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Biomedical subjects

W Epstein

Publications and source records attributed to W Epstein.

At least 19 recordsLinked to original sources
PubMed2001

Internalization: a metaphor we can live without.

Shepard has supposed that the mind is stocked with innate knowledge of the world and that this knowledge figures prominently in the way we see the world. According to him, this internal knowledge is the legacy of a process of internalization; a process of natural selection over the evolutionary history of the species. Shepard has developed his proposal most fully in his analysis of the relation between kinematic geometry and the shape of the motion path in apparent motion displays. We argue that Shepard has made a case for applying the principles of kinematic geometry to the perception of motion, but that he has not made the case for injecting these principles into the mind of the percipient. We offer a more modest interpretation of his important findings: that kinematic geometry may be a model of apparent motion. Inasmuch as our recommended interpretation does not lodge geometry in the mind of the percipient, the motivation of positing internalization, a process that moves kinematic geometry into the mind, is obviated. In our conclusion, we suggest that cognitive psychologists, in their embrace of internal mental universals and internalization may have been seduced by the siren call of metaphor.

Biomechanical Phenomena↗
PubMed2000

Substrate-binding clusters of the K+-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis.

The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K(+) with high affinity and specificity. Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K(+) binding from the analysis of mutants with reduced affinity for K(+) (Buurman, E., Kim, K.-T., and Epstein, W. (1995) J. Biol. Chem. 270, 6678-6685). K(+) binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K(+) binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K(+) affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K(+), an indication that a residue is in or very near a K(+) binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K(+) and thus reflects the effective distance between a residue and the site of binding or passage of K(+) in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K(+) as it moves through the KdpA subunit to cross the membrane.

Adenosine Triphosphatases↗
PubMed2000

Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex.

Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate. Similarities to mutations in the GYG motif of K(+) channels are discussed.

Adenosine Triphosphatases↗
PubMed2000

Modulation of KdpD phosphatase implicated in the physiological expression of the kdp ATPase of Escherichia coli.

The KdpD sensor kinase and the KdpE response regulator control the expression of the kdpFABC operon, encoding the KdpFABC high-affinity K+ transport system of Escherichia coli. Low turgor pressure has been postulated to be the environmental stimulus to express KdpFABC. KdpD has autokinase, phosphotransferase and, like many sensor kinases, response regulator (phospho-KdpE) specific phosphatase activity. To determine which of these activities are altered in response to the environmental stimulus, we isolated and analysed six kdpD mutants that cause constitutive expression of KdpFABC. In three of the mutants, phosphatase activity was undetectable and, in two, phosphatase was reduced. Kinase activity was unaffected in four of the mutants, but elevated in one. In one mutant, a pseudorevertant of a kdpD null mutation, kinase and phosphatase were both reduced to 20% of the wild-type level. These findings suggest that initiation of signal transduction by KdpD is mediated by the inhibition of the phospho-KdpE-specific phosphatase activity of KdpD, leading to an accumulation of phospho-KdpE, which in turn activates the expression of the KdpFABC system. The data also suggest that levels of activity in vitro may differ from what occurs in vivo, because in vitro conditions cannot replicate those in vivo.

Bacterial Proteins↗
PubMed1999

The Kdp-ATPase of Escherichia coli mediates an ATP-dependent, K+-independent electrogenic partial reaction.

Charge transport by the K+ transporting Kdp-ATPase from Escherichia coli was investigated using planar lipid membranes to which liposomes reconstituted with the enzyme were adsorbed. To study reactions in the absence of K+, given some contamination of solutions with K+, we used a mutant of Kdp whose affinity for K+ was 6 mM instead of the wild-type whose affinity is 2 microM. Upon rapid release of ATP from caged ATP, a transient current occurred in the absence of K+. In the presence of K+, a stationary current was seen. On the basis of their structural similarity, we propose a kinetic model for the Kdp-ATPase analogous to that of the Na+K+-ATPase. In this model, the first, K+-independent step is electrogenic and corresponds to the outward transport of a negative charge. The second, K+-translocating step is probably also electrogenic and corresponds to transport of positive charge to the intracellular side of the protein.

Adenosine Triphosphatases↗
PubMed1998

Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli.

Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF. The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them into the cytoplasmic membrane. In the strains that do not express KdpC or KdpA the other two subunits did not copurify on dye-ligand affinity columns after solubilization with non-ionic detergent. In the mutant lacking KdpB the other two subunits copurified under the same conditions. It is concluded that KdpC forms strong interactions with the KdpA subunit, serving to assemble and stabilise the Kdp complex. A structure in which KdpC could be one of the connecting links between the energy-delivering subunit KdpB and the K+-transporting subunit KdpA is suggested by these data.

Adenosine Triphosphatases↗
PubMed1998

Expression of the Kdp ATPase is consistent with regulation by turgor pressure.

The kdpFABC operon of Escherichia coli encodes the four protein subunits of the Kdp K+ transport system. Kdp is expressed when growth is limited by the availability of K+. Expression of Kdp is dependent on the products of the adjacent kdpDE operon, which encodes a pair of two-component regulators. Studies with kdp-lac fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464-468, 1981). More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H. Asha and J. Gowrishankar, J. Bacteriol. 175:4528-4537, 1993). We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured. In all cases, Kdp expression correlated with the K+ concentration when growth began to slow. Making the reasonable but currently untestable assumptions that the reduction in growth rate by K+ limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure.

Adenosine Triphosphatases↗
PubMed1997

Allergic contact dermatitis caused by Lithraea molleoides and Lithraea brasiliensis: identification and characterization of the responsible allergens.

BACKGROUND: Allergic contact dermatitis caused by species of Lithraea genus (Anacardiaceae) is frequent in South America. Nevertheless, it has been scarcely reported in the literature, hitherto the responsible allergens have not been studied in some species. OBJECTIVE: The purpose of this study was to identify and characterize the allergenic compounds of Lithraea molleoides and brasiliensis, and to investigate the existence of cross-reactions with Toxicodendron allergens. METHODS: Twenty-seven South American subjects (17 with previous Lithraea dermatitis and 10 controls without any plant dermatitis) and four North American subjects who are highly sensitive to poison oak were tested with both purified Lithraea molleoides and brasiliensis extracts and poison oak urushiol. Lithraea extracts were analyzed by gas liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS). RESULTS: All 17 Lithraea-sensitive subjects reacted to poison oak urushiol and 13 of them also reacted to Lithraea molleoides and/or brasiliensis extracts. All 4 poison oak sensitive subjects reacted to poison oak urushiol and to Lithraea extracts. In both groups of sensitive subjects, the responses to poison oak urushiol were stronger and occurred at lower concentration than those to Lithraea extracts. The allergenic fraction in both Lithraea species consisted of: 3-pentadecylcatechol, 3-pentadecenylcatechol, 3-heptadecenylcatechol and 3-hepta-dec-dienilcatechol. CONCLUSION: We concluded that Lithraea molleoides and brasiliensis allergens are closely related to urushiol, although their eliciting potential seems to be lower in comparison with poison oak urushiol, even for Lithraea-sensitive subjects.

Adolescent↗
PubMed1996

Two-dimensional crystals of the Kdp-ATPase of Escherichia coli.

A variant form of the Kdp-ATPase of Escherichia coli was overproduced to a level approaching 37% of the protein in the inner membrane of this organism. Membranes from overproducing cells were prepared with an inside-out orientation. Incubation of the membranes on ice for 1-2 weeks in the presence of sodium vanadate resulted in the formation of two-dimensional crystals of the Kdp-ATPase. The calculated projection map of the p1 crystal form showed three prominent density peaks at a resolution of 22 A. This technique is a useful and simple method to obtain low-resolution structures of membrane proteins.

Adenosine Triphosphatases↗
PubMed1995

Genetic evidence for two sequentially occupied K+ binding sites in the Kdp transport ATPase.

Substrate binding sites in Kdp, a P-type ATPase of Escherichia coli, were identified by the isolation and characterization of mutants with reduced affinity for K+, its cation substrate. Most of the mutants have an altered KdpA subunit, a hydrophobic subunit not found in other P-type ATPases. Topological analysis of KdpA and the locations of the residues changed in the mutants suggest that KdpA has 10 membrane-spanning segments and forms two separate and distinct sites where K+ is bound. One site is formed by three periplasmic loops of the protein and is inferred to be the site of initial binding. The other site is cytoplasmic. We believe K+ moves from the periplasmic site through the membrane to the cytoplasmic site where it becomes "occluded," i.e. inexchangeable with K+ outside the membrane. Membrane-spanning parts of KdpA probably form the path for transmembrane movement of K+. The kinetics of cation transport in the mutants indicate that each of the two binding sites contributes to the observed Km for cations as well as to the marked discrimination between K+ and Rb+ characteristic of wild-type Kdp. Energy coupling in Kdp, mediated by the KdpB subunit, is performed by a different subunit from the one that mediates transport.

Adenosine Triphosphatases↗
PubMed1995

TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli.

The corrected sequence of the trkH gene of Escherichia coli predicts that the TrkH protein is a hydrophobic membrane protein of 483 amino acid residues, of which 41% are identical to those of the homologous and functionally analogous TrkG protein. These two proteins form the transmembrane component of the Trk system for the uptake of K+. Each protein alone is sufficient for high-level Trk activity. When Trk is assembled with the TrkG protein, Rb+ and K+ are transported with a Km near or below 1 mM; however, the Vmax for Rb+ is only about 7% of that for K+. When Trk is formed with TrkH, the affinities for both for K+ and Rb+ are somewhat lower, and the Vmax for Rb+ is only 1% of that for K+ transport. The kinetics of transport in strains with wild-type alleles at trkG and at trkH suggest that both products participate in transport.

Amino Acid Sequence↗
PubMed1994

Interdependence of K+ and glutamate accumulation during osmotic adaptation of Escherichia coli.

Escherichia coli responds to an increase in medium osmolarity by accumulating K+ and glutamate. At low osmolarity a large fraction of cytoplasmic K+ serves to balance charge on macromolecular anions. That fraction of K+ is here referred to as "bound," as distinguished from "free" K+ that serves to balance charge of small anions. At higher osmolarity where cytoplasmic K+ increases markedly, the bound fraction decreases but the absolute amount of bound K+ expressed per unit of dry weight increases. The increase in bound K+ can be explained largely by the reduction of cytoplasmic putrescine at high osmolarity. At high osmolarity, glutamate is the major cytoplasmic anion, equal to at least 70% of free cytoplasmic K+. A sudden increase in the osmolarity of the medium stimulates glutamate synthesis with a lag of only about a minute; glutamate synthesis is almost totally dependent on K+ uptake. The high rate of flow of nitrogen through the glutamate pool under control conditions of growth at low osmolarity indicates that glutamate accumulation immediately after shift to high osmolarity must be due to inhibition of utilization of glutamate in the synthesis of other nitrogen-containing compounds rather than stimulation of glutamate synthesis. In agreement with this reasoning we find the kinetics of glutamate accumulation to be independent of the specific path of synthesis, whether by glutamate dehydrogenase or by glutamate synthase. Synthesis of glutamate appears to be required to attain normal values of the electrical membrane potential after shift to high osmolarity.

Acclimatization↗
PubMed1994

Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.

We measured enzymic and non-enzymic antioxidants in human epidermis and dermis from six healthy volunteers undergoing surgical procedures. Epidermis was separated from dermis by curettage and antioxidants were measured by high-performance liquid chromatography (HPLC) or standard spectrophotometric methods. The concentration of every antioxidant (referenced to skin wet weight) was higher in the epidermis than in the dermis. Among the enzymic antioxidants, the activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase were higher in the epidermis compared to the dermis by 126, 61 and 215%, respectively. Catalase activity in particular was much higher (720%) in the epidermis. Glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, which provide reduced nicotinamide adenine dinucleotide phosphate (NADPH), also showed higher activity in the epidermis than the dermis by 111% and 313%, respectively. Among the lipophilic antioxidants, the concentration of alpha-tocopherol was higher in the epidermis than the dermis by 90%. The concentration of ubiquinol 10 was especially higher in the epidermis, by 900%. Among the hydrophilic antioxidants, concentrations of ascorbic acid and uric acid were also higher in the epidermis than in the dermis by 425 and 488%, respectively. Reduced glutathione and total glutathione were higher in the epidermis than in the dermis by 513 and 471%. Thus the antioxidant capacity of the human epidermis is far greater than that of dermis. As the epidermis composes the outermost 10% of the skin and acts as the initial barrier to oxidant assault, it is perhaps not surprising that it has higher levels of antioxidants.

Aged↗
PubMed1993

The representational framework in perceptual theory.

The representational stance in perceptual theory is described and its proper domain of application is examined. A decision rule for invoking representation is proposed but, although the rule is principled, its implementation is problematic. Despite the widespread appeal of the representational stance, detailed working out of the position within perceptual theory is still needed.

Female↗
PubMed1992

Attentional demands of processing shape in three-dimensional space: evidence from visual search and precuing paradigms.

The hypothesis that representation of projective shape is preattentive whereas representation of objective shape in three-dimensional space requires allocation of attention was tested in 2 visual search and 2 precuing experiments. In the visual search experiments, the slope for projective shape search was expected to approach 0 and that for objective shape search was expected to be a positive monotonic function of set size. In the precuing experiments, the effects of precuing were expected to be largely limited to the task requiring representation of objective shape. The overall pattern of results conformed to expectations. The findings are interpreted in the context of a model of shape-at-a-slant processing set out by Epstein and Lovitts (1985) and Epstein and Babler (1989, 1990).

Adult↗