Biomedical subjects
M W Pierce
Publications and source records attributed to M W Pierce.
Estazolam treatment of insomnia in generalized anxiety disorder: a placebo-controlled study.
Estazolam, a triazolobenzodiazepine with an intermediate elimination half-life, has been shown previously to be an effective and safe hypnotic in insomniacs without concomitant psychiatric illness. Our study of the efficacy of estazolam in patients with insomnia associated with generalized anxiety disorder began when 108 patients meeting criteria for generalized anxiety disorder (mean total score of Hamilton Rating Scale for Anxiety [HAM-A] = 22.0 +/- 3.1 [SD]) and insomnia were given single-blind placebo for 7 nights. Nine patients whose anxiety and/or insomnia improved were dropped as placebo responders. The remaining 99 patients were randomly allocated (1:1) to double-blind treatment with either estazolam 2.0 mg or matching placebo for 7 nights. Hypnotic efficacy, as determined by patient-completed sleep questionnaires, was statistically significant for estazolam 2.0 mg versus placebo for all sleep indices (p less than 0.01). Patients treated with estazolam 2.0 mg showed significantly greater improvement in anxiety than those receiving placebo on the mean total score of HAM-A ([placebo, -3.4; estazolam, -7.1; p less than 0.001] and without the insomnia item [placebo, -2.7; estazolam, -5.5; p less than 0.001]). Anxiety scores on the State-Trait Anxiety Inventory showed greater improvement in the estazolam group, but without statistical significance (p = 0.237). Estazolam 2.0 mg is an effective hypnotic in patients with generalized anxiety disorder and appears to have a favorable anxiolytic action.
Efficacy of estazolam. The United States clinical experience.
The hypnotic efficacy of estazolam, a triazolobenzodiazepine, has been established in well-controlled sleep laboratory and outpatient clinical trials. Estazolam 1.0 mg and 2.0 mg significantly improves sleep latency, total sleep time, number of nocturnal awakenings, depth of sleep, and sleep quality in adults with chronic insomnia. Long-term studies indicate that estazolam 2.0 mg remains an effective hypnotic for at least six weeks of continuous nightly administration with no evidence of clinically significant tolerance. Estazolam is an effective treatment for situational insomnia and significantly improves sleep in patients with insomnia associated with moderately severe anxiety or depression.
Safety of estazolam. The United States clinical experience.
The safety profile of estazolam, a new triazolobenzodiazepine hypnotic medication, has been developed in 1,320 normal volunteers and patients with insomnia. No clinically significant effects of estazolam on vital signs or laboratory values were detected. Drug-specific adverse effects such as somnolence, dizziness, hypokinesia, and abnormal coordination occurred, but these are expected extensions of benzodiazepine pharmacologic activity. No consistent effects on psychomotor performance, including memory, were seen at the recommended hypnotic doses in insomniac subjects. These data, combined with the evidence for hypnotic activity, indicate that estazolam is a safe and effective treatment for insomnia.
Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells.
Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.
Molecular cloning of the alpha subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: chromosomal localization and homology to the alpha subunits of integrins.
The cell-surface glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct alpha subunit noncovalently associated with a common beta subunit. Leu-CAMs play crucial roles in cell-cell and cell-matrix interactions. We describe the isolation and analysis of two partial cDNA clones encoding the alpha subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig alpha chain was used for immunoscreening a lambda gt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig alpha subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1 alpha chain. RNA gel blots revealed that mature Mo1 alpha chain mRNA is approximately 5 kilobases in guinea pigs and humans. Southern analysis of DNA from hamster-human hybrids localized the human Mo1 alpha chain to chromosome 16, which has been shown to contain the gene for the alpha chain of lymphocyte function-associated antigen 1. A comparison of the Mo1 alpha chain coding region revealed significant homologies with carboxyl-terminal portions of the alpha subunits of fibronectin, vitronectin, and platelet IIb/IIIa receptors. These data suggest that the alpha subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the alpha subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related.
Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3).
Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion-promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+-binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins.
Identification of insulin receptor tyrosine residues autophosphorylated in vitro.
To identify the autophosphorylation sites on the human insulin receptor (IR), partially purified human IR was incubated in vitro in the presence of insulin and manganese [gamma-32P]ATP so as to achieve near-maximal activation of the histone 2b kinase activity. Approximately 70% of all beta subunit [32P]phosphotyrosine resides on two tryptic peptide segments identified by microsequencing as IR precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, R., Petruzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated peptide 5) and 1315-1329 (tyrosine at 1316, 1322, designated peptide 8), which were recovered in approximately equal amounts. Half of the remaining unidentified [32P]phosphotyrosine residues reside on another tryptic peptide of Mr 4000-5000. Assignment of [32P]phosphotyrosine to specific residues required subdigestion and Edman degradation of 32P peptides covalently coupled to solid supports. Peptide 5 was recovered in triple and double phosphorylated forms in a molar ratio of about 2:1. Tyr-1146 contained 32P in both forms of peptide 5; in the double phosphorylated form, phenylthiohydantoin-[32P]phosphotyrosine was recovered at both Tyr-1150 and Tyr-1151, in a ratio of about 1:2. Thus, the double phosphorylated peptide 5 is presumably a mixture of Tyr-P-1146/1150 and Tyr-P-1146/1151, predominantly the latter. Peptide 8 was recovered only as the double phosphorylated form. We conclude that autophosphorylation of human IR in vitro leads to the phosphorylation of at least 6 of the 13 tyrosine residues on the beta subunit intracellular extension. Five of these tyrosines are clustered in two domains; one domain is in the structurally unique C-terminal tail and contains Tyr-1316 and -1322 which are both phosphorylated. The second domain is located in the segment of the tyrosine kinase region homologous to the major in vitro autophosphorylation site of pp60 v-src and contains Tyr-1146, which is fully phosphorylated, and Tyr-1150 and -1151; although the majority of IR beta subunits exhibit phosphorylation of both tyrosine 1150 and 1151, up to 20-25% of Tyr-1150 remains unphosphorylated at complete kinase activation.
Control by insulin and insulin-related growth factor 1 of protein synthesis in a cell-free translational system from chick-embryo fibroblasts.
Insulin and insulin-related growth factor 1 (IGF-1) increase by 1.5-1.6-fold the rate of [3H]leucine incorporation into protein in primary monolayer cultures of chick-embryo fibroblasts (CEF); half-maximal hormone concentrations are 10 and 0.25 nM respectively. To investigate the mechanism of this effect, a rapid method is used to prepare a lysate from CEF which is active in protein synthesis. Lysate derived from cells treated for 30-150 min with insulin synthesized protein at 1.8-3.0-fold greater rate than did controls; the increased rate persisted for 20 min in vitro. Pactamycin (0.5 microM), an inhibitor of peptide-chain initiation, inhibited protein synthesis by 50% in lysates derived from insulin-treated and control cells. Thus insulin and IGF-1 cause an increase in the protein-synthesis rate in vivo, which persists in cell-free protein-synthesizing lysates of CEF.
Human complement receptor type 3.
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Leukocytes from four patients with complete or partial Leu-CAM deficiency contain the common beta-subunit precursor and beta-subunit messenger RNA.
Deficiency of a family of three leukocyte adhesion molecules (Leu-CAM) is associated with recurrent and life-threatening bacterial infections in man. Each of the three antigens, Mo1, LFA-1, and Leu M5 has a distinct alpha subunit noncovalently associated with a common beta subunit that appears to be required for the expression of these antigens on the cell surface. To investigate the molecular basis of Leu-CAM deficiency, we studied leukocytes from four unrelated patients suffering from complete or partial Leu-CAM deficiency using immunoprecipitation of metabolically labeled proteins, RNA extraction, and Northern blot analysis. We found that B cells from all four patients synthesized a normal sized beta subunit precursor that either failed to "mature" or matured only partially to the membrane expressed form. B cells from all four patients also had a single normal sized beta subunit mRNA of approximately 3.4 kb. Leu-CAM deficiency, in these unrelated patients, is not due to the absence of the beta chain gene or to aberrant splicing of its mRNA and are consistent with a defective beta subunit gene resulting in abnormal posttranslational processing of the synthesized molecule.
N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa.
Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.
High molecular weight microtubule-associated proteins: purification by electro-elution and amino acid compositions.
The high molecular weight microtubule-associated proteins (MAPs) consist of MAPs 1A, 1B, 1C, 2A, and 2B. The proteins have been prepared from rat brain by using taxol and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The individual proteins were purified by electro-elution from stained fixed gels and shown to migrate at their original mobility without proteolysis. Amino acid compositions of purified MAP 1 and 2 component proteins are reported and compared.