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R A Byrd

Publications and source records attributed to R A Byrd.

At least 19 recordsLinked to original sources
PubMed1998

The amino-terminal domain of human STAT4. Overproduction, purification, and biophysical characterization.

The multifunctional signal transducer and activator of transcription (STAT) proteins relay signals from the cell membrane to the nucleus in response to cytokines and growth factors. STAT4 becomes activated when cells are treated with interleukin-12, a key cytokine regulator of cell-mediated immunity. Upon activation, dimers of STAT4 bind cooperatively to tandem interferon-gamma activation sequences (GAS elements) near the interferon-gamma gene and stimulate its transcription. The amino-terminal domain of STAT4 (STAT4(1-124)) is required for cooperative binding interactions between STAT4 dimers and activation of interferon-gamma transcription in response to interleukin-12. We have overproduced this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and purified it to homogeneity for structural studies. The circular dichroism spectrum of hSTAT4(1-124) indicates that it has a well ordered conformation in solution. The translational diffusion constant of hSTAT4(1-124) was determined by nuclear magnetic resonance methods and found to be consistent with that of a dimer. The rotational correlation time (tauc) of hSTAT4(1-124) was estimated from 15N relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric protein. These results, together with the number of signals observed in the two-dimensional 1H-15N heteronuclear single quantum coherence spectrum of uniformly 15N-labeled protein, indicate that hSTAT4(1-124) forms a stable, symmetric homodimer in solution. Cooperativity in native STAT4 probably results from a similar or identical interaction between the amino-terminal domains of adjacent dimers bound to DNA.

Amino Acid Sequence↗
PubMed1998

Insights into the structure of hepatocyte growth factor/scatter factor (HGF/SF) and implications for receptor activation.

The modular structure of HGF/SF offers a reductionist or 'divide and rule' approach to the analysis of structure and function. Domain deletion experiments have established that the N domain, kringle 1 and kringle 2 are essential for HGF/SF activity and that truncated variants containing the N domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) can exhibit partial agonistic or antagonistic activity depending on target cells. Comparative modelling has been used to predict the 3D structures of the six domains of HGF/SF. More recently, NMR methods have shown that the N domain has a novel fold, the charge distribution of which suggests a heparin binding site. Crystals of NK1 indicate the relationship of this domain to the kringle 1, offering further insights into the mechanism of domain interactions and receptor activation.

Binding Sites↗
PubMed1998

Solution structure of the human Hck SH3 domain and identification of its ligand binding site.

SH3 domains are protein binding domains that occur widely among signal transduction proteins. Here, we present the NMR-determined solution structure of the SH3 domain from the cytoplasmic protein tyrosine kinase, Hck. Hck is involved in a number of cell signal transduction pathways, frequently in pathways associated with immune response. SH3 domains bind proteins via a left-handed polyproline type II helix on the target protein. We have assessed the structural impact of binding to a ligand through addition of a peptide corresponding to a proline-rich region of a Hck target, the GTPase activating protein of the Ras pathway. Ligand binding effects small structural changes and stabilizes the SH3 domain structure. Also, we have compared the solution structure of the Hck SH3 domain to the crystal structure of Hck, in which the SH3 domain exhibits an intramolecular binding to an interdomain linker region. These structures are interpreted as the apo- and holo- forms of the Hck SH3 domain.

Amino Acid Sequence↗
PubMed1998

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS: We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

Amino Acid Sequence↗
PubMed1997

Sequential assignments and secondary structure of the RNA-binding transcriptional regulator NusB.

The NusB protein is involved in transcriptional regulation in bacteriophage lambda. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM-containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six alpha-helices: R10-Q21, 127-F34, V45-L65, Q79-S93, Y100-F114 and D118-L127. The structure of NusB makes it a member of a newly emerging class of alpha-helical RNA-binding proteins.

Amino Acid Sequence↗
PubMed1995

NMR solution structure of a nonanucleotide duplex with a dG mismatch opposite a 10S adduct derived from trans addition of a deoxyadenosine N6-amino group to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene: an unusual syn glycosidic torsion angle at the modified dA.

A nonanucleotide, d(G1G2T3C4[BaP]A5C6G7A8G9), in which (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (7-hydroxyl group and epoxide oxygen are trans) is covalently bonded to the exocyclic N6-amino group of deoxyadenosine (dA5) through trans addition at C10 of the epoxide (to give a 10S adduct) has been synthesized. The solution structure of the duplex, d(G1G2T3C4[BaP]A5C6G7A8G9).d(C10T11C12G13G14G15A16C17C18+ ++), containing a dG mismatch opposite the modified dA (designated 10S-[BaP]dA.dG 9-mer duplex) has been investigated using a combination of 1D and 2D (including COSY, PECOSY, TOCSY, NOESY, and indirect detection of 1H-31P HETCOR) NMR spectroscopies. The NMR results together with restrained molecular dynamics/energy minimization calculations show that the modified dA5 adopts a syn glycosidic torsion angle whereas all other nucleotide residues adopt anti glycosidic torsion angles. The sugar ring of dA5 is in the C3'-endo conformation, and the sugar rings of the other residues are in the C2'-endo conformation. The hydrocarbon attached at dA5 orients toward the 3' end of the modified strand (i.e., dC6 direction) and intercalates between and parallel to bases of dG13 and dG14 of the complementary strand directly opposite dC6 and dA5, respectively. The edge of the hydrocarbon bearing H11 and H12 is positioned between the imino protons of dG13 and dG14 in the interior of the duplex, whereas H4 and H5 at the opposite edge are positioned near the sugar H1' and H2" protons of dG13 and facing the exterior of the duplex. The mismatched AG base pair is stabilized by dAsyn-dGanti base pairing in which the imino proton and the O6 of dG14 are hydrogen bonded to N7- and the single N6-amino proton, respectively, of the modified dA5. The modified DNA duplex remains in a right-handed helix, which bends at the site of intercalation about 20 to 30 degrees away from the helical axis and toward the direction of the modified strand.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Developmental toxicity of dinitroaniline herbicides in rats and rabbits. I. Trifluralin.

The potential developmental toxicity of trifluralin was evaluated in rats and rabbits. Pregnant rats and rabbits were dosed once daily by gavage on Gestation Days 6-15 and 6-18, respectively. Doses for rats were 0, 100, 225, 475, or 1000 mg/kg; doses for rabbits were 0, 100, 225, or 500 mg/kg. Cesarean sections were performed on rats and rabbits on Gestation Days 20 and 28, respectively. In rats, maternal toxicity was indicated in the 475 and 1000 mg/kg treatment groups by depression of body weights and food consumption. Fetal viability and morphology were not adversely affected at any dose level. Developmental toxicity was indicated at the 1000-mg/kg dose level by depression of fetal weight. The NOAEL for maternal toxicity in the rat was 225 mg/kg; the NOAEL for developmental toxicity in the rat was 475 mg/kg. In rabbits, maternal toxicity was indicated at the 225 and 500 mg/kg dose levels by abortions and/or deaths in conjunction with anorexia and cachexia. Developmental toxicity was indicated at the 500 mg/kg dose level by depressed fetal viability and weight. Fetal morphology was not adversely affected at any dose level. The NOAELs for maternal and developmental toxicity in the rabbit were 100 and 225 mg/kg, respectively. Based on these data, trifluralin did not exhibit selective toxicity toward the developing conceptus.

Aniline Compounds↗
PubMed1994

Developmental toxicology studies of fluoxetine hydrochloride administered orally to rats and rabbits.

Pregnant Fischer 344 rats were given fluoxetine orally at dose levels of 0, 2, 5, or 12.5 mg/kg on Gestation Days (GD) 6-15; pregnant Dutch Belted rabbits were given 0, 2.5, 7.5, or 15 mg/kg orally on GD 6-18. Cesarean sections were performed on rats and rabbits on GD 20 and 28, respectively. In rats, maternal toxicity was indicated at 12.5 mg/kg by depression of weight gain and food consumption. Fetal viability, weight, and morphology were not affected at any dose level. Maternal and developmental No Observed Adverse Effect Levels (NOAELs) in the rat were 5 and 12.5 mg/kg, respectively. In rabbits, weight loss occurred at 2.5, 7.5, and 15 mg/kg. Food consumption was also depressed at 7.5 and 15 mg/kg; abortions and maternal mortality occurred secondarily to anorexia and cachexia at 15 mg/kg. Fetal viability, weight, and morphology were not affected at any dose level. A NOAEL for maternal effects was not established in the rabbit; the NOAEL for developmental effects in the rabbit was 15 mg/kg. Based on these data, fluoxetine did not exhibit any toxicity toward the developing rat or rabbit conceptus at doses that were maternally toxic.

Administration, Oral↗
PubMed1994

Developmental toxicology studies of vancomycin hydrochloride administered intravenously to rats and rabbits.

Pregnant CD rats were given vancomycin intravenously in doses of 0, 40, 120, or 200 mg/kg on Gestation Days (GD) 6-15; pregnant New Zealand white rabbits were given 0, 40, 80, or 120 mg/kg intravenously on GD 6-18. Cesarean sections were performed on rats and rabbits on GD 20 and 28, respectively. In rats, maternal toxicity was indicated in the 120- and 200-mg/kg treatment groups by cortical tubular nephrosis. Maternal body weight gain and food consumption and fetal viability, weight, and morphology were not adversely affected by vancomycin. Maternal and developmental no observed adverse effect levels (NOAELs) in the rat were 40 and 200 mg/kg, respectively. In rabbits, maternal toxicity was indicated by cortical tubular nephrosis in the 80- and 120-mg/kg treatment groups; a single death and depression of body weight gain and food consumption occurred in the 120-mg/kg treatment group. Developmental toxicity was indicated by depression of fetal weight in the 120-mg/kg treatment group; fetal viability and morphology were not adversely affected by vancomycin. Maternal and developmental NOAELs in the rabbit were 40 and 80 mg/kg, respectively. Based on these data, vancomycin did not exhibit selective toxicity toward the developing rat or rabbit conceptus.

Animals↗
PubMed1994

Quantification of polysaccharide in Haemophilus influenzae type b conjugate and polysaccharide vaccines by high-performance anion-exchange chromatography with pulsed amperometric detection.

A sensitive method for the quantification of polysaccharide (PS) in Haemophilus influenzae type b (Hib) conjugate and PS vaccines has been developed. It is based on measurement of the Hib PS subunit after depolymerization of the PS in sodium hydroxide to produce the subunit, which is characterized by chemical composition and 31P n.m.r. analyses as ribitol-ribose-phosphate. The Hib vaccines were first treated with 0.1 M sodium hydroxide. The Hib PS subunit in the treated vaccines was then analysed directly by high-performance anion-exchange chromatography using a CarboPak PA-1 column, and quantified by pulsed amperometric detection. The PS contents of three conjugate vaccines and three PS vaccines from different manufacturers were determined. Their values were in the expected ranges. This method is particularly useful for vaccines with a sugar stabilizer such as lactose which would interfere with the colorimetric orcinol assay currently used for determination of the PS. The method can measure 0.1 microgram of PS and its sensitivity is at least 30-fold higher than that of the orcinol assay. It may be used for stability studies of conjugate vaccines since a breakdown as low as 5% of the PS from the PS-protein conjugates would be detected.

Chromatography, Ion Exchange↗
PubMed1994

Simultaneous differential staining of cartilage and bone in rodent fetuses: an alcian blue and alizarin red S procedure without glacial acetic acid.

Differential staining of cartilage and bone has several applications including developmental toxicology studies of new chemical candidates for pharmaceutical, industrial, and environmental use. It has been more common to stain fetal bone only using the dye alizarin red S; however, failure to evaluate the cartilaginous portion of the skeleton may result in the failure to identify toxicologically important alterations in skeletal morphology. Previously, differential staining of fetal cartilage and bone was best achieved by combining alizarin red S for staining bone with alcian blue to stain cartilage in glacial acetic acid solution; however, occupational hazards posed by the use of glacial acetic acid make these methods undesirable. Replacement of the glacial acetic acid with potassium hydrogen phthalate eliminates these hazards without compromising the quality of the stained specimen.

Acetates↗
PubMed1994

Reproductive and developmental toxicity of the dopamine agonist pergolide mesylate in mice.

Pergolide (Permax, LY127809, CAS 66104-23-2) a dopamine agonist for the treatment of Parkinson's disease, was evaluated for reproductive and developmental toxicity. Pergolide was administered in the diet at levels of 0, 5, 15, or 50 ppm to male and female ICR mice. In the F0 generation, the males were treated for 9 weeks prior to mating and throughout mating. The females were treated for 2 weeks prior to mating and throughout mating, gestation, and location (postnatal segment only). Females assigned to the teratology segment were killed on gestation day 18 for evaluation of fetal viability, weights, and morphology. Females assigned to the postnatal component were allowed to deliver and maintain their offspring throughout a 21-day lactation period. One male and one female were selected from each litter to continue as the F1 generation. Possible exposure of the F1 generation to pergolide ended at weaning. Growth of the F1 animals was monitored and reproductive performance evaluated. Treatment-related effects in the F0 generation were consistent with the pharmacologic effects of a dopamine agonist. These effects included pregnancy blockage at the 50-ppm dietary level and dose-related body weight depression in lactating dams and suckling progeny at the 15- and 50-ppm dietary levels. An increase in progeny mortality at the 50-ppm dietary level was attributed to lactation failure of the treated dams. The F1 mice of the 15- and 50-ppm groups remained smaller than the control mice until termination at approximately 20 weeks of age, although weight gains following weaning were not depressed and no impairment of mating performance or fertility was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A review of the toxicology of the antibiotic MICOTIL 300.

MICOTIL 300 is a new macrolide antibiotic for the treatment of Bovine Respiratory Disease complex. As with other macrolides used in human and veterinary medicine, overdoses of MICOTIL do not produce pathognomonic lesions. The toxicity dose response varies among laboratory animal and domestic livestock species. However, clinical evidence of MICOTIL toxicity due to large doses is generally a manifestation of the positive chronotropic and negative inotropic cardiovascular effects. No adverse environmental effects are expected from the use of MICOTIL in cattle.

Animals↗
PubMed1992

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.

Carbohydrate Conformation↗
PubMed1990

In vivo metabolism of 3-deoxy-3-fluoro-D-glucose.

Recent studies have demonstrated that 3-deoxy-3-fluoro-D-glucose (3-FG) is metabolized to 3-deoxy-3-fluoro-D-sorbitol (3-FS), via aldose reductase, and 3-deoxy-3-fluoro-D-fructose (3-FF), via the sorbitol dehydrogenase reaction with 3-FS, in rat cerebral tissue (Kwee, I. L., Nakada, T., and Card, P. J. (1987) J. Neurochem. 49, 428-433). However, the biochemistry of 3-FG in other mammalian organs has not been investigated making the application of 3-FG as a metabolic tracer uncertain. To address this issue we investigated 3-FG metabolism and distribution in isolated cell lines and in rabbit tissues in vivo with 19F NMR and gas chromatography-mass spectrometry. In general, the production of 3-FS is well correlated with the known distribution of aldose reductase in all the systems studied. Further metabolism of 3-FS to 3-FF was verified to occur in cerebral tissue. Surprisingly, two new fluorinated compounds were found in the liver and kidney cortex. These compounds are identified as 3-deoxy-3-fluoro-D-gluconic acid, which is produced via glucose dehydrogenase activity on 3-FG, and 3-deoxy-3-fluoro-D-gluconate-6-phosphate. Based on enzyme studies, it is argued that the 3-deoxy-3-fluoro-D-gluconate-6-phosphate is derived directly from 3-deoxy-3-fluoro-D-gluconic acid and not as a product of pentose phosphate activity. Direct oxidation and reduction are the major metabolic routes of 3-FG, not metabolism through glycolysis or the pentose phosphate shunt. Thus, 3-FG metabolism coupled with 19F NMR appears to be very useful for monitoring aldose reductase and glucose dehydrogenase activity in vivo.

Aldehyde Reductase↗