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Biomedical subjects

L Brand

Publications and source records attributed to L Brand.

At least 37 records · Page 2Linked to original sources
PubMed1996

Steady-state and time-resolved fluorescence measurements for studying molecular interactions: interaction of a calcium-binding probe with proteins.

The binding of 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl] 6-methoxy-8-bis[carboxymethyl] aminoquinoline, the fluorescent calcium probe Quin2, to serum albumin and several other proteins has been investigated. Changes in fluorescence emission spectra and fluorescence anisotropy revealed interactions between Quin2 and several proteins including human serum albumin, bovine serum albumin, aldolase, phosphoglucose isomerase, glyceraldehyde-3-phosphate dehydrogenase, and alkaline phosphatase. Protein-probe interactions were inhibited by the presence of calcium. Binding was also measured by resonance energy transfer and gel permeation chromatography. Equilibrium binding constants for Quin2 were quantitated by the application of the recently-developed "SPECTRABIND' program to spectroscopic data (D. Toptygin and L. Brand, Anal. Biochem., 224 (1995) 330-338). Binding of Quin2 to human serum albumin is discussed in terms of the published X-ray crystal structure of human serum albumin (X.M. He and D.C. Carter, Nature, 358 (1992) 209-215).

Aminoquinolines↗
PubMed1996

Profluorescent protease substrates: intramolecular dimers described by the exciton model.

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.

Amino Acid Sequence↗
PubMed1996

Solution conformations of a biantennary glycopeptide and a series of its exoglycosidase products from sequential trimming of sugar residues.

Linkages between sugar residues in branched oligosaccharides exhibit various degrees of flexibility. This flexibility, together with other forces, determines the overall solution conformation of oligosaccharides. We used the method of time-resolved resonance energy transfer to study the solution conformations of a biantennary glycopeptide and its partially trimmed products by exoglycosidases. The N-terminal of the glycopeptide was labeled with 2-naphthyl acetic acid as a fluorescent donor. Either terminal sugar residue, Gal6', on the branch bearing 6-linked Man (antenna 6'), or Neu5Ac on the branch bearing 3-linked Man (antenna 6) was labeled with 5-dimethylaminophthalene-1-sulfonyl as an acceptor. The distance and distance distributions between the terminals were measured. In the intact biantennary glycopeptide, the donor-acceptor distance distribution of antenna 6' is bimodal with a majority of the population in the extended conformation and that of antenna 6 in one very broad population. The Neu5Ac on antenna 6 is oriented toward the N-terminal at low temperature and adopts a more extended form at high temperature. The removal of individual sugar residues along one of the two antenna in the biantennary oligosaccharide has a small effect on the distance distribution of the remaining antenna for both antennae 6 and 6'. Together with previous studies of the triantennary glycopeptides (Rice, K. G., Wu, P. G., Brand, L., and Lee, Y.C. (1993) Biochemistry 32, 7264-7270), our results suggest that both steric hindrance and inter-residue hydrogen bonding are very important in the folding pattern in oligosaccharide structures.

Animals↗
PubMed1996

Fall-related occupational injuries on farms.

We assessed risk factors for fall-related farm injuries in a population-based, case-control study. Cases had to reside in a defined geographic region served by a single medical center. Multiple sources reported cases, and a special farm census enabled random selection of controls, The annual risk of farm fall injury was 7.5 (95% CI: 5.7, 10.0) per 1,000 person-years. The crude incidence rate was higher in men, while the rate based on hours of farmwork was higher in women. In a multivariate analysis of risk factors, three factors were significantly associated with the risk. The risk of fall injury increased 2% (95% CI: 1%, 4%) per hour worked. Residents of farms with some farm workers not living on the farm had a fall injury rate 2.5 (95% CI: 1.0, 6.2) times greater than residents of other farms. Residents of farms with registered cows had one-third (95% CI: 0.14, 0.93) the risk of residents of other farms. To identify environmental hazards for fall injuries, researchers from several disciplines may need to collaborate in the design and conduct of studies that include injury site investigations.

Accidental Falls↗
PubMed1996

Dopamine receptor binding of 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)- 4-oxobutyl]-1,2,3,6-tetrahydropyridine (HPTP), an intermediate metabolite of haloperidol.

The neuroleptic agent haloperidol (HP) is biotransformed to metabolites such as 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium (HPP+). In this study, radioligand binding studies were performed using [3H]SCH23390 as a dopamine D1 receptor ligand and [3H]spiperone as a D2 ligand. Ki values for D1 receptors were 35.8 microM and 54.9 microM for HP and HPTP, respectively. Corresponding values for D2 receptors were 39.1 nM and 329.8 nM. These results indicate similar low affinities in the micromolar range for both HP and HPTP at the dopamine D1 receptor, a much higher affinity of both HP and HPTP for the D2 receptor than for the D1 receptor, and that HPTP binds to D2 receptors with a 9-fold lower affinity than HP. The data are consistent with observations in mice that HPTP is a much less potent acute neuroleptic agent than HP.

Animals↗
PubMed1995

Analysis of equilibrium binding data obtained by linear-response spectroscopic techniques.

A new computational approach for the analysis of equilibrium binding data obtained by spectroscopic methods is described. The approach is aimed at systems involving multiple interactions, where the changes in the spectroscopic signal cannot be attributed to a single interaction. A family of absorption or fluorescence spectra or decay curves obtained in a course of multiple titrations can be decomposed into basic components in an infinite variety of different ways. Model equilibrium equations are used as constraints to determine the basic components associated with the actual chemical species and the concentrations of these species. No assumption is made regarding the parametric form of the component spectra or decays. Binding constants are evaluated simultaneously with the resolved spectra and/or decays in a nonlinear least-square procedure. A key benefit of the method presented is the reduction in the number of fitting parameters. The conditions necessary and sufficient for the existence of a unique solution are considered. The application of the method is demonstrated with experimental data involving titrations of horse liver alcohol dehydrogenase with NADH and isobutyramide.

Alcohol Dehydrogenase↗
PubMed1995

Incidence of farm-work-related acute injury in a defined population.

To determine occurrence and sources of farm-work-related injury, we conducted a population-based, prospective study in a large clinic and hospital serving a defined rural area. The population at risk was identified through a unique demographic and medical records linkage system and a special agricultural census. Cases were 510 individuals who sought inpatient or outpatient care from May 1990 through April 1992 from a physician or chiropractor for harm resulting from acute exposure to energy. One per 31 farm residents was treated annually for a farm-work-related injury. Eight percent of these cases were hospitalized. Animals were the most frequent source of injury. Severity did not differ between cases associated with animals, machinery, falls, or chemicals. Thirty-eight percent of farm-work-related injury cases occurred in nonfarm residents. Injury risk was 2.5 times greater among dairy farm residents than among nondairy farm residents, 352.0 vs. 141.0 cases per 10,000 person-years, respectively. Adult male farm residents had 556.9 injuries per 10,000 person-years and 21.3 injuries per million hours of farm work.

Accidents, Occupational↗
PubMed1995

Machine-related occupational injuries in farm residents.

Farm machinery is an important contributor to the high rates of occupational injury in agriculture. As part of a population-based case-control study, we studied risk factors for machine-related farm injuries. Case patients were farm residents residing in a geographically defined area of central Wisconsin who experienced a farm injury associated with a tractor, farm implement, or other machine which required medical or chiropractic care from May 1990 through April 1992. Controls were selected from an ad hoc census of farm residents in the same area. Telephone interviews regarding demographic characteristics, safety behaviors, and farming practices were completed for 97.8% of 90 case patients and for 82.8% of 221 control subjects. Personal characteristics significantly associated with an increased risk of machine-related injury included the number of hours worked per week and working primarily as a farmer. Dairy farms, farms with nonresident workers, and large farms were associated with an increased risk of injury while farms with registered cows and farms where cows were fed in the barn even in summer experienced fewer injuries. Based on a logistic regression model, the independent risk factors for machine-related farm injury included hours worked per week (2% increased risk/nonresident workers on farm (odds ratio) (OR) = 2.32; 95% confidence interval (CI): 1.07 to 5.06), cows fed in barn in summer (OR = 0.28; 95% CI: 0.12 to 0.64), and registered cows on farm (OR = 0.36; 95% CI: 0.17 to 0.79). Farm safety practices did not appreciably influence the risk of machine-related farm injury.

Accidents, Occupational↗
PubMed1995

A social skills group for boys with Asperger's syndrome.

A social skills group was conducted for 8 boys aged 8 to 12 years on a weekly basis for 14 sessions. In this descriptive paper we discuss the planning for the group, the teaching techniques used, the actual group process and a subjective assessment of outcomes. Possible directions for future interventions are also presented.

Autistic Disorder↗
PubMed1994

Conformational flexibility in a staphylococcal nuclease mutant K45C from time-resolved resonance energy transfer measurements.

Thermal fluctuations exist in native proteins and other macromolecules in solution. Some may play a role in ligand or receptor binding, control rates of enzymatic catalysis, or define a range of conformations a segment can adopt in solution. We apply the method of time-resolved resonance energy transfer to study the conformational flexibility of a staphylococcal nuclease mutant, K45C, where lysine 45 located at a flexible loop is replaced by a cysteine. We labeled the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) and used the TNB group covalently attached to the protein as an energy acceptor from a single tryptophan at residue 140 as the donor. Conformational flexibility occurring on the time scale of nanoseconds or longer is dispersed as an apparent distance distribution in time-resolved resonance energy transfer measurements. Below room temperature the apparent distance distribution was fitted with a symmetric Lorentzian model with a full width at half maximum height of about 6 A, indicating substantial degrees of heterogeneity between residues 45 and 140. At room or higher temperature where the protein is in its native state, the apparent distance distribution is asymmetric, indicating the presence of static disorders. Segments in the protein that contribute to the static disorder can be converted to mobile ones with the addition of denaturing guanidinium chloride.

Energy Transfer↗
PubMed1994

Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer equilibrium by fluorescence anisotropy.

Enzyme I (EI), the first protein of the bacterial phosphotransferase system (PTS), exists in a monomer/dimer (M/D) equilibrium. We have proposed that the two species are functionally different and that their interconversion may regulate sugar transport via the PTS. The C-terminal Cys of Escherichia coli EI was reacted with pyrene maleimide (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995), and the pyrene conjugate used to characterize the M/D equilibrium by fluorescence anisotropy. The properties of unlabeled and pyrene-labeled EI are indistinguishable. Values for the apparent association constant, K'eq, and the steady-state anisotropy of the monomer and the dimer were obtained under a variety of conditions. K'eq increases 23-fold, from 0.45 x 10(5) to 10.7 x 10(5) M-1, as the temperature increases from 6 to 30 degrees C; the association appears to be entropically driven. Under all conditions tested, the K'eq for phospho-EI is 6-12-fold less than for dephospho-EI. For phospho-EI, PEP and Mg2+ induce a 240-fold increase of K'eq when both ligands are present. Based on these data, EI was preincubated under conditions that change K'eq, and the initial activities of the different species were determined at 37 degrees C in a PTS sugar phosphorylation assay with PEP as the phosphoryl donor. The initial rate depends on the M/D ratio; it is maximal when EI is 100% dimer, and zero when EI is 100% monomer. In the latter case, the rate gradually increases in the assay mixture. The results have important implications for how the PTS regulates sugar transport and other physiological phenomena.

Biological Transport↗
PubMed1994

Sugar transport by the bacterial phosphotransferase system. Characterization of the Escherichia coli enzyme I monomer/dimer transition kinetics by fluorescence anisotropy.

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) exists in a monomer/dimer (M/D) equilibrium. These two forms are functionally different, and their interconversion may be a means of regulating the PTS. The M/D equilibrium was studied by fluorescence anisotropy of a pyrene derivative (Chauvin, F., Brand, L., and Roseman, S. (1994) J. Biol. Chem. 269, 20263-20269). In this paper, the kinetics of the transition is investigated. The following apparent rate constants were found for the M/D transition of phospho-Enzyme I in the presence of Mg2+ and PEP at 6 degrees C: k*A = 3.4 x 10(3) M-1 s-1 and k*D = 1.04 x 10(3) s-1. The association rate is especially slow, 2-3 orders of magnitude slower than the average dimerization rate determined for other proteins. Furthermore, the rate of quaternary structure changes matches that of enzymatic activity changes, as well as that of tertiary structure changes (Chauvin, F., Toptygin, D., Roseman, S., and Brand, L. (1992) Biophys. Chem. 44, 163-173). Finally, the effect of two ligands is shown; PEP increases the relaxation rate by 3-fold at 23 degrees C, and Mg2+ addition causes a 4-fold increase in the relaxation rate.

Biological Transport↗
PubMed1994

Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P. (1993) Biochemistry 32, 1816-1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns). The Förster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1994

The allosteric interaction between D-galactose and the Escherichia coli galactose repressor protein.

The Escherichia coli galactose repressor protein (GalR) inhibits transcription of the gal operon upon binding to two operator sites (1-7). This DNA binding activity is inhibited when D-galactose or D-fucose binds to GalR (8-14). Fluorescence spectroscopy was used to characterize the single tryptophan of GalR and to investigate the interaction between galactose and GalR. Fluorescence quenching experiments place both tryptophan residues of the GalR dimer in similar, solvent-exposed locations. Galactose is shown to enhance the intrinsic tryptophan fluorescence of GalR, the source of which is not explained by a change in decay times, but is due to an increase in the pre-exponential factor of the longest of the three fluorescence decay times. It is shown that the beta-anomer of D-galactose is the likely form that binds to GalR. An increase in pH from 6.3 to 9.5 causes the equilibrium association constant (K alpha) describing the galactose-GalR interaction to decrease 10-fold. The interaction is cooperative below pH 9.5. Over the pH range of 6.3 to 9.5, the tryptophan solvent exposure of GalR increases. Galactose binding also induces an increase in exposure. These results, and others presented in this paper, show that both pH and galactose cause global alterations in the structure of GalR.

Allosteric Regulation↗
PubMed1994

Resonance energy transfer: methods and applications.

Resonance energy transfer is widely used in studies of biomolecular structure and dynamics. It provides information about distances on the order of 10 to 100 A and is thus suitable for investigating spatial relationships of interest in biochemistry. The information available from energy transfer studies has been enhanced by the advances in instrumental methods and procedures of data analysis related to fluorescence decay studies. Some practical aspects of the method are reviewed. These include sample preparation, Förster type distance determination, methods of detecting energy transfer and calculating transfer efficiency, time-resolved measurements, and data analysis. Applications of resonance energy transfer, including qualitative measurements as well as microscopy, average distance estimation, and distance distribution analysis, are surveyed.

Carbohydrates↗
PubMed1993

Modification of oligosaccharide antenna flexibility induced by exoglycosidase trimming.

We have investigated the solution conformation of a triantennary glycopeptide using resonance energy transfer [Rice et al. (1991) Biochemistry 30, 6646-6655]. Triantennary glycopeptide was derivatized with a donor fluorophore on the N-terminus and with an acceptor fluorophore attached individually to each terminal galactose residue, resulting in three isomeric donor-acceptor pairs. Time-resolved energy-transfer experiments revealed two distinct donor-acceptor distance populations for two of the three antennae of the oligosaccharide. An extended conformation and a folded conformation were detected for the two flexible antennae whereas the third antenna on the oligosaccharide was rigid, containing only an extended conformer. The ratios of the extended to folded conformers of the two flexible antennae were reversibly modulated by temperature, allowing the calculation of delta H and delta S for the conformational change [Wu et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9355-9359]. In the present study, we have trimmed the fluorescent glycopeptides with exoglycosidases which specifically removed the unmodified antennae of the oligosaccharide. The resulting single-chain isomeric glycopeptides each contained identical core sugar residues and a terminally located donor and acceptor, but differed only in the linkage configuration of the sugar residues. Analysis of these glycopeptides by time-resolved energy transfer indicated that each antenna of the oligosaccharide is, by itself, maintained exclusively in the extended conformation. Temperature modulation failed to induce antenna folding as was previously observed for the complete triantennary structure. These data suggest that interantenna interactions modulate the conformation of individual antenna of complex oligosaccharides.

Carbohydrate Conformation↗
PubMed1993

Dynamic fluorescence measurements of two-state systems: applications to calcium-chelating probes.

A fluorescence technique for characterizing ligand binding is evaluated. This technique uses the combination of steady-state and time-resolved methods to recover molar concentrations and overcomes errors inherent in the use of either method alone. The technique is applicable to time-resolved measurements made either with time-domain or frequency-domain instrumentation. A straightforward single-frequency phase/modulation approach is presented to determine whether an experimental system can be described by a two-state system. The approach is based on a nonlinear transformation of the phase/modulation data that results in a linear model function. Here, the theory is applied to the fluorescent calcium-binding probes Quin-2 and Calcium Green, but is relevant to studies involving other interacting systems. The technique described is used to assess the fraction of bound ligand (Ca2+) and binding constants for these probes.

Aminoquinolines↗