Search PubMed⌕ Search

Biomedical subjects

A Maxwell

Publications and source records attributed to A Maxwell.

At least 73 records · Page 4Linked to original sources
PubMed1996

Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris.

The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter. Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter. As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system. Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues.

Alcohol Oxidoreductases↗
PubMed1996

Botulinum A exotoxin for glabellar folds: a double-blind, placebo-controlled study with an electromyographic injection technique.

BACKGROUND: Botulinum A exotoxin (BTX) has been used successfully to treat a variety of hyperkinetic movement disorders. BTX is also capable of reducing hyperkinetic facial lines including prominent glabellar frown lines. OBJECTIVE: The purposes of this study were to (1) confirm the efficacy of BTX in a double-blind, placebo-controlled investigation; (2) evaluate the use of an electromyogram attached to the injection needle to confirm intramuscular corrugator placement of the BTX; and (3) determine the optimum direction injection technique. METHODS: Length and depth of glabellar frown lines were measured before treatment and 4 and 12 weeks after injection of 10 units of BTX or saline solution. RESULTS: Patients treated with BTX had a highly significant reduction in depth and length of glabellar frown lines compared with control subjects. CONCLUSION: BTX appears to be effective and safe for reduction of glabellar frown lines.

Botulinum Toxins, Type A↗
PubMed1996

Mode of action of GR122222X, a novel inhibitor of bacterial DNA gyrase.

GR122222X is a potent inhibitor of the supercoiling reaction of bacterial DNA gyrase. We show that this compound binds stoichiometrically to inactivate the ATPase activity of a 43-kDa N-terminal fragment of the B subunit and competitively inhibits the binding of a radiolabelled coumarin drug to N-terminal fragments of GyrB. These and other data suggest that GR122222X has a mode of action similar, but not identical, to that of coumarin antibiotics.

DNA Topoisomerases, Type II↗
PubMed1995

Nucleotide binding to the 43-kilodalton N-terminal fragment of the DNA gyrase B protein.

The binding of ADPNP (5'-adenylyl beta,gamma-imidodiphosphate) to the 43-kDa N-terminal fragment of the DNA gyrase B protein is found to stabilize a dimer of the protein. Analysis of the kinetics of binding of ADPNP to the fragment suggests that protein dimers can contain 1 or 2 molecules of bound nucleotide. ATP, ADP, or coumarin drugs inhibit the binding of ADPNP. The rate of dissociation of ADPNP from the 43-kDa protein is found to be very slow and unaffected by the presence of other nucleotides. These data can be accommodated by a scheme in which the 43-kDa monomer forms a short-lived complex with ADPNP that can be converted into long-lived dimer complexes containing either 1 or 2 molecules of bound ADPNP; dimer formation with 2 bound ADPNPs is strongly favored. Coumarin drugs inhibit the binding of ADPNP to the 43-kDa fragment, with novobiocin binding to the protein with a stoichiometry of 1:1 and coumermycin binding with a stoichiometry of 0.5:1.

Adenosine Diphosphate↗
PubMed1995

3 beta-aminospirosolane steroidal alkaloids from Solanum triste.

The alkaloid fraction of the MeOH extract of the aerial parts of Solanum triste afforded the new steroidal alkaloid, (22R,25R)-3 beta-amino-5-spirosolene [2], and its previously synthesized dihydro derivative, (22R,24R)-3 beta-amino-5 alpha-spirosolane [3], which is reported here for the first time as a natural product. The structures were elucidated by spectral techniques including 1H-nmr, 13C-nmr, and 1H-1H COSY, HMQC, HMBC, and NOESY nmr experiments.

Chromatography, Thin Layer↗
PubMed1995

Skin resurfacing with the Ultrapulse carbon dioxide laser. Observations on 100 patients.

BACKGROUND: The Ultrapulse carbon dioxide (CO2) laser has been shown to be a highly effective treatment for photodamaged skin including improvement of rhytides, precancerous and benign skin lesions, as well as superficial benign pigmented lesions. OBJECTIVE: We describe our experiences using the Ultapulse CO2 laser in treating 100 patients with different severities of photodamaged skin. METHODS: All patients had moderately or severely photodamaged skin and were selected for treatment with the Ultrapulse CO2 laser to evaluate the efficacy and toxicity of this treatment. The patients were placed on a pretreatment regimen for 2-6 weeks prior to receiving treatment with the Ultrapulse CO2 laser. Patients were evaluated for pre- and posttreatment severity of photodamage. In addition, skin surface replicas were obtained in selected patients and evaluated by computer image analysis technology. RESULTS: At 1 month post-laser treatment, 68 patients received a moderate improvement, five patients achieved a marked improvement, and the remaining 27 patients showed minimal improvement. By 2 months post-laser treatment, 20 of the 27 patients who at 1 month showed only minimal improvement revealed a moderate to marked improvement from baseline. All the patients studied developed a transient erythema that lasted up to 6 weeks, and many of these patients showed a transient hyperpigmentation that persisted for up to 4 months. CONCLUSION: The Ultrapulse CO2 laser is confirmed as an effective treatment for skin resurfacing of photodamaged skin.

Adult↗
PubMed1994

Topoisomerases. In one gate, out the other.

The ability of type II DNA topoisomerases to pass one DNA double helix through another can be explained by a mechanism involving two gates in the enzyme structure.

Adenosine Triphosphate↗
PubMed1994

The complex of DNA gyrase and quinolone drugs with DNA forms a barrier to transcription by RNA polymerase.

The effects of DNA gyrase and quinolone drugs on in vitro transcription of a template containing a preferred gyrase cleavage site have been investigated. We have found that gyrase-quinolone complexes with DNA lead to blocking of transcription by Escherichia coli and bacteriophage T7 RNA polymerases. Either gyrase or quinolone alone has no effect on transcription. With DNA gyrase containing a point mutation in the gyrase A protein, known to confer quinolone resistance, blocking was found to occur only at much higher concentrations of the drug. Other agents that inhibit gyrase-catalysed supercoiling (novobiocin and 5'-adenylyl-beta,gamma-imidodiphosphate) do not arrest transcription in the presence of gyrase. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Analysis of transcription in the absence of drug suggests that RNA polymerase does not displace gyrase from the template. These results are discussed in the light of models for the bactericidal effects of quinolone drugs.

Adenylyl Imidodiphosphate↗
PubMed1994

Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein.

A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin. One crystal form has been obtained that is orthorhombic, P2(1)2(1)2(1), with unit cell dimensions a = 40.3 A, b = 47.7 A, c = 111.9 A. The asymmetric unit of this crystal form contains one molecule (Vm = 2.24 A3/Da). Complete native data have been collected to 2.5 A resolution. This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine. These crystals also exhibit P2(1)2(1)2(1) symmetry but have unit cell dimensions of a = 68.8 A, b = 68.6 A, c = 48.6 A. The Vm value of this crystal form is 2.39 A3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2.0 A resolution. Molecular replacement studies of both complexes are underway.

Binding Sites↗
PubMed1994

Domain structure of Escherichia coli DNA gyrase as revealed by differential scanning calorimetry.

The domain structure of DNA gyrase from Escherichia coli has been examined using differential scanning microcalorimetry. The intact enzyme (an A2B2 tetramer) shows at least four transitions with apparent Tm's at 44.8, 53.3, 58.6, and 60.7 degrees C. Comparison with the thermal stabilities of the two separate subunits and genetically-engineered protein fragments has been used to assign these transitions to individual domains within the intact gyrase proteins. The thermal unfolding of DNA gyrase and all individual fragments are irreversible under the conditions of the calorimetric experiment. Further evidence for the assignment of transitions to particular domains has been obtained by studying the effects of tight-binding ligands such as novobiocin on the thermal stabilities of the various protein fragments.

Adenosine Triphosphate↗
PubMed1994

Evidence for a conformational change in the DNA gyrase-DNA complex from hydroxyl radical footprinting.

We have used the technique of hydroxyl radical footprinting to probe the complex between DNA gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. We find that gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl-beta,gamma-imidodiphosphate or a quinolone drug causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. The significance of these results for the mechanism of DNA supercoiling by gyrase is discussed.

Base Sequence↗
PubMed1994

Psychosocial interest, medical interviews, and the recognition of depression.

OBJECTIVES: To measure primary care physicians' attitudes toward psychosocial issues, determine their relationship to the style of the medical interview, and assess whether attitudes and interview behaviors lead to correct diagnosis in patients with depression. DESIGN: Physicians were videotaped while interviewing four patients standardized with criteria symptoms of major depression. Physicians were unaware of the mental health focus of the study. SETTING: Patient examining rooms. PARTICIPANTS: Physicians were eligible for recruitment if they were board certified or eligible in family practice or internal medicine, practiced primary care medicine, and were listed in regional directories. Standardized patients were recruited from the community. MAIN OUTCOME MEASURES: Attitudes toward psychosocial issues (measured by the Physician Belief Scale), interview content (measured by review of the videotaped encounters), interview behaviors (measured by the Interaction Analysis System for Interview Evaluation), and a listing of depression in the differential diagnosis (determined by physician debriefing interviews). RESULTS: Forty-seven community-based practitioners participated. Forty-eight percent of interviews resulted in a diagnosis of depression. Physician Belief Scale scores were not significantly correlated with patient-centered interviewing, psychosocial questions, inquiry about depression symptoms, or a depression diagnosis. Longer interviews were more likely to result in a depression diagnosis. CONCLUSIONS: High interest in psychosocial issues was not associated with patient-centered interviewing behaviors, questions about psychosocial or depression symptoms, or depression diagnoses. However, certain patient-centered interviewing behaviors, particularly those defined as "affective," did lead to the recognition of depression.

Adult↗
PubMed1994

Immunogold localization of GyrA and GyrB proteins in Escherichia coli.

Immunogold preparations of Escherichia coli, using anti-GyrA and anti-GyrB antibodies to the subunits of DNA gyrase, showed clear labelling with both secondary antibody and protein A-gold conjugates. Both proteins were located mainly in the cytoplasm, with typically less than 10% in the nucleoid. This partitioning of gyrase proteins between nucleoid and cytoplasm was nonrandom and was consistently observed for a range of different cell preparations. Total gold particle counts were highly variable but suggested levels of at least 1000-3000 molecules per cell for both GyrA and GyrB. Sequential treatment with both anti-GyrA and anti-GyrB monoclonal antibodies resulted in simultaneous labelling of both proteins and revealed no clear association between the two groups of molecules. Treatment of cells with chloramphenicol caused marked changes in nucleoid conformation, but no reduction in cytoplasmic labelling of gyrase proteins. On the assumption that gyrase complexes within the nucleoid are not differentially masked from the monoclonal antibodies, the results obtained in this study suggest that most of the gyrase proteins are not associated with either central nucleoid DNA or cytoplasmic loops of peripheral single-stranded DNA, but are distributed randomly throughout the cytoplasm.

Antibodies, Monoclonal↗
PubMed1994

The 24 kDa N-terminal sub-domain of the DNA gyrase B protein binds coumarin drugs.

A number of lines of evidence suggest that the N-terminal sub-domain of the DNA gyrase B protein contains the binding site for the coumarin antibiotics. We have engineered a clone which encodes a 24 kDa protein which represents this domain. Bacteria which overproduce this protein show an elevated level of resistance to coumarins, suggestive of binding of the 24 kDa protein to the drugs in vivo. In vitro we find that the 24 kDa protein does not interact with the gyrase A or B proteins or with DNA, and fails to hydrolyse ATP or show significant binding to ATP, ADP or ADPNP. However, we show that the 24 kDa protein binds coumarin drugs as tightly as the intact B protein. A number of experiments suggest that the interaction of the coumarins with the protein is predominantly hydrophobic in nature.

Adenine Nucleotides↗
PubMed1993

Identifying the catalytic residue of the ATPase reaction of DNA gyrase.

We propose a mechanism for the hydrolysis of ATP by the DNA gyrase B protein in which Glu42 acts as a general base and His38 has a role in aligning and polarizing the glutamate residue. We have tested this mechanism by site-directed mutagenesis, converting Glu42 to Ala, Asp, and Gln, and His38 to Ala. In the presence of wild-type A protein, B proteins bearing the mutations Ala42 and Gln42 show no detectable supercoiling or ATPase activities, while Asp42 and Ala38 proteins have reduced activities. In the DNA cleavage and relaxation reactions of gyrase, which do not require ATP hydrolysis, wild-type and mutant proteins have similar activities. When the 43-kDa N-terminal fragment of the gyrase B protein (which hydrolyzes ATP) contained the mutations Ala42 or Gln42, ATP was bound but not hydrolyzed, supporting the idea that Glu42 is involved in hydrolysis but not nucleotide binding.

Adenosine Triphosphatases↗
PubMed1993

The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes ATP and binds coumarin drugs.

We have cloned and overexpressed a gene encoding a 43-kDa protein corresponding to the N-terminal fragment of the DNA gyrase B subunit. We show that this protein hydrolyzes ATP and binds coumarin drugs. The hydrolysis of ATP shows distinctly non-Michaelis-Menten kinetics and is consistent with a scheme in which the active form of the protein is a dimer, a conclusion supported by molecular weight studies. The coumarin drugs bind very tightly to the 43-kDa fragment, with novobiocin binding to the protein monomer and coumermycin A1 apparently inducing the formation of a dimer. The implications of these results with respect to the mechanism of supercoiling by DNA gyrase and the inhibition of gyrase by coumarin drugs are discussed.

Adenosine Triphosphatases↗