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Biomedical subjects

D L Sackett

Publications and source records attributed to D L Sackett.

At least 19 recordsLinked to original sources
PubMed2000

The accuracy of patient history, wheezing, and laryngeal measurements in diagnosing obstructive airway disease. CARE-COAD1 Group. Clinical Assessment of the Reliability of the Examination-Chronic Obstructive Airways Disease.

CONTEXT: The accuracy of the clinical examination in detecting obstructive airway disease (OAD) is largely unknown because of a paucity of methodologically rigorous studies. OBJECTIVE: To determine the accuracy of patient history, wheezing, laryngeal height, and laryngeal descent in the diagnosis of OAD. DESIGN: Comparison study conducted from November 3, 1998, to December 4, 1998, evaluating 4 clinical examination elements for diagnosis of OAD vs the gold standard of forced expiratory volume in 1 second (FEV1) and FEV1-forced vital capacity (FVC) ratio less than the fifth percentile (adjusted for patient height, age, and sex). SETTING: Twenty-five sites, including primary care and referral practices, in 14 countries. PARTICIPANTS: A total of 309 consecutive patients were recruited (mean age, 56 years; 43% female), 76 (25%) with known chronic OAD, 114 (37%) with suspected chronic OAD, and 119 (39%) with neither known nor suspected OAD. MAIN OUTCOME MEASURES: Sensitivity, specificity, and likelihood ratios (LRs) for each of the 4 elements of the clinical examination compared with the gold standard. RESULTS: Mean FEV1 and FVC values were 2.1 L/s and 2.9 L; 52% had an FEV1 and FEV1-FVC ratio less than the fifth percentile. The LR for wheezing was 2.7 (95% confidence interval [CI], 1.7-4.2) and was not statistically significant in the multivariate model. The LR for laryngeal descent ranged from 0.9 (95% CI, 0.5-1.4) to 1.2 (95% CI, 0.4-3.4), depending on the cut point chosen, and did not enter the multivariate model. Only 4 of the history or physical examination elements we tested were significantly associated with the diagnosis of OAD on multivariate analysis: smoking for more than 40 pack-years (LR, 8.3), self-reported history of chronic OAD (LR, 7.3), maximum laryngeal height of 4 cm [corrected] or less (LR, 2.8), and age at least 45 years (LR, 1.3). Patients having all 4 findings had an LR of 220 (ruling in OAD); those with none had an LR of 0.13 (ruling out OAD). The area under the receiver operating characteristic curve for the model incorporating these 4 factors was 0.86. CONCLUSIONS: Further research is needed to validate our model, but in the meantime, our data suggest that less emphasis should be placed on the presence of individual symptoms or signs (such as wheezing or laryngeal descent) in the diagnosis of OAD.

Female↗
PubMed2000

Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations.

Measurement of the nuclear DNA content allows classification of human cancers as either diploid or aneuploid. To gain further insight into mechanisms of aneuploidy, we compared the cytogenetic profile of mismatch-repair-deficient diploid versus mismatch-repair-proficient aneuploid colorectal carcinoma cell lines using comparative genomic hybridization and spectral karyotyping. Aneuploid carcinomas revealed an average of 19 chromosomal imbalances per cell line. Such numerical aberrations were exceedingly scarce in the diploid tumors. This pattern of chromosomal aberrations is consistent with a mechanism involving the impairment of chromosome segregation fidelity during mitotic cell division. In support of this idea, we demonstrate the exclusive occurrence of centrosome amplification and instability in all of the aneuploid tumor cell lines analyzed. All diploid tumors contained centrosomes that were functionally and structurally indistinguishable from those in normal human fibroblasts. Due to the observed differences in centrosomes between these two classes of tumors, we incubated the cells with the microtubule depolymerizing drugs nocodazole and griseofulvin. Our results indicate that the aneuploid tumor cell lines have an increased sensitivity to these reagents and a delay in aster formation and microtubule regrowth. However, microtubule nucleation was initiated from one or two centers in both the diploid and aneuploid cells. These observations support the notion that the integrity of the centrosome plays a central role in the development of aneuploidy. Genes Chromosomes Cancer 27:183-190, 2000. Published 2000 Wiley-Liss, Inc.

Aneuploidy↗
PubMed1999

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at </=15 degrees C and yields of active tetramer increased as the concentration of unfolded subunits decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0 degrees C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 microM subunit, respectively) when incubated at 15 degrees C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl(2), and KCl.

Amino Acid Oxidoreductases↗
PubMed1999

Interactions of the sponge-derived antimitotic tripeptide hemiasterlin with tubulin: comparison with dolastatin 10 and cryptophycin 1.

The sponge-derived antimitotic tripeptide hemiasterlin was previously shown to inhibit tubulin polymerization. We have now demonstrated that hemiasterlin resembles most other antimitotic peptides in noncompetitively inhibiting the binding of vinblastine to tubulin (apparent K(i) value, 7.0 microM), competitively inhibiting the binding of dolastatin 10 to tubulin (apparent K(i) value, 2.0 microM), stabilizing the colchicine binding activity of tubulin, inhibiting nucleotide exchange on beta-tubulin, and inducing the formation of tubulin oligomers that are stable to gel filtration in the absence of free drug, even at low drug concentrations. The tubulin oligomerization reaction induced by hemiasterlin was compared to the reactions induced by dolastatin 10 and cryptophycin 1. Like dolastatin 10, hemiasterlin induced formation of a tubulin aggregate that had the morphological appearance primarily of ring-like structures with a diameter of about 40 nm, while the morphology of the cryptophycin 1 aggregate consisted primarily of smaller rings (diameter about 30 nm). However, the hemiasterlin aggregate differed from the dolastatin 10 aggregate in that its formation was not associated with turbidity development, and the morphology of the hemiasterlin aggregate (as opposed to the dolastatin 10 aggregate) did not change greatly when microtubule-associated proteins were present (tight coils and pinwheels are observed with dolastatin 10 but not with hemiasterlin or cryptophycin 1). Opacification of tubulin-dolastatin 10 mixtures was inhibited by hemiasterlin at 22 degrees C and stimulated at 0 degrees C, while cryptophycin 1 was inhibitory at both reaction temperatures.

Animals↗
PubMed1999

Biosynthesis of radiolabeled curacin A and its rapid and apparently irreversible binding to the colchicine site of tubulin.

Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [(14)C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0 degrees C, with a minimum k(f) of 4.4 x 10(3) M(-1) s(-1). We were unable to demonstrate any dissociation of the [(14)C]curacin A from tubulin. Consistent with these observations, the K(a) value was so high that an accurate determination by Scatchard analysis was not possible. The [(14)C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [(14)C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a K(a) of 3.2 x 10(-5) M(-1).

Antineoplastic Agents↗
PubMed1999

Low-dose and high-dose acetylsalicylic acid for patients undergoing carotid endarterectomy: a randomised controlled trial. ASA and Carotid Endarterectomy (ACE) Trial Collaborators.

BACKGROUND: Endarterectomy benefits certain patients with carotid stenosis, but benefits are lessened by perioperative surgical risk. Acetylsalicylic acid lowers the risk of stroke in patients who have experienced transient ischaemic attack and stroke. We investigated appropriate doses and the role of acetylsalicylic acid in patients undergoing carotid endarterectomy. METHODS: In a randomised, double-blind, controlled trial, 2849 patients scheduled for endarterectomy were randomly assigned 81 mg (n=709), 325 mg (n=708), 650 mg (n=715), or 1300 mg (n=717) acetylsalicylic acid daily, started before surgery and continued for 3 months. We recorded occurrences of stroke, myocardial infarction, and death. We compared patients on the two higher doses of acetylsalicylic acid with patients on the two lower doses. FINDINGS: Surgery was cancelled in 45 patients, none were lost to follow-up by 30 days, and two were lost by 3 months. The combined rate of stroke, myocardial infarction, and death was lower in the low-dose groups than in the high-dose groups at 30 days (5.4 vs 7.0%, p=0.07) and at 3 months (6.2 vs 8.4%, p=0.03). In an efficacy analysis, which excluded patients taking 650 mg or more acetylsalicylic acid before randomisation, and patients randomised within 1 day of surgery, combined rates were 3.7% and 8.2%, respectively, at 30 days (p=0.002) and 4.2% and 10.0% at 3 months (p=0.0002). INTERPRETATION: The risk of stroke, myocardial infarction, and death within 30 days and 3 months of endarterectomy is lower for patients taking 81 mg or 325 mg acetylsalicylic acid daily than for those taking 650 mg or 1300 mg.

Aged↗
PubMed1999

Systolic and diastolic learning: an analogy to the cardiac cycle.

It has been observed that the active-passive classification of adult learning can be viewed in terms of a systolic-diastolic model. This model represents an analogy to the cardiac cycle and the work done by the heart during these two phases of the cycle. The determinants of systolic and diastolic learning can be compared to the determinants of cardiac function: preload, afterload and contractility. Similarly, dysfunction in these two phases of learning can be compared to cardiac dysfunction from a pathophysiologic perspective.

Adult↗
PubMed1999

The coral-derived natural products eleutherobin and sarcodictyins A and B: effects on the assembly of purified tubulin with and without microtubule-associated proteins and binding at the polymer taxoid site.

We examined interactions with purified tubulin of synthetic sarcodictyins A and B and eleutherobin (coral-derived antimitotic agents) and of compound 1, an analogue of sarcodictyin A methylated at the C-3 oxygen atom (i.e., the methyl ketal analogue of sarcodictyin A and thus structurally similar to eleutherobin but lacking the C-3 sugar moiety). Eleutherobin was much more active than sarcodictyins A and B, which were somewhat more active than compound 1. Effects of eleutherobin did not differ greatly from those of paclitaxel and epothilone A. Eleutherobin and epothilone A were competitive inhibitors of the binding of radiolabeled paclitaxel to tubulin polymer (apparent Ki values of 2.1 and 2.6 microM, respectively). Tubulin assembly reactions induced by all compounds were similar to the paclitaxel-driven reactions in being enhanced by the addition of microtubule-associated proteins and/or GTP to the reaction mixture and by progressively higher reaction temperatures. Antiproliferative activity was studied in six human cancer cell lines, including two paclitaxel-resistant lines with point mutations in a beta-tubulin gene. Except for compound 1, effects on cell growth were generally in accord with effects on purified tubulin. Thus, sarcodictyins A and B had IC50 values in the 200-500 nM range; paclitaxel, <10 nM (except in the resistant lines); and eleutherobin and epothilone A, 10-40 nM. The antiproliferative activity of compound 1 was more comparable to that of eleutherobin than sarcodictyin A, despite its weak interaction with tubulin. The activities of the sarcodictyins, eleutherobin, and compound 1 in the mutant ovarian lines were similar to their activities in the parental line.

Alkaloids↗
PubMed1999

Purification, characterization, and drug susceptibility of tubulin from Leishmania.

Past work suggests that tubulin from kinetoplastid parasites may present an excellent drug target. To explore this possibility, tubulin was purified on a milligram scale from Leishmania mexicana amazonensis promastigotes by sonication, DEAE-Sepharose chromatography, and one cycle of assembly-disassembly. Purified leishmanial tubulin is recognized by commercially available anti-tubulin antibodies and displays concentration dependent assembly in vitro. The vinca site agents vinblastine, maytansine, and rhizoxin bind to leishmanial tubulin as assessed by the quenching of intrinsic tubulin fluorescence and the alteration of the proteins reactivity with the sulfhydryl-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid). They also interfere with the assembly of leishmanial tubulin at low micromolar concentrations. Electrophilic compounds such as phenyl arsenoxide and 4-chloro-3,5-dinitro-alpha,alpha,alpha-trifluorotoluene (chloralin), which are of interest as traditional and experimental antiparasitic agents, respectively, inhibit the assembly of leishmanial tubulin in vitro as well. Colchicine-site agents and trifluralin, on the other hand, have little or no effect on leishmanial tubulin in these assays. Maytansine, taxol, and the electrophiles block the growth of Leishmania donovani amastigote-like forms in vitro at low ( <1 microM) concentrations, while colchicine site agents, trifluralin, vinblastine, and rhizoxin are at least two orders of magnitude less toxic to the parasite.

Animals↗
PubMed1999

Cytotoxic and tubulin-interactive hemiasterlins from Auletta sp. and Siphonochalina spp. sponges.

Chemical and biological investigations of extracts from the sponge genus Auletta and two collections of Siphonochalina sp. have shown these organisms to be producers of the potent hemiasterlin class of antitumor agents. In addition to the previously known hemiasterlin (1) and hemiasterlin A (2), a new analogue, hemiasterlin C (3), was isolated and identified. The structures of 1 and 2 were assigned based on comparison to literature values, and 3 was identified on the basis of 1H NMR, 13C NMR, COSY, HSQC, and HMBC experiments. The cytotoxic and antitubulin activities of 1-3 were evaluated. In a comparative assay for inhibition of tubulin polymerization, the hemiasterlins were more potent than dolastatin 15 and equipotent with cryptophycin 1, but were somewhat less potent than dolastatin 10.

Animals↗
PubMed1999

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.

Amino Acid Oxidoreductases↗