Biomedical subjects

C R Hoffman

Publications and source records attributed to C R Hoffman.

13 recordsLinked to original sources

PubMed2006

Astrophysical reaction rate for the neutron-generator reaction 13C(alpha,n)16O in asymptotic giant branch stars.

The reaction 13C(alpha,n) is considered to be the main source of neutrons for the s process in asymptotic giant branch stars. At low energies, the cross section is dominated by the 1/2+ 6.356 MeV subthreshold resonance in (17)O whose contribution at stellar temperatures is uncertain by a factor of 10. In this work, we performed the most precise determination of the low-energy astrophysical S factor using the indirect asymptotic normalization (ANC) technique. The alpha-particle ANC for the subthreshold state has been measured using the sub-Coulomb alpha-transfer reaction ((6)Li,d). Using the determined ANC, we calculated S(0), which turns out to be an order of magnitude smaller than in the nuclear astrophysics compilation of reaction rates.

Journal Article↗

PubMed2005

Observation of proton radioactivity of the (21+) high-spin isomer in 94Ag.

We have observed direct one-proton decay of the (21+) isomer in the N=Z nuclide 94Ag into high-spin states in 93Pd by detecting protons in coincidence with gamma-gamma correlations and applying gamma gates based on known 93Pd levels. Two decay branches have been identified, with proton energies of 0.79(3) and 1.01(3) MeV and branching ratios of 1.9(5)% and 2.2(4)%, respectively. The corresponding partial half-life values are 21(6) and 18(4) s. The Q value of the direct proton decay of the (21+) isomer was found to be 5.78(3) MeV. The very small reduced widths of the observed proton decays might reflect dominating collective configurations in the (21+) isomer, and the fine structure of the proton spectrum might indicate a strong deformation of this state.

Journal Article↗

PubMed2005

29Na: defining the edge of the island of inversion for Z=11.

The low-energy level structure of the exotic Na isotopes (28,29)Na has been investigated through beta-delayed gamma spectroscopy. The N=20 isotones for Z=10-12 are considered to belong to the "island of inversion" where intruder configurations dominate the ground state wave function. However, it is an open question as to where and how the transition from normal to intruder dominated configurations happens in an isotopic chain. The present work, which presents the first detailed spectroscopy of (28,29)Na, clearly demonstrates that such a transition in the Na isotopes occurs between 28Na (N=17) and 29Na (N=18), supporting the smaller N=20 shell gap in neutron-rich sd shell nuclei. The evidence for inverted shell structure is found in beta-decay branching ratios, intruder dominated spectroscopy of low-lying states, and shell model analysis.

Journal Article↗

PubMed2005

p-sd Shell gap reduction in neutron-rich systems and cross-shell excitations in 20 O.

Excited states in 20O were populated in the reaction 10Be(14C,alpha) at Florida State University (FSU). Charged particles were detected with a particle telescope consisting of 4 annularly segmented Si surface barrier detectors and gamma radiation was detected with the FSU gamma detector array. Five new states were observed below 6 MeV from the alpha-gamma and alpha-gamma-gamma coincidence data. Shell model calculations suggest that most of the newly observed states are core-excited 1p-1h excitations across the N=Z=8 shell gap. Comparisons between experimental data and calculations for the neutron-rich O and F isotopes imply a steady reduction of the p-sd shell gap as neutrons are added.

Journal Article↗

PubMed2001

Pharmacokinetic parameters and mechanisms of inhibition of rat type 1 and 2 steroid 5alpha-reductases: determinants for different in vivo activities of GI198745 and finasteride in the rat.

The interaction of baculovirus expressed rat steroid 5alpha-reductase types 1 and 2 (r5AR1 and r5AR2) with 17beta-N-(2,5-bis(trifluoromethyl)phenyl)carbamoyl-4-aza-5alpha-androst-1-en-3-one (GI198745) was investigated at pH 7 and 37 degrees. This 5alpha-reductase inhibitor was found previously to be a time-dependent inhibitor of the two human 5alpha-reductase isozymes. In contrast, we demonstrate in the present study that although GI198745 is a potent time-dependent inhibitor of r5AR2, it is a classical rapid-equilibrium inhibitor of r5AR1. This type of behavior with human and rat 5alpha-reductases has been shown for the inhibitor 17beta-(N-tert-butylcarbamoyl)-4-aza-5alpha-androst-1-en-3-one (finasteride), a current therapy for benign prostatic hyperplasia. Inhibition of r5AR1 by GI198745 was competitive with testosterone and followed Michaelis-Menten kinetics with a K(i) value of 0.3 +/- 0.02 nM. Data for the inhibition of r5AR2 by GI198745 were consistent with a two-step mechanism, where K(i) is the dissociation constant for an initial enzyme-inhibitor complex and k(3) is the rate constant for the second slow step. The pseudo-bimolecular rate constant (k(3)/K(i)) for the association of GI198745 with r5AR2 was (2.0 +/- 0.4) x 10(7) M(-1) sec(-1). The high affinity of this inhibitor for r5AR2 was further demonstrated by the inability of the enzyme-inhibitor complex to dissociate after approximately 7 days of dialysis at 4 degrees. Both GI198745 and finasteride appear to inactivate r5AR2 by apparent irreversible modification, but are classical, reversible inhibitors of r5AR1. Therefore, we hypothesize that because of its pharmacokinetic parameters and increased potency against r5AR1, GI198745 is more effective than finasteride in preventing the growth of the rat prostate.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed2000

Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

Acid Anhydride Hydrolases↗

PubMed1999

Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.

ADAM Proteins↗

PubMed1997

Human recombinant phosphodiesterase 4B2B binds (R)-rolipram at a single site with two affinities.

The interactions between (R)-rolipram and purified human recombinant low-Km, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1-564) and an N-terminal truncated protein (amino acids 81-564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152-528) lacked high-affinity (R)-rolipram binding. The 152-528 and 81-564 proteins had similar Km's and kcat/Km's and differed less than 4-fold compared with the 1-564 protein. (R)-Rolipram inhibition plots were biphasic for the 1-564 and 81-564 proteins and fit to two states, a high-affinity (Ki = 5-10 nM) state and a low-affinity (Ki = 200-400 nM) state, whereas the 152-528 protein fit to a single state (Ki = 350-400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be <1 mol of (R)-rolipram per mole of 1-564 or 81-564 protein. Titration microcalorimetric studies revealed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-rolipram per mole of protein and a low-affinity state with a stoichiometry of 0.6 mol of (R)-rolipram per mole of protein for the 81-564 protein. A single low-affinity state with a stoichiometry of 0.9 mol of (R)-rolipram per mole of protein was seen using the 152-528 protein. The data indicate that purified HSPDE4B2B 1-564 and 81-564 proteins contain a single binding site for (R)-rolipram and suggest that the proteins exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the protein requires amino acid residues at the N-terminus (81-151) of the protein and catalytic domain (152-528), whereas the low-affinity binding state only requires residues in the catalytic domain (152-528). Phosphorylation at residues 487 and 489 of the 81-564 protein does not appear to alter the substrate kinetics or the stoichiometry and binding affinity of (R)-rolipram.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

Animals↗

PubMed1997

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

ADAM Proteins↗

PubMed1997

Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high- and low-affinity (R)-rolipram binding.

We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Refined crystal structures of unligated adenylosuccinate synthetase from Escherichia coli.

Crystal structures of unligated adenylosuccinate synthetase from Escherichia coli in space groups P2(1) and P2(1)2(1)2(1) have been refined to R-factors of 0.199 and 0.206 against data to 2.0 and 2.5 A, respectively. Bond lengths and angles deviate from expected values by 0.011 A and 1.7 degrees for the P2(1) crystal form and by 0.015 A and 1.7 degrees for the P2(1)2(1)2(1) crystal form. The fold of the polypeptide chain is dominated by a central beta-sheet, which is composed of nine parallel strands and a tenth antiparallel strand. Extending off from this central beta-sheet are four subdomains. The four subdomains contribute loops of residues that are disordered or have high thermal parameters. At least three of these loops (residues 42 to 52, 120 to 131 and 298 to 304) contribute essential residues to the putative active site of the synthetase. In the absence of ligands, much of the active site of the synthetase exists in an ill-defined conformational state. Two, nearly independent regions contribute residues to the interface between polypeptide chains of the synthetase dimer. A pair of helices (H4 and H5) interact with their symmetry-equivalent mates by way of residues that are not conserved amongst the known sequences of the synthetase. The second interface region involves conserved residues belonging to structural elements that connect strands of the central beta-sheet. Residues putatively involved in the binding of IMP lie at or near the interface between polypeptide chains of the dimer. Of the four sequence elements putatively common to all GTP hydrolases, the synthetase has only the guanine recognition element and a glycine-rich loop (P-loop). Although the base recognition element is essentially identical with those of the p21 ras and G alpha proteins, the P-loop of the synthetase is extended in size relative to the P-loops of other GTP hydrolases. The P-loop has two acid residues (Asp13 and Glu14), which are found in the P-loops of only the synthetase family. Glu14 may be involved in the stabilization of the enlarged P-loop of the synthetase, whereas Asp13 may play a role in catalysis and in the coordination of Mg2+. The structural elements of the p21 ras and G alpha proteins responsible for binding Mg2+ are either absent from the synthetase or unavailable for the coordination of metal cations.

Adenylosuccinate Synthase↗

PubMedDate not supplied

Short-term effects of a high-fiber, high-carbohydrate diet in very obese diabetic individuals.

The short-term effects of a weight-maintenance diet high in fiber and carbohydrate (HFHC) was studied in seven very obese individuals with type II diabetes mellitus. Such diets contained 68% of kcal as carbohydrate and total fiber content of 81 g (in contrast to 42% and 28 g during baseline). Fasting glucose concentrations, summed glucose concentrations, and 24-h glucosuria were unaffected in six of seven individuals. Fasting insulin levels decreased (-6.0 +/- 2.0 microU/ml, P less than 0.05), but meal-stimulated insulin concentrations were not altered. Triglyceride and HDL cholesterol concentrations were also unaffected. Total cholesterol concentrations fell in four individuals whose initial values exceeded 200 mg/dl. Basal glucose production rates were similar in the obese diabetic subjects, in five nonobese normal subjects, and in one obese normal individual (2.05 +/- 0.19 versus 2.63 +/- 0.30 and 1.85 mg/kg/min for the obese normal individual) while on baseline diets, and did not change with HFHC. During isoglycemic hyperinsulinemic glucose clamp procedures (maintenance of basal glucose concentrations), 40 mU/m2/min was infused intravenously for 2 h. Glucose disappearance rates increased significantly in the normal-weight control subjects, but did not increase during baseline diets in the obese type II diabetic subjects (4.65 +/- 0.80 versus 0.68 +/- 0.40 mg/kg/min). HFHC diet had no effect on glucose disappearance rates. Plasma insulin levels were 131.0 +/- 11.0, 120.0 +/- 11.0, and 120.0 +/- 5.0 microU/ml during these studies. These studies indicate that short-term HFHC diets without caloric restriction were ineffective in improving glycemic control or lessening insulin resistance in very obese patients with type II diabetes.

Adult↗