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Biomedical subjects

B Durham

Publications and source records attributed to B Durham.

At least 19 recordsLinked to original sources
PubMed2002

Is the radiation of chest pain a useful indicator of myocardial infarction? A prospective study of 541 patients.

In recent years it has been claimed that in those presenting with chest pain, the extent of pain radiation may be predictive of MI and that women's and men's pattern of pain radiation differs. This prospective study therefore investigated whether there were differences in pain radiation between those with and without MI and according to gender. Patients (n = 541) presenting to a CCU with an episode of chest pain were asked to indicate on a body map the region of pain radiation they experienced at the time of symptom onset. As expected, radiation to the left and/or to right shoulder/arm was significantly higher in the MI group. Women with MI experienced more chest pain radiating to the right arm/shoulder (P = 0.0005), upper right region (P = 0.0006) and arm/shoulder than those without MI. Additionally, women with MI also described more pain radiation in the front neck (P = 0.015) area, and the right shoulder/arm (P = 0.02) than their male counterparts. A third of these women also experienced more pain radiating to the back (P = 0.005). The premise of greater chest pain distribution amongst those with an MI could not be confirmed. Nevertheless, the study identified significant differences amongst women with MI, the discussion analyses the implications for practice.

Aged↗
PubMed2001

Exposure of bovine cytochrome c oxidase to high triton X-100 or to alkaline conditions causes a dramatic change in the rate of reduction of compound F.

The final step in the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a(3) in compound F, was investigated using a binuclear polypyridine ruthenium complex ([Ru(bipyridine)(2)](2)(1,4-bis[2-(4'-methyl-2, 2'-bipyrid-4-yl)ethenyl]benzene)(PF(6))(4)) as a photoactive reducing agent. In the untreated dimeric enzyme, the rate constant for reduction of compound F decreased from 700 s(-1) to 200 s(-1) as the pH was increased from 7.5 to 9.5. Incubation of dimeric enzyme at pH 10 led to an increase in the rate constant to 1650 s(-1), which was independent of pH between pH 7.4 and 10. This treatment resulted in a decrease in the sedimentation coefficient consistent with the irreversible conversion of the enzyme to a monomeric form. Similar results were obtained when the enzyme was incubated with Triton X-100 at pH 8.0. These treatments, which have traditionally been used to convert dimeric enzyme to monomeric form, have no effect on the steady-state activity. The data indicate that either the conversion of the bovine oxidase to a monomeric form or some structural change coincident with this conversion strongly influences the rate constant of this step in the catalytic cycle, perhaps by influencing the proton access to the heme-copper binuclear center.

Animals↗
PubMed2000

Use of a photoactivated ruthenium dimer complex to measure electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the cytochrome bc(1) complex.

Electron transfer between the Rieske iron-sulfur protein (Fe(2)S(2)) and cytochrome c(1) was studied using the ruthenium dimer, Ru(2)D, to either photoreduce or photooxidize cytochrome c(1) within 1 micros. Ru(2)D has a charge of +4, which allows it to bind with high affinity to the cytochrome bc(1) complex. Flash photolysis of a solution containing beef cytochrome bc(1), Ru(2)D, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 micros, followed by electron transfer from cytochrome c(1) to Fe(2)S(2) with a rate constant of 90,000 s(-1). Flash photolysis of reduced beef bc(1), Ru(2)D, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 micros, followed by electron transfer from Fe(2)S(2) to cytochrome c(1) with a rate constant of 16,000 s(-1). Oxidant-induced reduction of cytochrome b(H) was observed with a rate constant of 250 s(-1) in the presence of antimycin A. Electron transfer from Fe(2)S(2) to cytochrome c(1) within the Rhodobacter sphaeroides cyt bc(1) complex was found to have a rate constant of 60,000 s(-1) at 25 degrees C, while reduction of cytochrome b(H) occurred with a rate constant of 1000 s(-1). Double mutation of Ala-46 and Ala-48 in the neck region of the Rieske protein to prolines resulted in a decrease in the rate constants for both cyt c(1) and cyt b(H) reduction to 25 s(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.

Amino Acid Substitution↗
PubMed2000

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants.

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.

Amino Acid Sequence↗
PubMed2000

Parathyroid hormone-related protein in the aetiology of fibrous dysplasia of bone in the McCune Albright syndrome.

OBJECTIVE: Fibrous dysplasia, observed in bone lesions in the McCune Albright syndrome (MAS), is thought to result from abnormalities in cells of the osteogenic lineage associated with over-activation of the cAMP signalling pathway in affected cells. The aim of this study was to investigate the role of parathyroid hormone-related protein (PTHrP) in the aetiology of MAS, and to determine a possible therapeutic role for 1,25-dihydroxy vitamin D(3) (1,25(OH)(2)D(3)). DESIGN: The effects of 1,25(OH)(2)D(3) on PTHrP production and mRNA expression were determined in vitro. 1,25(OH)(2)D(3) therapy was administered to three patients with MAS. PATIENTS: Clinical data from four MAS patients (MAS1, 2, 3 and 4), and in vitro studies using bone from three MAS patients (MAS1, 2, and 3), are presented. MEASUREMENTS: Immunoradiometric assay and low-cycle number reverse transcriptase-linked PCR were used to determine PTHrP production and mRNA expression in vitro. Standard clinical biochemistry was recorded pre and post commencement of 1,25(OH)(2)D(3) treatment. RESULTS: We report the elevated secretion of PTHrP, and a concomitant rise in PTHrP mRNA expression, in cultured osteoblasts from three MAS patients. Treatment with 1,25(OH)(2)D(3) produced a dose-dependent decrease in PTHrP protein secretion and mRNA expression. Marked improvement in bone biochemistry in MAS1, 2 and 3 post treatment with 1,25(OH)(2)D(3) is documented. CONCLUSION: This study provides the first evidence suggesting that PTHrP may contribute to the aetiology of fibrous dysplasia in MAS. In addition, the therapeutic administration of 1,25(OH)92)D(3) may provide clinicians with an important new regime for symptomatic relief of bone pain and fracture in some patients with MAS.

Adolescent↗
PubMed2000

Are manual gestures, verbal descriptors and pain radiation as reported by patients reliable indicators of myocardial infarction? Preliminary findings and implications.

Patients experiencing an episode of acute chest pain need to be assessed promptly and effectively to ensure optimal management. The aim of this study was to investigate whether there were specific aspects of patients' symptom reports which could be viewed as additional indicators of myocardial infarction (MI) and contribute to the assessment process. The sample consisted of 267 patients who presented with an episode of acute chest pain. Methods of data collection were based on, or modified from, previous studies which had investigated the use of manual gestures, choice of verbal descriptors and extent of pain radiation in patients with and without MI. To determine whether these variables were of significance in the diagnosis, the responses of those with (n = 118) and without MI (n = 149) were compared. The results suggest that it is currently impossible to draw any conclusions as to whether the variables studied can be judged as reliable indicators of MI. The findings indicate that there are some differences between the groups particularly in the language used. Moreover, women with MI characterized their symptoms through stronger emotive words such as 'worrying' (P = 0.014) 'frightening' and 'intolerable' and also differed from their male counterparts in their reports regarding pain radiation. Implications for practice and for research are discussed.

Aged↗
PubMed2000

A detailed assessment of alterations in bone turnover, calcium homeostasis, and bone density in normal pregnancy.

The effects of pregnancy on bone turnover and the potential risk of developing an osteoporotic fracture in pregnancy are controversial. Utilizing biochemical markers of bone formation and resorption and dual-energy X-ray absorptiometry (DEXA), bone turnover before, during, and after pregnancy was studied in detail. Ten women (mean age 30 years; range 23-40) were recruited. Prepregnancy data were obtained and then a review was performed at 2-week intervals , once pregnancy was confirmed, until 14 weeks of gestation and thereafter monthly until term. Bone mineral density (BMD) was estimated by DEXA scanning of hip, spine, and forearm preconception and postpartum. In addition, BMD of the forearm at 14 weeks and 28 weeks gestation was obtained. All pregnancies had a successful outcome. Urinary free pyridinium cross-links, free pyridinoline (fPyr) and free deoxypyridinoline (fDPyr), were normal prepregnancy (mean [+/-SD]) 14.6 nmol/mmol (1.8) and 5.0 nmol/mmol (1.0) creat, respectively. By 14 weeks, they had increased to 20.8 nmol/mmol (4.3) and 6.1 nmol mmol (1.4) (both p < 0.02) and by 28 weeks to 26.3 nmol/mmol (5.6) and 7.4 nmol/mmol (1.6) (both p < 0.01). The ratio of fPyr to fDPyr remained constant. A similar significant increase was observed in N-telopeptide (NTx). Bone formation was assessed by measurement of carboxyterminal propeptide of type 1 collagen (P1CP) and bone-specific alkaline phosphatase (BSAP). Neither were altered significantly before 28 weeks, but subsequently mean P1CP increased from 110 microg/liter (23) to 235 microg/liter (84) at 38 weeks and mean BSAP increased from 11.1 U/liter (5.0) to 28.6 U/liter (11.1) (p < 0.01 for both variables). Lumbar spine (L1-L4) BMD decreased from a prepregnancy mean of 1.075 g/cm (0.115) to 1.054 g/cm2 (0.150) postpartum (p < 0.05). Total hip BMD decreased from a prepregnancy mean of 0.976 g/cm2 (0.089) to 0.941 g/cm2 (0.097) (p < 0.05). Forearm BMD at midradius, one-third distal and ultradistal decreased but did not reach statistical significance. As assessed by these bone markers, in the first 2 trimesters of pregnancy, bone remodeling is uncoupled with a marked increase in bone resorption. A corresponding increase in formation markers is not observed until the third trimester. Spinal BMD exhibits a significant decrease from prepregnancy to the immediate postpartum period with a mean reduction in BMD of 3.5 % in 9 months.

Absorptiometry, Photon↗
PubMed1999

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants.

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

Animals↗
PubMed1999

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

2,2'-Dipyridyl↗
PubMed1998

Single electron reduction of cytochrome c oxidase compound F: resolution of partial steps by transient spectroscopy.

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.

2,2'-Dipyridyl↗
PubMed1998

Human keratinocytes express transcripts for three isoforms of parathyroid hormone-related protein (PTHrP), but not for the parathyroid hormone/PTHrP receptor: effects of 1,25(OH)2 vitamin D3.

Parathyroid hormone-related protein (PTHrP) is strongly expressed in the epidermis and has been implicated in the regulation of growth and differentiation of keratinocytes. PTHrP has N-terminal sequence homology with parathyroid hormone (PTH) and binds to the type I PTH/PTHrP receptor, but earlier reports suggest that keratinocytes do not possess this cell surface receptor. In order to determine which PTHrP mRNA isoforms are expressed by keratinocytes and whether the type I receptor mRNA is present, we designed specific primers for reverse transcriptase-polymerase chain reaction. The interaction of PTHrP with other promoters of keratinocyte differentiation is unclear. In particular, 1,25(OH)2D3 is also fundamental in calcium homeostasis and induces changes in intracellular calcium. We therefore investigated the effect of 1,25(OH)2D3 on PTHrP mRNA expression and protein production in cultured human keratinocytes. Cells were incubated for 3 days at concentrations of 1.25(OH)2D3 of 10(-10)-10(-6) mol/L. PTHrP in culture supernatant, measured by two site immunoradiometric assay, was 915 +/- 98 PTHrP fmol/mg of cell layer protein in untreated cultures decreasing to 570 +/- 113 with 10(-8) mol/L and 402 +/- 24 with 10(-6) mol/L 1,25(OH)2D3 (mean +/- SEM, P < 0.01, n = 6). Transcripts for all three PTHrP isoforms (139, 141 and 173 amino acids) were detectable in keratinocyte mRNA. Corresponding to the decrease in PTHrP protein we demonstrated a reduction in all three PTHrP mRNA transcripts after 3 days' incubation with 1,25(OH)2D3 over a concentration range 10(-10)-10(-6) mol/L. Repeated studies failed to detect type I PTH/PTHrP receptor mRNA in human keratinocytes, either in control cultures or in the presence of 1,25(OH)2D3. We have shown that keratinocytes produce abundant PTHrP and that this is modulated by 1,25(OH)2D3, suggesting a physiological role. Further studies are required to investigate the relative expression of PTHrP isoforms, their role in keratinocyte signalling and the receptors involved.

Calcifediol↗
PubMed1997

trans-diaquabis(2,2'-bipyridine-N,N')-ruthenium(II) bis(hexafluorophosphate).

The Ru atom of the title complex, [Ru(C10H8N2)2-(H2O)2](PF6)2, is located on a crystallographic inversion center; the bipyridine ligands are bowed [dihedral angle between C5N planes is 162.68 (12) degrees] due to the interligand steric interactions of the trans bipyridyl units. The Ru--O distance is 2.116 (2) A and the mean Ru--N distance is 2.074 (2) A.

Crystallography, X-Ray↗
PubMed1997

Temperature and viscosity dependence of the electron-transfer reaction between plastocyanin and cytochrome c labeled with a ruthenium(II) bipyridine complex.

The temperature and viscosity dependence of the photo-induced electron-transfer reaction between plastocyanin and cytochrome c labeled at Lys13 with Ru(4,4'-dicarboxybipyridine)(bipyridine)(2+)2 have been investigated. In these studies, a short pulse of 450 nm light was used to excite the ruthenium complex which was oxidatively quenched by the iron center of cytochrome c. The resulting Fe(II) cytochrome c was then rapidly reoxidized by plastocyanin. The reactions were investigated over a temperature range of 3.5 to 37 degrees C under low ionic strength conditions such that protein/protein complex formation was favored. The enthalpy of activation was 7 kcal mol-1 and the entropy of activation was -20 cal mol-1 K-1. Increasing the viscosity by the addition of sucrose up to 70% resulted in a 4-fold decrease in the rate constant for electron transfer. The overall results suggest a rate-limiting step that involves either dissociation of the dominant protein/protein complex or surface diffusion of the associated proteins.

2,2'-Dipyridyl↗
PubMed1997

Pelvic ultrasound performed by emergency physicians for the detection of ectopic pregnancy in complicated first-trimester pregnancies.

STUDY OBJECTIVE: To determine the accuracy and application of pelvic ultrasound performed by emergency physicians in detecting ectopic pregnancy (EP) in complicated first-trimester pregnancies. METHODS: We studied consecutive pelvic ultrasounds performed in all women who presented with abdominal pain or vaginal bleeding during the first trimester of pregnancy over a 6-month period. Patients with clinical evidence of incomplete abortion were not included. We compared ultrasound results with subsequent ultrasound findings by the radiology department and correlated them with follow-up diagnoses and outcomes. RESULTS: Of the 136 eligible patients, a final diagnosis was rendered in 125; 11 were lost to follow-up. These ED ultrasound findings were recorded: established intrauterine pregnancy (IUP) with embryonic structures, 87 (70%); indeterminate scan revealing no distinct evidence of IUP or EP, 15 (12%); early intrauterine gestational sac of less than 6 weeks without embryonic structures, 12 (10%); EP, 8 (6%); blighted ovum, 2 (2%); and molar pregnancy, 1 (1%). The initial ED ultrasound determination was consistent with radiology department findings, final outcome, or both in 121 (96%) (95% confidence interval [CI], 91% to 97%). ED ultrasound accurately identified 87 pregnancies with intrauterine embryonic structures, including 5 patients with fetal demise (95% Cl, 97% to 100%). Diagnosis of pregnancy location in these 87 patients effectively ruled out EP, with a negative predictive value of 100%. The sensitivity and specificity of ED ultrasound in the detection of EP were 90% and 88%, respectively. CONCLUSION: Pelvic ultrasonography performed by emergency physicians can be used to rule out EP and make an accurate diagnosis in most patients with complicated first-trimester pregnancies during the initial ED visit. The remaining patients at risk can be identified and a diagnosis made by means of follow-up ultrasound and serial hCG determinations.

Adult↗
PubMed1997

Biochemical markers of bone turnover in seronegative spondylarthropathy: relationship to disease activity.

To investigate bone turnover in patients with seronegative spondylarthropathy, a bone formation marker, type 1 procollagen carboxy-terminal propeptide (P1CP), and resorption markers, the pyridinium cross-links of collagen [urinary free (f) PYR and DPYR], were measured. The median f-PYR, f-DPYR and P1CP (+/-interquartile range) were 15.8 (6.00) nmol/mmol creatinine, 3.8 (2.2) nmol/mmol creatinine and 101.5 (38) micrograms/1, respectively. There was a positive correlation between resorption markers and acute-phase reactants such as C-reactive protein (r = 0.42 for PYR, r = 0.42 for DPYR, P < 0.05), and a negative correlation observed between P1CP and the erythrocyte sedimentation rate (r = -0.64, P < 0.05). In the subgroup of patients with an elevated CRP concentration, the concentration of PYR and DPYR was significantly increased (f-PYR 25.7 vs 15.8 and f-DPYR 6.6 vs 3.8, P < 0.01 for f-PYR, P < 0.05 for f-DPYR). This study suggests than an elevation in acute-phase response in patients with seronegative spondylarthropathy is associated with increased concentration of bone resorption markers with a tendency for reduction in bone formation markers. This may represent uncoupling of bone formation and resorption, leading to bone loss in such patients.

Acute-Phase Reaction↗
PubMed1996

Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site.

A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constants kf and kd of the high-affinity complex between yeast iso-1-cytochrome c (yCc) and cytochrome c peroxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C, C102T variant of yCc, and has the same reactivity with CMPI as native yCc. kd and kf were measured by photoreducing a small concentration of Ru-39-Cc in the presence of the oxidized yCcIII: CMPI complex, which must dissociate before Ru-39-CcII can bind to CMPI and reduce the radical action. The value of kd for the 1:1 high-affinity complex is very small at low ionic strength, < 5 s-1 but is increased significantly by binding yCc to a second low-affinity site. However, the low-affinity yCc binding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM. The value of kd increases to 4000 s-1 at 150 mM ionic strength, while kf decreases from > 3 x 10(9) M-1 s-1 at low ionic strength to 1.3 x 10(9) M-1 s-1 at 150 mM ionic strength. These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength. The interaction between yCc and CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constant kf without allowing product dissociation to be rate-limiting. The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.

Binding Sites↗
PubMed1996

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase.

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.

2,2'-Dipyridyl↗