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PubMed2003

Conservation of electrostatic properties within enzyme families and superfamilies.

Electrostatic interactions play a key role in enzyme catalytic function. At long range, electrostatics steer the incoming ligand/substrate to the active site, and at short distances, electrostatics provide the specific local interactions for catalysis. In cases in which electrostatics determine enzyme function, orthologs should share the electrostatic properties to maintain function. Often, electrostatic potential maps are employed to depict how conserved surface electrostatics preserve function. We expand on previous efforts to explain conservation of function, using novel electrostatic sequence and structure analyses of four enzyme families and one enzyme superfamily. We show that the spatial charge distribution is conserved within each family and superfamily. Conversely, phylogenetic analysis of key electrostatic residues provide the evolutionary origins of functionality.

Amino Acid Sequence↗
PubMed2005

The thermodynamic landscape of testosterone binding to cytochrome P450 3A4: ligand binding and spin state equilibria.

Human cytochrome P450 (CYP) 3A4 catalyzes the oxygen-dependent metabolism of greater than 60% of known drugs. CYP3A4 binds multiple ligands simultaneously, and this contributes to complex allosteric kinetic behavior. Substrates that bind to this enzyme change the ferric spin state equilibrium of the heme, which can be observed by optical absorbance and electron paramagnetic resonance (EPR) spectroscopy. The ligand-dependent spin state equilibrium has not been quantitatively understood for any ligands that exhibit multiple binding. The CYP3A4 substrate testosterone (TST) has been shown previously by absorbance spectroscopy to induce spin state changes that are characteristic of a low spin to high spin conversion. Here, EPR was used to examine the equilibrium binding of TST to CYP3A4 at [CYP3A4] > K(D), which allows for characterization of the singly occupied state (i.e., CYP3A4.TST). We also have used absorbance spectroscopy to examine equilibrium binding, where [CYP3A4] < K(D), which allows for determination of K(D)'s. The combination of absorbance and EPR spectroscopy at different CYP3A4 concentrations relative to K(D) and curve fitting of the resultant equilibrium binding titration curves to the Adair-Pauling equations, and modifications of it, reveals that the first equivalent of TST binds with higher affinity than the second equivalent of TST and its binding is positively cooperative with respect to ligand-dependent spin state conversion. Careful analysis of the EPR and absorbance spectral results suggests that the binding of the second TST induces a shift to the high spin state and thus that the second TST binding causes displacement of the bound water. A model involving six thermodynamic states is presented and this model is related to the turnover of the enzyme.

Computer Simulation↗
PubMed2001

Quantum chemical characterization of the reactions of guanine with the phenylnitrenium ion.

Density functional calculations at the B3LYP/6-311+G(2d,p)//pBP/DN level predict all cationic adducts combining guanine, at either its N2, O6, N7, or C8 positions, with phenylnitrenium ion, at either its N, 2, or 4 positions, to be lower in energy than the separated reactants. This relative stability of all adducts is preserved after addition of aqueous solvation free energies computed at the SM2 level, although some leveling of the adduct relative energies one to another is predicted. Cations having the lowest relative energies in solution correspond structurally to those adducts most commonly found when guanine reacts with larger, biologically relevant nitrenium ions in vitro and in vivo. One of these, the N-C8 adduct, is stabilized both by a rearomatized phenyl ring and by the operation of an anomeric effect not found in any of the others. On the basis of energetic analysis, direct conversion of an N-N7 cation to an N-C8 cation according to a previously proposed mechanism is unlikely; however, an alternative rearrangement converting a 2-N7 cation to an N-C8 cation via the intermediacy of a five-membered ring may be operative in nitrenium ions with aromatic frameworks better able than phenyl to stabilize endocyclic cationic charge.

Guanine↗
PubMed2000

Association of aberrant p53 and p21(WAF1) immunoreactivity with the outcome of oral verrucous leukoplakia in Taiwan.

The expression of p53 and p21WAF1 in 53 oral verrucous leukoplakias (OVLs), mostly non-dysplastic lesions, was investigated to ascertain the role of such events in malignant conversion. Immunohistochemical analysis revealed aberrant p53 and p21WAF1 immunoreactivity in 51% (27 cases) and 75% (40 cases), respectively. After an average follow-up period of three and a half years, histopathological examination revealed that 22 (42%) cases had developed oral squamous cell carcinoma (OSCC), 14 (26%) cases had undergone recurrence, and 17 (32%) cases were free of disease. The oncogenic potential of this subset of premalignant lesions warrants attention. A significant difference in the frequency of OSCC progression/recurrence was noted in lesions bearing aberrant immunoreactivity of either p53 (93% vs 42%; P=0.00008) or p21WAF1 (80% vs 32%; P=0.002) in comparison with lesions without immunoreactivity. This study suggested that the aberrant immunoreactivity of p53 and p21WAF1 may represent important alterations of OVL and could affect the outcome of this lesion.

Adult↗
PubMed2000

A survey of interfacial forces used during filing of root canals.

The pattern of dentine removal during endodontic instrumentation is influenced by many factors including the interfacial forces applied by the operator. The aim of this study was to investigate the influence of operators and different sizes and types of instruments on the magnitude of these interfacial forces. Single-rooted teeth were mounted on a cantilevered aluminium beam to which two pairs of single element strain gauges were joined in a half-bridge configuration and mounted at right angles to each other. The strain gauges were connected to an analogue-to-digital converter fitted in a micro-computer via conditioning amplifiers. This enabled strains to be recorded over a period of time. Twenty operators instrumented root canals using a series of hand instruments for 1 min each. The mean interfacial forces used by operators demonstrated a wide variation ranging from 9.06 g to 149.42 g (range of forces from 0-331 g) but there was a consistency in the relative magnitude for each operator. The 20 operators could be divided into 13 groups which were significantly different (alpha = 0.05) from each other. There were significant differences (alpha = 0.05) between the forces used for each of the K-Flex files (15, 25, 35, 45, and 70), the force increasing with the file size. There was also a significant difference (alpha = 0.05) in the forces used between the Flexofile (#25) and the #25 K-Flex and Hedström files. However, there was no significant difference between the K-Flex and Hedström files.

Amplifiers, Electronic↗
PubMed2004

Biological abnormality of impaired reading is constrained by culture.

Developmental dyslexia is characterized by a severe reading problem in people who have normal intelligence and schooling. Impaired reading of alphabetic scripts is associated with dysfunction of left temporoparietal brain regions. These regions perform phonemic analysis and conversion of written symbols to phonological units of speech (grapheme-to-phoneme conversion); two central cognitive processes that mediate reading acquisition. Furthermore, it has been assumed that, in contrast to cultural diversities, dyslexia in different languages has a universal biological origin. Here we show using functional magnetic resonance imaging with reading-impaired Chinese children and associated controls, that functional disruption of the left middle frontal gyrus is associated with impaired reading of the Chinese language (a logographic rather than alphabetic writing system). Reading impairment in Chinese is manifested by two deficits: one relating to the conversion of graphic form (orthography) to syllable, and the other concerning orthography-to-semantics mapping. Both of these processes are critically mediated by the left middle frontal gyrus, which functions as a centre for fluent Chinese reading that coordinates and integrates various information about written characters in verbal and spatial working memory. This finding provides an insight into the fundamental pathophysiology of dyslexia by suggesting that rather than having a universal origin, the biological abnormality of impaired reading is dependent on culture.

Asian People↗
PubMed1996

Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+.

Previous reports indicated that arachidonic acid is incorporated into the isolated perfused rabbit heart in preference to other fatty acids, and that incorporation of arachidonic acid, but not other fatty acids, is inhibited during Mg2+ depletion. In this study, we have not been able to demonstrate an arachidonic acid-specific fatty acyl-CoA synthetase in rat or rabbit heart by hydroxyapatite chromatography. Kinetic evidence was consistent with a single enzyme, as the slopes of pseudo-Hill plots were not significantly different from -1. The single fatty acyl-CoA synthetase present appears to prefer C18:0 unsaturated fatty acids to arachidonate, and had about the same affinity for C10:0 -C14:0 saturated fatty acids as for arachidonate. At 35 microM arachidonate, enzyme velocity increased as the total Mg2+ was increased from 3 to 80 mM. Calculated [MgATP] indicated that the MgATP complex was not rate-limiting. At low concentrations, Mn2+ and Ni2+ supported activity, but Cu2+ and Zn2+ did not. Low Ca2+ concentrations activated only oleic acid conversion. Kinetic analysis indicated that the Vmax of the enzyme was increased with increasing concentrations of ionized Mg2+ for both oleic acid and arachidonic acid. The data are constant with the hypothesis that Mg2+ has a direct effect on fatty acyl-CoA synthetase activity, and suggest that preference for oleic acid and arachidonic acid can be influenced by the ionic milieu.

Adenosine Triphosphate↗
PubMed1990

Expressive language recovery in severely brain-injured children and adolescents.

The spontaneous expressive language abilities of 9 severely brain-injured children and adolescents and their age-matched normal controls were examined seven times over a 12-month period following injury. Analysis of conversational language samples revealed a relatively stable pattern of language performance for the normal subjects over this time interval. The brain-injured subjects, as a group, demonstrated improvement on the majority of measures, but only a few reached the level of their control subjects and interindividual variability was considerable. Results suggest that the prognosis for clinically significant improvement in severely brain-injured subjects is good; however, deficits in expressive skills remain apparent up to at least 12 months following injury.

Adolescent↗
PubMed1991

The effects of digital quantization error on speech intelligibility and perceived speech quality.

The effects of digital quantization error upon speech intelligibility and perceived speech quality, for normally hearing subjects, were investigated for digitized speech processed to simulate 6-, 8-, 10-, 12-, 14-, and 16-bit integer conversion and 2-, 3-, 4-, 5-, 6-, and 7-bit floating-point conversion. For the integer data, there were no significant differences in speech intelligibility for 8- to 16-bit conversion. Only 6-bit integer conversion at 55 dB SPL resulted in a significant degradation in speech intelligibility. For the floating-point data, there were no significant differences in speech intelligibility for 2- to 7-bit floating-point conversion. However, results of the perceived quality experiment appeared to be more sensitive to differences among the various conditions. Speech processed using 12-, 14-, and 16-bit integer conversion was judged to be superior to speech processed using the 6-, 8-, and 10-bit integer conditions. Speech processed using 5-, 6-, and 7-bit floating-point conversion was judged to be superior to speech processed using 2-, 3-, and 4-bit floating-point conversion.

Acoustics↗
PubMed2000

Risk for speech disorder associated with early recurrent otitis media with effusion: two retrospective studies.

The goals of this two-part series on children with histories of early recurrent otitis media with effusion (OME) were to assess the risk for speech disorder with and without hearing loss and to develop a preliminary descriptive-explanatory model for the findings. Recently available speech analysis programs, lifespan reference data, and statistical techniques were implemented with three cohorts of children with OME and their controls originally assessed in the 1980s: 35 typically developing 3-year-old children followed since infancy in a university-affiliated pediatrics clinic, 50 typically developing children of Native American background followed since infancy in a tribal health clinic, and (in the second paper) 70 children followed prospectively from 2 months of age to 3 years of age and older. Dependent variables included information from a suite of 10 metrics of speech production (Shriberg, Austin, Lewis, McSweeny, & Wilson, 1997a, 1 997b). Constraints on available sociodemographic and hearing status information limit generalizations from the comparative findings for each database, particularly data from the two retrospective studies. The present paper reports findings from risk analysis of conversational speech data from the first two cohorts, each of which included retrospective study of children for whom data on hearing loss were not available. Early recurrent OME was not associated with increased risk for speech disorder in the pediatrics sample but was associated with approximately 4.6 (CI = 1.10-20.20) increased risk for subclinical or clinical speech disorder in the children of Native American background. Discussion underscores the appropriateness of multifactorial risk models for this subtype of child speech disorder.

Age Factors↗
PubMed2001

A survey of interfacial forces used during endosonic instrumentation of root canals.

AIM: The aim of this in-vitro study was to measure interfacial forces acting between root canal dentine and ultrasonic files during endosonic instrumentation. METHODOLOGY: Single-rooted teeth were mounted on a cantilevered aluminium beam to which two pairs of single element strain gauges were joined in a half-bridge configuration mounted at right angles to each other. The strain gauges were connected to an analogue-to-digital converter fitted in a microcomputer via a conditioning amplifier. This enabled strains to be recorded as a function of interfacial forces over a period of time. Twenty operators instrumented root canals using sizes 15, 20 and 25 ultrasonically energized K-type files. The lateral forces generated were calculated. RESULTS: The mean interfacial forces used varied widely between operators and files, ranging from 18 g to 149 g, but there was a consistency in the relative magnitude for each operator. The average force used by the operators increased with file size; the differences between file sizes were significant (P < 0.05). CONCLUSIONS: The range of forces measured is broader than previously reported and may have a bearing on possible uncontrolled dentine removal, even during ultrasonically activated irrigation.

Analog-Digital Conversion↗
PubMed1994

Use of intracavity saline instillation and transvaginal ultrasonography to detect tamoxifen-associated endometrial polyps.

There is sometimes a discrepancy between the apparent thickness of postmenopausal endometria, as determined by transvaginal ultrasonography, and the examination of endometrial biopsies. We describe a case which showed that tamoxifen (20 mg/day over 12 months) decreased impedance to blood flow in the uterine arteries and increased the apparent thickness of the endometrium. Conversely, the analysis of biopsies suggested the presence of an atrophic endometrium. The introduction of sterile saline into the uterine cavity during a repeat ultrasound scan revealed the presence of a large, free-floating endometrial polyp, which was subsequently difficult to see by hysteroscopy.

Journal Article↗
PubMed2002

Conversion to digital technology improves efficiency in the pediatric echocardiography laboratory.

BACKGROUND: Although the videotape method (VTM) is commonly used to record and intrepret ecocardiographic images, many pediatric echocardiographers are considering acquiring to, interpreting from, and storing their images to digital disk using the single-beat digital acquisition method (SBM). The paucity of image redundancy using SBM should translate into improved laboratory efficiency compared with VTM, but hard data are lacking. The purpose of this study was to test the hypothesis that the time to acquire images to videotape using VTM and to disk using SBM would be the same for normal hearts and corresponding congenital heart diseases, but interpretation times would be shorter using SBM. METHODS: We measured the times to acquire and interpret 403 echocardiograms using standard VTM from Children's Hospital in Cincinnati, Ohio, and 352 echocardiograms acquired using SBM from Children's Hospital in San Diego, Calif. Diagnostic categories at each site included: (1) normal, (2) simple shunt or isolated valve disease, and (3) multiple-lesion disease. RESULTS: As a group, SBM echocardiograms included more hemodynamic measurements and took more time to acquire (P <.037), but less time to read (P <.001) than corresponding images acquired using VTM. Using SBM, it took more time to acquire normals and isolated valve or shunt lesions, whereas the average time to acquire multiple-lesion disease was the same using both VTM and SBM. With SBM, in contrast, interpretation times were significantly less for all corresponding diagnoses. CONCLUSION: SBM studies took longer to acquire because more hemodynamic measurements were acquired, but they were read in less time than corresponding VTM studies even though all videotapes were replayed in search fast-forward mode. Pediatric echocardiographers can increase their laboratory efficiency by converting from VTM to SBM.

Analog-Digital Conversion↗
PubMed2001

Cephalometric digitization: A determination of the minimum scanner settings necessary for precise landmark identification.

This study focused on the interpretation of scanned cephalometric images after the digitization of the original analog film, with a variety of standard scanner settings. The purpose was to determine the minimum allowable resolution, grayscale, and color settings of these digital images, without compromising the precision of orthodontic landmark identification. Forty-nine orthodontic residents and faculty at Saint Louis University identified 13 landmarks in 3 separate trials on either an original lateral cephalogram or 1 of 6 digitized radiographic images. They also subjectively assessed the quality of the image. Ability to identify landmarks did not differ significantly among the groups tested. Conversely, opinions of image quality differed significantly among the groups. Participants found the quality of the grayscale images without color to be poorer than that of the original film but could detect differences only at resolution extremes. Prediction of ability to identify landmarks was substantially greater for higher resolution images. In conclusion, the scanner settings used in the digitization of a cephalometric film did not matter significantly when standard settings were used. In addition, subjective opinion of image quality was predictive of how precisely landmarks were identified when the resolution was relatively high.

Analog-Digital Conversion↗
PubMed1998

Phosphotyrosine 1173 mediates binding of the protein-tyrosine phosphatase SHP-1 to the epidermal growth factor receptor and attenuation of receptor signaling.

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important for this interaction (Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, A. V., Ullrich, A., and Böhmer, F. D. (1997) J. Biol. Chem. 272, 5966-5973). We mapped the EGFR phosphotyrosine 1173 as the major binding site for SHP-1 by a combination of phosphopeptide activation, phosphopeptide competition, and receptor YF mutant analysis. Mutational conversion of the EGFR sequence 1171-1176 AEYLRV into the high affinity SHP-1 binding sequence LEYLYL of the erythropoietin receptor (EpoR) led to a highly elevated SHP-1 binding to the mutant EGFR (EGFR1171-1176EpoR) and in turn to an enhanced dephosphorylation of the receptor. SHP-1 expression interfered with EGF-dependent mitogen-activated protein kinase stimulation, and this effect was more pronounced in case of EGFR1171-1176EpoR. Reduced SHP-1 binding to the EGFR Y1173F mutant resulted in a reduced receptor dephosphorylation by coexpressed SHP-1 and less interference with EGF-dependent mitogen-activated protein kinase stimulation. The effects of receptor mutations on SHP-1 binding were, however, stronger than those on receptor dephosphorylation by SHP-1. Therefore, receptor dephosphorylation may be the result of the combined activity of receptor-bound SHP-1 and SHP-1 bound to an auxiliary docking protein.

Amino Acid Sequence↗
PubMed2003

Ganciclovir release rates in vitreous from different formulations of 1-O-hexadecylpropanediol-3-phospho-ganciclovir.

PURPOSE: To determine the optimal formulation of lipid prodrug, 1-O-hexadecyloxypropyl-phospho-ganciclovir (HDP-P-GCV), for intravitreal delivery. METHODS: Equal concentrations of crystalline or liposomal HDP-P-GCV were exposed to rabbit whole vitreous, core vitreous, peripheral vitreous, human plasma, and heat inactivated rabbit vitreous, and the samples were incubated at 37 degrees C for one week. Aliquots were taken at day 1, 2, 3, and 7 and subjected to HPLC analysis for conversion to GCV. RESULTS: The resultant concentration of GCV from crystalline HDP-P-GCV in vitreous was 198 +/- 49 microM (n = 3) at day 1 and 1253 +/- 248 microM (n = 3) at day 7. The resultant concentration of GCV from the liposomal formulation of HDP-P-GCV in vitreous was much lower, yielding a concentration of 66 +/- 7 microM (n = 3) at day 1 and 243 +/- 39 microM (n = 3) at day 7 (P < 0.001, t Test). When the crystalline HDP-P-GCV was incubated with heat-inactivated vitreous, the detectable GCV concentrations were low (22 microM) and did not increase over time. The concentration of GCV detected from the crystalline HDP-P-GCV in the core vitreous was 19.69 +/- 3.84 microM (n = 3) at day 1 and 1537.36 +/- 177.14 microM (n = 3) at day 7. The concentration of GCV released from crystalline HDP-P-GCV in peripheral vitreous was 32.86 +/- 5.07 microM (n = 3) at day 1 and 1805.78 +/- 327.94 microM (n = 3) at day 7. Detectable GCV concentration from both core and peripheral vitreous samples increased over time, however, the magnitude of GCV release from peripheral vitreous samples was higher (P < 0.05, t Test). CONCLUSION: In vitreous, HDP-P-GCV as a crystalline formulation was converted to GCV more rapidly than liposomal formulation of HDP-P-GCV. Vitreous cells may play an important role in the metabolism of either formulation of HDP-P-GCV delivered into vitreous.

Animals↗
PubMed2001

Communication and cooperation in networked environments: an experimental analysis.

Interpersonal communication and cooperation do not happen exclusively face to face. In work contexts, as in private life, there are more and more situations of mediated communication and cooperation in which new online tools are used. However, understanding how to use the Internet to support collaborative interaction presents a substantial challenge for the designers and users of this emerging technology. First, collaborative Internet environments are designed to serve a purpose, so must be designed with intended users' tasks and goals explicitly considered. Second, in cooperative activities the key content of communication is the interpretation of the situations in which actors are involved. So, the most effective way of clarifying the meaning of messages is to connect them to a shared context of meaning. However, this is more difficult in the Internet than in other computer-based activities. This paper tries to understand the characteristics of cooperative activities in networked environments--shared 3D virtual worlds--through two different studies. The first used the analysis of conversations to explore the characteristics of the interaction during the cooperative task; the second analyzed whether and how the level of immersion in the networked environments influenced the performance and the interactional process. The results are analyzed to identify the psychosocial roots used to support cooperation in a digital interactive communication.

Adult↗