted system.

Abstract: Long-term exposure of various cell types to beta-adrenergic agonists such as isoproterenol leads to an attenuated responsiveness ("desensitization") of the adenylate cyclase system to further challenge with these agonists. The turkey erythrocyte model system was used earlier to show that a covalent modification of the receptor (phosphorylation) is associated with this process. The functionality of the "desensitized" beta-adrenergic receptor was assessed by implanting purified beta-adrenergic receptor preparations from control and desensitized turkey erythrocytes into phospholipid mixtures and then fusing them with receptor-deficient cells (Xenopus laevis erythrocytes). Desensitized beta-adrenergic receptors showed a 40 to 50 percent reduction in their ability to couple to the heterologous adenylate cyclase system, comparable to the reduction in their functionality observed in their original membrane environment. These results demonstrate the utility of recently developed receptor reconstitution techniques for assessing the functionality of purified receptors and show a direct link between a covalent modification of a membrane-bound receptor and its impaired functionality in a reconstituted system.

Immunopathogenetic roles of leukotrienes in human diseases.

Abstract: The recent definition of the pathways of generation and structures of diverse products of the lipoxygenation of arachidonic acid has established the identity of a new family of mediators of hypersensitivity and inflammation. Studies of the effects of these mediators have shown that leukotrienes C, D, and E, the constituents of the slow-reacting substance of anaphylaxis (SRS-A), are extremely potent smooth muscle contractile and vasoactive factors. Leukotriene B is a highly active stimulus of neutrophil and eosinophil functions and suppresses the immunological capabilities of T lymphocytes. The development of specific and sensitive radioimmunoassays has permitted the detection of elevated concentrations of leukotrienes in tissues or exudates in several diseases, including asthma, diverse allergic states, adult respiratory distress syndrome, psoriasis, spondyloarthritis, and gout. The application of selective inhibitors and antagonists of leukotrienes will clarify their pathogenetic contributions in human diseases and may yield new therapeutic approaches.

Human purine nucleoside phosphorylase cDNA sequence and genomic clone characterization

Abstract: The isolation of a cDNA clone containing the complete coding region for human purine nucleoside phosphorylase (PNP) has been described previously. In this report we present the nucleotide sequence of this cDNA clone and compare the derived amino acid sequence, encoding a protein of 32 kilodaltons, with the published amino acid composition. Using a fragment of the cDNA clone as a probe, human PNP genomic clones from a bacteriophage lambda library have been isolated and the structural organization of the wild type PNP gene determined.

Identification of calmodulin-binding proteins in chicken embryo fibroblasts.

Abstract: We recently reported the detection of multiple classes of calmodulin-binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium-modulated proteins. The fact that, in some cases, the same calmodulin-binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium-modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin-binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.

Two anonymous X-specific human sequences detecting restriction fragment length polymorphisms in region Xq26→qter

Abstract: Two anonymous X-specific sequences isolated from a genomic library of flow-sorted X chromosomal DNA were selected for study because they revealed restriction fragment length polymorphisms in the region Xq26 → qter. One sequence, DXS10, detected a two-allele TaqI polymorphic system with allele frequencies of 0.33 and 0.67. The other, 4D-8, defined an Mspl polymorphism with allele frequencies of 0.18 and 0.82. DXS10 is tightly linked to the hypoxanthine phosphoribosyltransferase (HPRT)locus with recombination distance θ=0 cM at LOD=5.55 (95 % probability limit θ<15 cM). DXS10 maps to Xq26 but is not contained within the HPRTlocus itself. 4D-8 shows no detectable linkage to the HPRTlocus, with maximum likelihood estimate for θ=50 cM and a LOD score of −2.61 at θ= 5 cM. These two polymorphisms provide additional chromosomal loci for gene mapping by linkage at the distal end of the long arm of the human X chromosome.

Defective polymorphonuclear leukocyte chemotaxis in homosexual men with persistent lymph node syndrome.

Abstract: Persistent lymph node syndrome, epidemiologically related to acquired immune deficiency syndrome, is characterized by reactive lymphadenopathy and an increased incidence of localized bacterial, viral, and fungal infections. Seven typical patients were found to have a localized infection every 4.5 ± 2.0 months (mean ± SD) since onset of adenopathy. Because recurrent bacterial infections may be associated with defective polymorphonuclear leukocyte (PMNL) function, studies of PMNL function in these patients were undertaken. The patients' PMNLs had diminished chemotactic responses to high concentrations of two structurally distinct chemotactic factors, leukotriene B4 and N-formylmethionylleucylphenylalanine. The patients' PMNLs also had deficient degranulating responses to the former but not to the latter. Diminished PMNL function may contribute to the observed increased incidence of localized infections, which are a major source of morbidity, in these patients.

A second type II restriction endonuclease from Thermus aquaticus with an unusual sequence specificity

Abstract: A type II restriction endonuclease activity free of TaqI was prepared from Thermus Aquaticus YT. The fraction contains two endonucleolytic components with apparently different specificities, however the major activity is sufficiently dominant to allow partial digestion analysis of the position of recognition sites. A precise determination of the location of cleavage sites in pBR322 DNA and a computer-aided search for regions of homology in the vicinity of the cut sites indicate that this enzyme recognizes the nonpalindromic sequences GACCGA or CACCCA. Other related sequences are not cleaved, in particular, GACCCA and CACCGA, indicating that the enzyme requires the identity of nucleotides in the first and fifth positions, a type of specificity that has not been previously reported. The position of cleavage is located outside of the site and is represented as: (Formula: see text).

Developmental Expression of the Rat Somatostatin Gene

Abstract: The developmental expression of the somatostatin (SRIF) gene was investigated in rat brain and stomach, two SRIF-rich tissues. The accumulation of raRNA encoding SRIF was determined in these organs during fetal and early (1–4 weeks) postnatal development using a sensitive radiodensitometric cDNA hybridization assay and a cloned preprosomatostatin cDNA. A single band of mRNA which hybridized specifically to the rat SRIF cDNA was detected in both tissues examined throughout ontogenesis, suggesting that the same SRIF gene is expressed in these tissues in the developing as well as in the adult rat. Whereas SRIF mRNA was undetectable in fetal stomach arid rose gradually only after birth, brain SRIF mRNA was already detectable by day 7 of embryonic life and reached concentrations corresponding to those in the adult brain by embryonic day 20. These marked differences may reflect basic differences in the developmental regulation of SRIF gene expression in neural vs. nonneural tissues or may be related to the onse...

Mechanisms of Regulating the Respiratory Burst in Leukocytes

Abstract: When leukocytes encounter opsonized microorganisms or a variety of inflammatory stimuli, their utilization of oxygen is substantially enhanced. This phenomenon was first observed as increased oxygen uptake by the stimulated cells (Baldridge and Gerard, 1933; Sbarra and Karnovsky, 1959) and was correlated with the production of hydrogen peroxide (Iyer et al., 1961). Concomitant with the alterations in respiration, enhanced glucose oxidation via the hexose monophosphate shunt occurs as well (Sbarra and Karnovsky, 1959). In recent years, it has become clear that oxygen utilization in activated phagocytic cells can proceed by one electron reduction steps, and that the initial product is probably superoxide anion ( 2 - ) (Babior et al., 1973). Two molecules of 2 - can then interact in a dismutation reaction, resulting in the formation of hydrogen peroxide (H2O2). These reactions are outlined in Eqs. (1) and (2): $$ {O_2} + {e^ - } \to O_2^ - $$ (1) $$ 2O_{_2}^ - + 2{H^ + } \to O_2^ - + {H_2}{O_2} $$ (2)