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R Bone

Publications and source records attributed to R Bone.

At least 37 records · Page 2Linked to original sources
PubMed1989

Structural analysis of specificity: alpha-lytic protease complexes with analogues of reaction intermediates.

To better understand the structural basis of enzyme specificity, the structures of complexes formed between alpha-lytic protease, an extracellular serine protease of Lysobacter enzymogenes, and five inhibitory peptide boronic acids (R2-boroX, where R2 is methoxysuccinyl-Ala-Ala-Pro- and boroX is the alpha-aminoboronic acid analogue of Ala, Val, Ile, Norleu, or Phe) have been studied at high resolution by X-ray crystallography. The enzyme has primary specificity for Ala in the P1 position of peptide substrates with catalytic efficiency decreasing with increasing side-chain volume. Enzyme affinity for inhibitors with boroVal, boroIle, and boroPhe residues is much higher than expected on the basis of the catalytic efficiencies of homologous substrates. Covalent tetrahedral adducts are formed between the active-site serine and the boronic acid moieties of R2-boroAla, R2-boroVal R2-boroIle, and R2-boroNorleu. Though R2-boroVal is a slowly bound inhibitor and R2-boroAla is rapidly bound [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry 27, 7682-7688], there appear to be no structural differences that could account for slow binding. The removal from solution of 20% more hydrophobic surface on binding accounts for the improved affinity of alpha-lytic protease for R2-boroVal relative to R2-boroAla. The high affinity of the enzyme for R2-boroIle derives from the selective binding of the L-allo stereoisomer of the boroIle residue, which can avoid bad steric interactions in the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Structural plasticity broadens the specificity of an engineered protease.

The substrate specificity of alpha-lytic protease has been changed dramatically, with a concomitant increase in activity, by replacing an active-site Met with Ala. The substrate specificity of both this mutant and another similar mutant are extraordinarily broad. X-ray crystallographic analysis shows that structural plasticity, a combination of alternate side-chain conformations and binding-site flexibility, allows both large and small substrates to be well accommodated.

Crystallography↗
PubMed1989

Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity.

The alpha-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia coli by fusing the promoter and signal sequence of the E. coli phoA gene to the proenzyme portion of the alpha-lytic protease gene. Following induction, active enzyme was found both within cells and in the extracellular medium, where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicating that the large amino-terminal pro region is necessary for activity. The implications for protein folding are discussed. Furthermore, inactivation of the protease by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the alpha-lytic protease, suggesting that the enzyme is self-processing in E. coli.

Cloning, Molecular↗
PubMed1989

Detection of clonally expanded salivary gland lymphocytes in Sjögren's syndrome.

Recurrent swelling of the parotid and submandibular salivary glands occurs in some patients with Sjögren's syndrome (SS). Using Southern blot methods, we analyzed immunoglobulin and T cell antigen receptor (TCAR) gene rearrangements in DNA obtained from salivary gland lymphocytes of 9 SS patients. Based on histologic appearance of the biopsy specimens, these patients were diagnosed as having myoepithelial sialadenitis. We found oligoclonal rearrangements of the kappa gene (4 patients) and lambda gene (1 patient), and oligoclonal rearrangement of the TCAR beta chain in 2 additional SS patients. Patients with Ig gene rearrangements did not show rearrangements of their TCAR gene, nor did patients with TCAR rearrangements exhibit Ig rearrangements. The observed oligoclonal rearrangements probably derive from 5-10% of the salivary gland B cells or T cells. Three of these SS patients developed non-Hodgkin's lymphoma 2-8 years after the initial biopsy. Our results suggest that minor populations of B cells or T cells may clonally expand in the salivary gland tissues of SS patients with pseudolymphoma, and that such lymphocyte expansions may be controlled by the endogenous immune response and/or medications. However, continued lymphoproliferation in these salivary gland tissues may eventually lead to emergence of a neoplastic clone that escapes immunologic control and develops into a non-Hodgkin's lymphoma as a result of a multistep process.

Adult↗
PubMed1988

Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids.

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1987

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid.

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.

Boronic Acids↗
PubMed1986

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs.

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.

Adenine Nucleotides↗
PubMed1986

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate.

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.

Adenine Nucleotides↗
PubMed1986

Expression of histocompatibility antigen HLA-DR by salivary gland epithelial cells in Sjögren's syndrome.

Recent studies have suggested that the induction of HLA-DR antigens on epithelial cells plays an important role in the pathogenesis of autoimmune endocrine-exocrine disorders. We found that salivary gland epithelial cells (i.e., acinar and ductal cells) in salivary gland biopsy specimens from patients with primary Sjögren's syndrome (keratoconjunctivitis sicca) expressed high levels of HLA-DR antigen, which were detected by staining frozen tissue sections with monoclonal antibodies and immunoperoxidase technique. In contrast, salivary gland epithelial cells from normal subjects did not express this antigen. Lymphocytes eluted from the salivary gland biopsy specimens of patients who had Sjögren's syndrome produced a soluble factor that stimulated HLA-DR synthesis by a salivary gland-derived cell line (Sal-1). These tissue culture supernatants contained gamma-interferon, and their ability to induce HLA-DR synthesis was blocked by monoclonal anti-gamma-interferon antibody. These results demonstrate the presence of HLA-DR antigen on salivary gland epithelial cells and suggest that local production of gamma-interferon plays a role in this induction.

Cells, Cultured↗
PubMed1986

Upper aerodigestive neoplasm perpetuated in the nude mouse.

The ability to study and characterize squamous cell cancer of the upper aerodigestive system would be greatly facilitated by an in vivo model. The subsequent opportunity to observe cellular kinetics, membrane antigenicity and hence, potential response to chemotherapy, immunotherapy, radiotherapy, or a combination of these treatment modalities would most likely have early and significant clinical relevance. A multidisciplinary team of basic scientists and clinicians have developed a nude mouse colony for such investigational research. Melanoma and pulmonary squamous and pulmonary adenocarcinoma have been grown and successfully transferred. In addition, head and neck squamous cell carcinoma from multiple sites have also been successfully colonized. We present our experience with this interesting in vivo model and discuss problems in creating a nude mouse colony, techniques of successful tumor inoculation, ongoing maintenance of successful cell lines, and theoretical advantages for potential clinical investigations using this dynamic biologic system.

Adenocarcinoma↗
PubMed1982

Plasma nicotine levels in pipe smokers.

The lower morbidity and mortality of pipe smokers compared with cigarette smokers may be due to different inhaling patterns of pipe smokers. Plasma nicotine levels were determined in three groups of pipe smokers: (1) primary pipe smokers, (2) persons who smoke both pipes and cigarettes, and (3) secondary pipe smokers, ie, former cigarette smokers who now smoke pipes only. Primary pipe smokers inhaled minimally, as evidenced by low plasma nicotine concentrations after pipe smoking. In contrast, persons who smoke cigarettes and also pipes had high plasma nicotine levels after pipe smoking (mean, 22 ng/mL) and, hence, must have inhaled. Secondary pipe smokers (former cigarette smokers) had mostly low levels of plasma nicotine (mean, 6 ng/mL), suggesting that not all cigarette smokers continue to inhale when converting to pipe smoking. Pipe smoking may be safer than cigarette smoking in certain individuals.

Adult↗
PubMed1980

Aerodynamic size distribution of cromolyn sodium at ambient and airway humidity.

The aerodynamic size distribution of cromolyn sodium was determined at ambient and high (98%) humidity using the single particle aerodynamic relaxation time analyzer. This instrument measures the aerodynamic size single, suspended solid particles and liquid droplets in the respirable size range. The measurement accommodates all properties such as density, shape and surface characteristics that affect aerodynamic behavior. Particle morphology under ambient humidity conditions was studied using scanning electron microscopy. Count median aerodynamic diameter increased from 1.40 to 1.52 micron (p < 0.05), and the mass median aerodynamic diameter increased from 2.31 to 3.02 micron (p < 0.005) when measured at ambient and high humidities. The total mass of 0.99 mg of active ingredient aerosolized at ambient humidity in the size range compatible with pulmonary deposition was approximately 5% of the total mass of active ingredient packaged in the capsule. The low mass of particle aerosolized in the size range appropriate for pulmonary deposition may explain the low estimated deposition of cromolyn reported by other investigators.

Aerosols↗
PubMed1979

Immunocompetence of the guinea pig's middle ear.

Immunocompetence of the middle ear was studied in guinea pigs in two series of experiments. The first, afferent phase, consisted of grafting iso and allografts in the guinea pig's middle ear to sensitize the animals which were later to receive a skin graft on the back. The second, efferent phase, first sensitized the animals with a skin graft on their back and later in the middle ear. The results show a rather low capacity of the middle ear to elicit a second-set rejection of the skin grafts. This lack of sensitization is explained by the authors by the concept of tolerance.

Animals↗
PubMed1978

Aerodynamic size distribution of metered-dose bronchodilator aerosols.

The aerodynamic size distributions of 9 commonly used bronchodilator aerosols were determined by using a new instrument, the single-particle, aerodynamic relaxation time analyzer, which can measure the aerodynamic size of single particles and droplets in real time independent of density, shape, and other physical characteristics. The following commercial preparations were studied: Duo-Medihaler, Medihaler-Epi, Medihaler-Epi Medihaler-Iso, Norisodrine Aerotrol, Alupent, Metaprel, Isuprel Mistometer, Bronkometer, and Asthma-Meter. Count median diameters ranged from 0.62 to 0.82 micrometer, and mass median aerodynamic diameters ranged from 2.8 to 4.3 micrometer. The heterodisperse nature of the aerosols was demonstrated by the relatively large geometric standard deviations, which ranged from 1.5 to 2.1. None of these aerosols had a log-normal distribution; rather, the distribution curves were multimodal.

Aerosols↗
PubMed1977

Foreign body removal with the flexible fiberoptic bronchoscope.

Intrabronchial foreign bodies are occasionally encountered in adults but most available information in humans consists only of reports evaluating one or two cases. We surveyed our experience and found seven patients with intrabronchial foreign bodies who had been examined with the flexible fiberoptic bronchoscope, six of whom were managed successfully. Nine cases have been identified in the literature. Although there were no major complications in either our cases or those in the literature, several problems were identified which under other circumstances could have caused significant complications. We assessed all of these cases for situations which presented potential risk to the patient. These risks were grouped into the following problem categories: 1. selection of appropriate bronchoscope, 2. availability of appropriate instruments, 3. control of the foreign body, and 4. unexpected foreign bodies. Endoscopists planning to use the FFB in foreign body removal should be aware of the problems and hazards which may ensue if improperly managed, and should attempt to gain experience either in the animal laboratory or in models prior to approaching patients with foreign bodies with the flexible fiberoptic bronchoscope.

Adolescent↗