Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.     

A novel soluble tissue factor variant with an altered factor VIIa binding interface

Tissue factor (TF) residues Lys20 and Asp58 form part of a binding epitope previously shown by alanine scanning to be critical for high affinity interactions with factor VIIa (FVIIa). To explore the possibility of enhancing the affinity of a TF-based antagonist for FVIIa, we created libraries in which residues at 20, 58, and adjacent positions were varied in constructs containing the soluble extracellular domain of TF (sTF) fused to the bacteriophage M13 tail coat protein. TF variants monovalently displayed on phage were then sorted on the basis of binding to FVIIa. Sorting of preliminary libraries, in which position 58 and/or 20 and surrounding residues were randomized, led to the selection of TF proteins of essentially wild-type sequence. Therefore, we devised a strategy wherein TF position 20 was held fixed as alanine and 5 specific residues near to, and including, position 58 were randomized to effectively obtain alternative sequences at this interface. The consensus sequence reached with this library included wild-type residues at positions 61, 62, 65, and 66 but exclusively tryptophan at position 58. Analyses of the soluble K20A,D58W (A20W58) TF protein indicated that it binds FVIIa with an affinity comparable with wild-type sTF but is defective as a cofactor for FVIIa-dependent factor X activation. Further experiments designed to elucidate the mechanism of binding suggest that the new binding interactions involve more than the simple addition of hydrophobic surface area.     

Inverse planning with constraints to generate smoothed intensity-modulated beams

Highly conformal dose distributions can be generated by intensity-modulated radiotherapy. Intensity-modulated beams (IMBs) are generally determined by inverse-planning techniques designed to maximize conformality. Usually such techniques apply no constraints on the form of the IMBs which may then develop fine-scale modulation. In this paper we present a technique for generating smoother IMBs, which yields a dose distribution almost identical to that without the constraint on the form of the IMBs. The method applies various filters successively at intervals throughout the iterative inverse planning. It is shown that the IMBs so determined using a simple median window filter have desirable properties in terms of increasing the efficiency of delivery by the dynamic multileaf collimator method and may be 'more like conventional beams' than unconstrained, highly modulated IMBs.     

Answering questions with an electroencephalogram-based brain-computer interface

OBJECTIVE: To demonstrate that humans can learn to control selected electroencephalographic components and use that control to answer simple questions. METHODS: Four adults (one with amyotrophic lateral sclerosis) learned to use electroencephalogram (EEG) mu rhythm (8 to 12Hz) or beta rhythm (18 to 25Hz) activity over sensorimotor cortex to control vertical cursor movement to targets at the top or bottom edge of a video screen. In subsequent sessions, the targets were replaced with the words YES and NO, and individuals used the cursor to answer spoken YES/NO questions from single- or multiple-topic question sets. They confirmed their answers through the response verification (RV) procedure, in which the word positions were switched and the question was answered again. RESULTS: For 5 consecutive sessions after initial question training, individuals were asked an average of 4.0 to 4.6 questions per minute; 64% to 87% of their answers were confirmed by the RV procedure and 93% to 99% of these answers were correct. Performances for single- and multiple-topic question sets did not differ significantly. CONCLUSIONS: The results indicate that (1) EEG-based cursor control can be used to answer simple questions with a high degree of accuracy, (2) attention to auditory queries and formulation of answers does not interfere with EEG-based cursor control, (3) question complexity (at least as represented by single versus multiple-topic question sets) does not noticeably affect performance, and (4) the RV procedure improves accuracy as expected. Several options for increasing the speed of communication appear promising. An EEG-based brain-computer interface could provide a new communication and control modality for people with severe motor disabilities.     

Direct current shocks to the heart generate free radicals: an electron paramagnetic resonance study

OBJECTIVES: We sought to demonstrate that direct current (DC) shocks to the heart generate free radicals. BACKGROUND: Although it is a lifesaving maneuver, defibrillation is known to have myocardial toxicity. The mechanism of this toxicity is unknown. If DC shocks generate free radicals, free radicals could be a mechanism of myocardial injury. METHODS: In a canine model, DC shocks of 10 to 100 J were delivered to the epicardium of both beating and fibrillating hearts, and 200-J transthoracic shocks were administered in dogs with beating hearts. Ascorbate free radical (AFR) concentration was measured in arterial blood and blood continuously withdrawn from the coronary sinus. In some dogs, the antioxidant enzymes superoxide dismutase (15,000 U/kg) and catalase (55,000 U/kg) (SOD/Cat) were administered before shocks. RESULTS: Ascorbate free radicals were generated by DC shocks. A peak AFR increase of 14 +/- 2% (mean +/- SEM) was seen 5 to 6 min after 100-J epicardial shocks. A peak AFR increase of 7 +/- 5% occurred after transthoracic shocks. There was a significant linear relation between the shock energy and peak percent AFR increase: %AFR increase = 0.18 (Shock energy) + 2.9 (r = 0.73, p < 0.0001). Shocks delivered to hearts in ventricular fibrillation (30 s) resulted in generation of AFR equal to but not greater than that observed during similar shocks delivered to beating hearts in sinus rhythm. Multiple successive shocks (100 J delivered twice or five times) did not result in a greater AFR increase than single 100-J shocks, indicating that peak, not cumulative, energy is the principal determinant of AFR increase. Animals receiving SOD/Cat before shock administration showed significant attenuation of the AFR increase. CONCLUSIONS: Direct current epicardial and transthoracic shocks generate free radicals; antioxidant enzymes reduce the free radical generation by shocks.     

Use of an automated sequencer to determine the sequence specificity of DNA damage

When searching on Scots pines, females of the aphid parasitoid Pauesia silvestris responded to differences in mortality risks, host distribution and host quality by changing foraging tactics. They foraged more successfully (i.e. they laid more eggs per unit time) on the pine aphid Cinara pini than on Cinara pineaTherefore, the former species was considered to be of higher quality. However, P. silvestris suffered from a high mortality (19.5%) from ant aggression when foraging for C. piniwhile mortality was zero on pines with C. pineaAll females that were killed were foraging on the bark, while females searching on needles were safe from ant attacks. When searching for C. pineaP. silvestris spent significantly more time on needles if the aphid colonies were ant-attended. On pines with C. piniin contrast, females spent more time on bark in ant-attended colonies. The high adult mortality risk on bark was counterbalanced by a significantly higher foraging success in ant-attended colonies.     

A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo

Gut endocrine cells possess the capacity to take up and decarboxylate biogenic amine precursors. Decarboxylation is mediated by aromatic L-amino acid decarboxylase (AADC) which is encoded by mRNAs differing in their 5' untranslated regions (UTR) depending on the usage of alternative first exons, 1a and 1b, each with its own acceptor site (-13 and -8 bases relative to the translation start site, respectively). We describe here a novel splice variant of exon 1a-AADC mRNA in rat antral mucosa. Both exon 1a and 1b mRNAs were expressed in rat antral mucosa, but the 1a form was spliced into the acceptor site usually associated with exon 1b (-8). An enteroendocrine cell line (STC-1) expressed exon 1a or exon 1b mRNAs spliced into the -8 acceptor site of exon 2. Transient transfection of a range of cell lines with reporter constructs revealed that all three 5' UTRs efficiently supported expression of the Luciferase reporter. There is therefore a novel, functional 5' UTR of AADC mRNA in rat antral mucosa; alternative AADC splice variants could provide the capacity for control at the level of mRNA translation.     

Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity

The objective of this study was to compare the results of three nociceptive tests, tail-flick, hot-plate and electrical stimulation vocalisation, reflecting the responses from different sites in the CNS. A subcutaneous morphine dose (5 mg/kg) was administered to three parallel groups of rats in which the nociceptive response was measured by one of the three methods. The baseline decreased during the period of measurement for the hot-plate test, but remained stable for the other methods. The spinally mediated tail-flick response was more sensitive to the morphine effects as compared to the supraspinally mediated hot-plate and electrical stimulation vocalisation responses. The electrical stimulation vocalisation-test demonstrated more even effect-time profiles and less variability among the rats than did the tail-flick and the hot-plate methods. In the tail-flick group, 59% of the observations attained the cut-off latency at this morphine dose, leading to underestimation of the peak effect, the area under the effect curve (AUEC), and the variability among the rats. In the hot-plate group, 13% of the observations were at the cut-off latency, and 2% in the electrical stimulation vocalisation group. Different ways of presenting the data are discussed. In conclusion, the test selected for measuring the nociceptive response will influence the effect-time profile and subsequently any pharmacodynamic parameters describing it.     

Size- and scale-dependent chemical attraction contribute to an ontogenetic shift in sociality

Caribbean spiny lobsters, Panulirus argus, reside solitarily during the first months postsettlement, but shift to gregarious shelter use in later juvenile stages, at sizes as small as 15 mm in carapace length. To determine whether receptivity to or production of a chemical attractant among spiny lobster conspecifics is dependent upon body size or spatial scale, we conducted a series of overnight Y-maze shelter choice experiments. We placed a test lobster in an experimental arena and allowed it to choose between two shelters, which differed only in that water flowing by one shelter contained sea water that had passed through a header tank containing a conspecific. We varied the size of the lobster in the arena, the size and number of lobsters in the header tank, and the size of the experimental arena. Lobsters of all sizes tested released odours that attracted conspecifics; however, a single small lobster could attract other conspecifics only in the small arena. Lobsters greater than 15 mm in carapace length were attracted to shelters from which conspecific odours were emanating, while smaller lobsters were not. The results of this study suggest that: (1) the earliest benthic stages (less than 15 mm in carapace length) are unresponsive to conspecific odours, but lobsters greater than 15 mm in carapace length are attracted by conspecific odours; and (2) large lobsters produce a sufficient quantity of scent to attract conspecifics over distances of at least a few metres, whereas small lobsters (15-30 mm in carapace length) cannot. Body size- and spatial scale-dependent attraction could contribute to the shift from solitary to gregarious shelter use among Caribbean spiny lobsters. Copyright 1998 The Association for the Study of Animal Behaviour.     

A novel mtDNA deletion in an infant with Pearson syndrome

Pearson syndrome is a multisystem mitochondrial disorder of infancy that is associated with deletions in the mitochondrial DNA (mtDNA) genome. We report a study on a male infant with Pearson syndrome. Assessment of oxidative phosphorylation activity indicated combined respiratory-chain defects in muscle, liver and fibroblasts; in particular, activity of complex I was reduced. Analysis of the patient's mtDNA identified a novel heteroplasmic 2.461 kb deletion, present at levels greater than 50% of the total mtDNA in the tissues examined. The deletion spanned nucleotides 10368 to 12828 and was flanked by a 3 bp GCC direct repeat sequence. Gene sequences affected are subunits 3, 4, 4L and 5 of complex I, and tRNAs for arginine, histidine, serine and leucine. Our findings correlate with the multiorgan involvement observed in Pearson syndrome.     

Characterization of an exocellular protein phosphatase with dual substrate specificity from the yeast Yarrowia lipolytica

In previous work, the major endocellular protein phosphatase activity has been identified in the secretory yeast Yarrowia lipolytica as a PP2A. The aim of the present work was to seek the presence of one protein phosphatase excreted in the exocellular medium and to study its activity during yeast growth in media supplemented or not supplemented with inorganic phosphate. Protein phosphatase was purified and activity was assayed by following the dephosphorylation of three substrates, [32P]casein, phosphotyrosine and a synthetic tyrosine-phosphorylated peptide. Phosphatase activity recovered in the medium after 25 h culture was greatly enhanced by Pi-deficiency. After several purification steps, the enzyme preparation presents an apparent electrophoretic homogeneity on SDS-PAGE with associated phosphoseryl/threonyl and phosphotyrosyl activities. The kinetic properties exclude contamination by a copurified protein and it is concluded that the two activities are carried by the same single proteic species. It was characterized by gel filtration as a 33 kDa protein with one single subunit demonstrated by SDS-PAGE. An absolute requirement for reducing-agents is observed suggesting that the enzyme contains at least one essential reactive cysteinyl residue. Optimum pH value is 6.1, apparent K(m) for phosphotyrosine was calculated to be 760 microM and Hill coefficient 3.2 indicating a rather high cooperativity. These results showed that the involvement of alkaline and/or acid phosphatase was unlikely. In conclusion, a protein phosphatase distinct from endocellular PP2A is secreted by Yarrowia lipolytica and characterized as a phosphotyrosine protein phosphatase with associated phosphoseryl/threonyl activity.     

Antigen-pulsed CD8alpha+ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node

Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8alpha homodimers. The functions of the two subsets remain controversial, although it has been suggested that the lymphoid-derived (CD8alpha+) subset induces tolerance, whereas the myeloid-derived (CD8alpha-) subset has been shown to prime naive T cells and to generate memory responses. To study their capacity to prime or tolerize naive CD4(+) T cells in vivo, purified CD8alpha+ or CD8alpha- dendritic cells were injected subcutaneously into normal mice. In contrast to CD8alpha- dendritic cells, the CD8alpha+ fraction failed to traffic to the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8alpha+ dendritic cells could be detected. Such responses may have been initiated via transfer of peptide-major histocompatibility complex complexes to migratory host CD8alpha- dendritic cells after injection. These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8alpha+ and CD8alpha- dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the response to immunogenic CD8alpha- dendritic cells by CD8alpha+ dendritic cells.     

Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor

Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.     

Stump-socket interface pressure as an aid to socket design in prostheses for trans-femoral amputees--a preliminary study

We describe the effects on cell function of treating porous bioactive glass (BG) such that its surface is a composite of carbonated hydroxyapatite and serum protein. The effects on bone cell function of porous hydroxyapatite (HA) ceramic and porous glass treated to become amorphous calcium phosphate only also were studied subsequent to their having adsorbed a serum protein layer. Substrates treated for different durations were seeded with MC3T3-E1 cells and cultured for 3-17 days. Whereas cells seeded on any substrates, BG and HA produced collagen types I and III, bone sialoprotein, and osteopontin, there were significant differences between HA and BG, and among the various surface conditions created on BG. Covering the glass surface with hydroxyapatite and serum protein enhanced expression of high alkaline phosphatase activity, high rates of cell proliferation, and production of mineralized extracellular matrix. The enhancement may be due to the adsorption of a high quantity of fibronectin from the serum onto the reacted bioactive glass surface.     

Studies of interface deformations in single- and multi-layered liquid baths due to an impinging gas jet

An impinging gas jet on a molten bath having a slag layer on top is encountered in various metal processing operations. The impinging region was studied using a physical model consisting of an air jet and water bath. Kerosene and corn oil were used as the second layer to investigate the role of the slag layer properties on interface shape and bath circulation. The interface shapes were measured both photographically and by using a surface-tracking resistance probe. The limiting condition at which the jet breaks through the kerosene or corn oil layer and reaches the water layer was determined experimentally. A phenomenological model for the prediction of penetration depth is developed for both short and long jet heights for liquid baths with and without a second liquid layer on top.