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J Fitch

Publications and source records attributed to J Fitch.

At least 19 recordsLinked to original sources
PubMed2004

Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy.

The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.

Cytochromes c2↗
PubMed2004

Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.

Cytochromes c2↗
PubMed2003

Impaired fracture healing in the absence of TNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption.

UNLABELLED: TNF-alpha is a major inflammatory factor that is induced in response to injury, and it contributes to the normal regulatory processes of bone resorption. The role of TNF-alpha during fracture healing was examined in wild-type and TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice. The results show that TNF-alpha plays an important regulatory role in postnatal endochondral bone formation. INTRODUCTION: TNF-alpha is a major inflammatory factor that is induced as part of the innate immune response to injury, and it contributes to the normal regulatory processes of bone resorption. METHODS: The role of TNF-alpha was examined in a model of simple closed fracture repair in wild-type and TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice. Histomorphometric measurements of the cartilage and bone and apoptotic cell counts in hypertrophic cartilage were carried out at multiple time points over 28 days of fracture healing (n = 5 animals per time point). The expression of multiple mRNAs for various cellular functions including extracellular matrix formation, bone resorption, and apoptosis were assessed (triplicate polls of mRNAs). RESULTS AND CONCLUSIONS: In the absence of TNF-alpha signaling, chondrogenic differentiation was delayed by 2-4 days but subsequently proceeded at an elevated rate. Endochondral tissue resorption was delayed 2-3 weeks in the TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice compared with the wild-type animals. Functional studies of the mechanisms underlying the delay in endochondral resorption indicated that TNF-alpha mediated both chondrocyte apoptosis and the expression of proresorptive cytokines that control endochondral tissue remodeling by osteoclasts. While the TNF-alpha receptor ablated animals show no overt developmental alterations of their skeletons, the results illustrate the primary roles that TNF-alpha function contributes to in promoting postnatal fracture repair as well as suggest that processes of skeletal tissue development and postnatal repair are controlled in part by differing mechanisms. In summary, these results show that TNF-alpha participates at several functional levels, including the recruitment of mesenchymal stem, apoptosis of hypertrophic chondrocytes, and the recruitment of osteoclasts function during the postnatal endochondral repair of fracture healing.

Animals↗
PubMed2001

The efficacy and resource utilization of remifentanil and fentanyl in fast-track coronary artery bypass graft surgery: a prospective randomized, double-blinded controlled, multi-center trial.

UNLABELLED: We compared (a) the perioperative complications; (b) times to eligibility for, and actual time of the following: extubation, less intense monitoring, intensive care unit (ICU), and hospital discharge; and (c) resource utilization of nursing ratio for patients receiving either a typical fentanyl/isoflurane/propofol regimen or a remifentanil/isoflurane/propofol regimen for fast-track cardiac anesthesia in 304 adults by using a prospective randomized, double-blinded, double-dummy trial. There were no differences in demographic data, or perioperative mortality and morbidity between the two study groups. The mini-mental status examination at postoperative Days 1 to 3 were similar between the two groups. The eligible and actual times for extubation, less intense monitoring, ICU discharge, and hospital discharge were not significantly different. Further analyses revealed no differences in times for extubation and resource utilization after stratification by preoperative risk scores, age, and country. The nurse/patient ratio was similar between the remifentanil/isoflurane/propofol and fentanyl/isoflu-rane/propofol groups during the initial ICU phase and less intense monitoring phase. Increasing preoperative risk scores and older age (>70 yr) were associated with longer times until extubation (eligible), ICU discharge (eligible and actual), and hospital discharge (eligible and actual). Times until extubation (eligible and actual) and less intense monitoring (eligible) were significantly shorter in Canadian patients than United States' patients. However, there was no difference in hospital length of stay in Canadian and United States' patients. We conclude that both anesthesia techniques permit early and similar times until tracheal extubation, less intense monitoring, ICU and hospital discharge, and reduced resource utilization after coronary artery bypass graft surgery. IMPLICATIONS: An ultra-short opioid technique was compared with a standard fast-track small-dose opioid technique in coronary artery bypass graft patients in a prospective randomized, double-blinded controlled study. The postoperative recovery and resource utilization, including stratification of preoperative risk score, age, and country, were analyzed.

Aged↗
PubMed2000

Charged-particle multiplicity near midrapidity in central Au+Au collisions at sqrt[SNN]=56 and 130 GeV.

We present the first measurement of pseudorapidity densities of primary charged particles near midrapidity in Au+Au collisions at sqrt[s(NN)] = 56 and 130 GeV. For the most central collisions, we find the charged-particle pseudorapidity density to be dN/deta|(|eta|<1) = 408+/-12(stat)+/-30(syst) at 56 GeV and 555+/-12(stat)+/-35(syst) at 130 GeV, values that are higher than any previously observed in nuclear collisions. Compared to proton-antiproton collisions, our data show an increase in the pseudorapidity density per participant by more than 40% at the higher energy.

Journal Article↗
PubMed1999

Selective blockade of membrane attack complex formation during simulated extracorporeal circulation inhibits platelet but not leukocyte activation.

OBJECTIVE: Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation. METHODS: In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation. RESULTS: Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit. CONCLUSIONS: The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

Antibodies, Monoclonal↗
PubMed1998

Purification and primary structure analysis of two cytochrome c2 isozymes from the purple phototrophic bacterium Rhodospirillum centenum.

The isolation and amino acid sequences of two cytochromes c-552 from the thermotolerant bacterium Rhodospirillum (R.) centenum have been determined. They are very similar to one another with 85% identity. They are homologous to the cytochromes c2 from purple bacteria with approximately 67% identity to that from Rhodopseudomonas (Rps.) palustris compared to only 42% identity with others of the c2 subclass. In addition, they share an unusual six-residue insertion with Rps. palustris cytochrome c2 not found in any other cytochrome. The relationship with Rps. palustris is thus highly significant. The redox potentials of the R. centenum isozymes are 293 and 316 mV. Although the proteins have strongly different iso-electric points, both have three conserved lysine residues at the proposed site of electron transfer. These results suggest that they may be functionally interchangeable.

Amino Acid Sequence↗
PubMed1998

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Animals↗
PubMed1997

Influence of conserved amino acids on the structure and environment of the heme of cytochrome c2. A resonance Raman study.

Resonance Raman spectra using Soret excitations of oxidized and reduced Rhodobacter capsulatus cytochrome c2 at pH 7.5 were studied. The spectra of oxidized cytochrome c2 show three components for the v10 mode at 1638, 1633, and 1629 cm(-1). The intensities of these components are sensitive to the excitation wavelength. This effect is explained in the context of a conformational equilibrium of the ferriheme between a nearly planar structure and two ruffled structures. In the case of reduced cytochrome c2, the absolute frequencies as well as the excitation-dependent frequency dispersion of the v10 mode (1618-1621 cm(-1)) indicate a displacement of the conformational equilibrium of heme toward the more planar structures. To measure the influence of some key amino acid residues on the heme-protein interaction of cytochrome c2, four site-directed mutants of Rb. capsulatus cytochrome c2 have been studied by resonance Raman spectroscopy and their spectra compared with the spectra obtained for the wild type cytochrome. The mutants studied are K14E/K32E, P35A, W67Y, and Y75F. The spectral changes induced by the mutations are interpreted in terms of alterations in the structure and/or environment of the cytochrome c2 heme in the framework of the expected role of the different amino acid residues in the stability and redox potential.

Amino Acids↗
PubMed1997

Acoustic measures of dysphonic severity across and within voice types.

The purpose of the study was to explore an interaction between pathologic voice type and the acoustic prediction of dysphonic severity. One hundred and two phonatory samples, representing a wide range of laryngeal conditions, were categorized by listeners into three voice types: breathy, rough, and hoarse. A second group of trained listeners rated the severity of the samples on a 7-point scale. Twenty-five frequency- and time-domain measures were used to predict perceptual severity. Multiple regression analyses showed that the most useful measure for the prediction of severity across voice types was noise-to-harmonic ratio (NHR). The severity of individual voice types was predicted with differential results on the basis of both short- and long-term measures of perturbation.

Adolescent↗
PubMed1997

Influence of cholesterol screening and nutritional counseling in reducing cholesterol levels in children. The American Heart Association.

The purpose of this study was to determine whether cholesterol screening and nutritional counseling can reduce cholesterol concentrations in populations of otherwise unrecognized hypercholesterolemic children. A large pediatric practice in Parma Heights, Ohio, has conducted cholesterol surveillance of children over 2 years of age since 1986. The importance of cholesterol and other recognized risk factors for the progression of atherosclerosis is discussed with all families, and the American Heart Association's Step-One diet is recommended. The present study examines data from a cohort of 894 children (473 boys, 421 girls) who had cholesterol concentrations above 185 mg/dL (4.79 mmol/L) (the 90th percentile) at baseline and, after counseling, had a repeat measurement an average of 2.2 years later. Their mean ages were 7 years at the first testing and 9.2 years at the second. Children who had cholesterol concentrations above 200 mg/dL (5.18 mmol/L) (the 95th percentile) had lipoprotein profiles done, and if their LDL cholesterol exceeded 130 mg/dL (3.37 mmol/L) (the 95th percentile), they were referred to a nutritionist, and family members were advised to have their blood lipids analyzed. Mean cholesterol concentration for all 894 children over this time period decreased by 9.4% (19.5 mg/dL [0.51 mmol/L]; 95% CI = 17.5 mg/dL [0.45 mmol/L] to 21.5 mg/dL [0.56 mmol/L]; P < 0.001). A similar decrease of 8.6% (16.6 mg/dL [0.43 mmol/L); 95% CI = 14.0 mg/dL [0.36 mmol/L] to 19.3 mg/dL [0.50 mmol/L); P < 0.001) was observed for the 463 children who had initial cholesterol concentrations between 185 and 200 mg/dL (4.79 and 5.18 mmol/L) and who therefore received a less intense intervention. Cholesterol concentrations in groups of otherwise unidentified hypercholesterolemic children can be significantly reduced as a result of cholesterol screening and nutritional counseling in a pediatric practice setting.

Age Factors↗
PubMed1996

Stability study of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry.

To estimate the stability of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants, charge state distributions and hydrogen/deuterium exchange rates were monitored by electrospray ionization mass spectrometry. The relative stability of the mutants was observed with the order: V11 insert > Y75F > wild-type = K32E > K12D = K14E > or = K52E > K14E/K32E > W67Y > P35A > I57N > G34S. (A preliminary account has been presented for mutants G34S and P35A [Jaquinod et al. (1995) Rapid Commun. Mass Spectrom. 9, 1135-1140].) This approach is shown to be a useful tool for rapid characterization of mutational effects on protein conformation.

Cytochrome c Group↗
PubMed1996

Amplification of the inflammatory response: adhesion molecules associated with platelet/white cell responses.

Cardiopulmonary bypass (CPB) causes leukocyte and platelet activation, resulting in upregulation of the adhesion receptor CD11b/CD18 on leukocytes and upregulation of P-selectin, the adhesion receptor that binds the activated platelet to polymorphonuclear neutrophils (PMNs) and monocytes. Our laboratory has studied the expression of activation-dependent adhesion receptors during in vivo CPB. Both PMN and monocyte CD11b were upregulated during CPB but with differing time courses. Peak PMN CD11b levels occurred at the end of the hypothermic phase of bypass, whereas monocyte CD11b levels increased steadily throughout the course of CPB, peaked at 2-4 h after CPB, and remained significantly elevated as late as 18-24 h post CPB. The percentage of P-selectin-positive platelets increased significantly during bypass, peaking around the end of bypass and remaining elevated in the early post-bypass period. The level then returned to normal by 18 h post-bypass. Monocyte-platelet binding paralleled the increase in P-selectin-positive platelets during bypass and similarly remained elevated in the post-bypass period. PMN-platelet binding also increased but peaked early during CPB. Upregulation of these adhesive receptors and formation of platelet-leukocyte conjugates may influence the prothrombotic activity of monocytes and the proinflammatory activity of PMNs in the post-CPB period. Our laboratory has developed an in vitro model of extracorporeal circulation, and recirculation of blood on this circuit results in significant activation of PMNs and monocyte CD11b expression, increasing progressively over time. Likewise, the percentage of P-selectin-positive platelets increased and was paralleled by the formation of leukocyte-platelet conjugates comparable to the pattern found in vivo. Generation of the complement fragments C5a and the C5b-9 membrane-attack complex may contribute to platelet P-selectin expression and formation of leukocyte-platelet conjugates during CPB. The in vitro model has been used to test the cellular effects of complement inhibition employing a monoclonal antibody that blocks cleavage of C5 into C5a and C5b to determine the role of early vs. late complement components in the cellular activation induced by CPB. Preliminary results demonstrate that blockage of the formation of C5a and the C5b-9 membrane-attack complex during simulated extracorporeal circulation effectively inhibits platelet and PMN activation and the formation of leukocyte-platelet conjugates.

Blood Platelets↗
PubMed1995

Sentence/vowel correlation in the evaluation of dysphonia.

Considering the widespread use of the sustained vowel in the evaluation of voice disorders, our objective was to study the degree to which the vowel is representative of voice in continuous speech. Phonatory samples were recorded from 20 normal subjects and 60 patients representing commonly occurring voice problems (nodules, unilateral paralysis, and functional). The phonatory samples were evaluated by 25 listeners on the basis of 12 bipolar, semantic differential scales. Factor analysis produced two perceptual dimensions with similar loadings for both vowels and sentences. A correlation of 0.78 was obtained between vowels and sentences on factor 1 scores, "vocal severity," and a correlation of 0.77 was obtained between vowels and sentences on factor 2 scores, "pitch/quality." Six of the 80 pairs of sentences and vowels differed by two scale points or more on vocal severity. Results suggest that although a relatively strong relationship exists between the two measures, sustained vowel sounds may not be an adequate clinical index to the dysphonic severity of continuous speech.

Adult↗
PubMed1995

Acoustic prediction of severity in commonly occurring voice problems.

The relative effectiveness of three acoustic measures (jitter, shimmer, and harmonic/noise ratio) in predicting the dysphonic severity of a diverse clinical population singly and together was investigated. Phonatory samples were recorded from 20 normal subjects and 60 patients representing 3 laryngeal groups (nodules, paralysis, and functional). The phonatory samples were evaluated by 22 listeners using a 7-point equal-appearing interval scale. Shimmer produced a bivariate correlation of 0.54 with dysphonic severity; harmonic/noise ratio correlated -0.32 with dysphonic severity; and jitter produced no significant correlation with severity. The combination of acoustic variables through multiple regression analysis produced a correlation of 0.56, with only shimmer and average F0 contributing to the correlation. For this particular clinical population, therefore, findings indicated that (a) none of the variables was strongly correlated with dysphonia ratings, and (b) a combination of acoustic predictors was no more successful than a single predictor of dysphonic severity, namely, shimmer.

Adolescent↗
PubMed1995

Pathologic voice type and the acoustic prediction of severity.

We hypothesized that acoustic measures would predict dysphonic severity with differential results for pathological voice types. An instructional program based upon synthesized voice signals was developed to facilitate an awareness of prototypical voice types. Eighty phonatory samples representing normal subjects as well as patients with unilateral vocal fold paralysis, vocal nodules, and functional dysphonia were analyzed acoustically on the basis of four measures: average fundamental frequency (F0), jitter, shimmer, and harmonic/noise ratio (H/N ratio). Following training, 29 listeners classified 62% of the phonatory samples on the basis of breathy, hoarse, rough, and normal. Dysphonic severity of rough voices was predicted more successfully by H/N ratio (r2 = .73) than by shimmer (r2 = .43). Dysphonic severity of breathy voices was predicted only by the combined features of less jitter, more shimmer, and lower H/N ratio (r2 = .74). No combination of acoustic variables was successful in the prediction of the hoarse voice type.

Adolescent↗