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At least 793 records · Page 44Linked to original sources
PubMed2000

Effect of grade on disease-free survival and overall survival in FIGO stage I adenocarcinoma of the endometrium.

OBJECTIVE: To analyse the effect of differentiation on disease-free survival (DFS) and overall survival (OS) in patients with stage I adenocarcinoma of the endometrium. PATIENTS AND METHODS: From 1979 to 1995, 350 patients with FIGO stage IA-IC with well (G1), moderately (G2) or poorly (G3) differentiated tumors were treated with surgery and high dose-rate brachytherapy with or without external radiation. Median age was 65 years (39-86 years). RESULTS: The 5-year DFS was 88+/-3% for the G1 tumors, 77+/-4% for the G2 tumors, and 67+/-7% for the G3 tumors (P=0.0049). With regard to the events contributing to DFS, the 5-year cumulative percentage of local relapse was 4.6% for the G1 tumors, 9.0% for the G2 tumors, and 4.6% (P=0.027) for the G3 tumors. Cumulative percentage of metastasis was 1.4, 6.3 and 7.2% (P<0.001), respectively, whereas percentages of death were 6.0, 7.9 and 20.7% (P<0.001). The 5-year OS was 91+/-3, 83+/-4 and 76+/-7%, respectively (P=0.0018). In terms of multivariate hazard ratios (HR), the relative differences between the three differentiation groups correspond to an increase of 77% of the risk of occurrence of either of the three events considered for the DFS (HR=1.77, 95% CI [0.94-3.33]), (P=0.078) for the G2 tumors and of 163% (HR=2.63, 95% CI [1.27-5.43]), (P=0.009) for the G3 tumors with respect to the G1 tumors. The estimated relative hazards for OS are, respectively, in line with those for DFS: HR=1.51 (P=0.282) for the G2 tumors; and HR=3.37 (P=0.003) for the G3 tumors. CONCLUSION: Patients with grade 1 tumors are those least exposed to either local relapse, metastasis, or death. In contrast patients with grade 2 tumors seem to be at higher risk of metastasis, whereas patients with grade 3 tumors appear at higher risk of death. Since we have looked at the first of three competing events (local relapse, metastasis and death), this suggests that patients with grade 3 tumors probably progress to death so fast that local relapse, if any, cannot be observed.

Adenocarcinoma↗
PubMed1998

Age-related decrease in the overall extent of perikaryal projections in rabbit spinal ganglion neurons.

The overall extent of the perikaryal projections of sensory neurons from spinal ganglia of young adult and aged rabbits was estimated by a stereological method using the electron microscope. The extent of perikaryal projections was significantly smaller in the aged animals. This age-related decrease did not seem to depend on factors intrinsic to the neuron, but on the absence of a satellite cell covering over extensive portions of the nerve cell body surface. This decrease may influence the organization of the subplasmalemmal cytoskeleton, metabolic exchange between the nerve cell body and its environment and perhaps also neuronal metabolism.

Aging↗
PubMed2000

Modified partial hyperfractionation in radiotherapy for bulky uterine cervical cancer: reduction of overall treatment time.

PURPOSE: The purpose of this study was to assess the feasibility and toxicity of modified fractionation of external beam radiation with the intention of reducing the overall treatment time (OT) by 1 week in cervical cancer. METHODS AND MATERIALS: Thirty-one patients (Group 1, n = 31) with bulky cervical cancer (>/= 4 cm with Stage II and III, >/= 5 cm with Stage IB2) were entered into the twice a day (b.i. d.) protocol (18 Gy/10 fx in 2 weeks followed by 18 Gy/12 fx, b.i.d. in 6 days, then midline block at 36 Gy with 45 Gy to the whole pelvis and 51-59 Gy to the parametrium). These patients underwent high-dose-rate brachytherapy with 4 Gy/fx x 7 to point A, biweekly. During the same period, patients with non-bulky tumors (Group 2, n = 31) received conventional treatment and similar brachytherapy. RESULTS: The OT of Group 1 was 7 weeks or less in 61.3%, 7.1-8 weeks in 29%, and more than 8 weeks in 9.7% (19.4%, 51.6%, and 29% in Group 2, respectively, p = 0.003). Incidences of acute complications and treatment breaks were similar in both groups. Late complication (rectal bleeding) occurred only in Group 1 (13%, 4/31), but was self-limited. Locoregional failures occurred within 2 years after completion of radiation therapy in both groups (16% and 13% in Group 1 and 2, respectively, with minimum and median follow-ups of 2 years and 34 months). CONCLUSION: Partial hyperfractionation on the third week of radiation permitted patients to finish their treatment with shorter OT without excessive acute complications and with acceptable grade 2 late rectal bleeding complications. This treatment scheme may be an effective method for the improvement of local control of bulky cervical cancer.

Adult↗
PubMed2003

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) negatively affect overall survival in carcinoma of the cervix treated with radiotherapy.

PURPOSE: The purpose of this study was to examine a variety of biomarkers in carcinoma of the cervix to better characterize (1). the natural history of the disease, (2). response to radiotherapy (RT), and (3). potential for new therapeutic strategies. MATERIALS AND METHODS: Fifty-five patients with Stage IB-IVA carcinoma of the cervix, treated with definitive intent RT, and on whom tumor tissue blocks were available were included in this study. Charts were reviewed for clinical parameters and disease status. Immunohistochemistry was performed for epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), CD34, topoisomerase II alpha (topo-II), and cyclooxygenase-2 (COX-2). Univariate and multivariate Cox proportional hazards modeling was performed with disease-free survival (DFS) and overall survival (OS) as the end points. Biomarkers were evaluated for correlation between various prognostic factors. RESULTS: In this series of 55 patients with carcinoma of the cervix treated with definitive RT, only stage was significant on univariate analysis for DFS (p < 0.0001). On univariate analysis, increasing FIGO stage (p < 0.0001) and membranous staining of EGFR (p < 0.037) indicated diminished OS. On multivariate analysis for DFS, COX-2, VEGF, and stage were significant (p = 0.012, p = 0.014, and p = 0.03, respectively), with increased expression indicating a worse prognosis. For OS, multivariate analysis revealed that VEGF, EGFR, and FIGO stage were significant (p = 0.005, p = 0.011, and p < 0.0001, respectively). Significant direct correlations were identified between VEGF and CD34 (p = 0.04), COX-2 and topo-II (p = 0.04), COX-2 and grade (p = 0.04), and tumor size and clinical stage (p = 0.04). CONCLUSION: Multivariate analysis revealed that increased staining for VEGF and COX-2 indicated diminished DFS, and VEGF and EGFR identified patients at increased risk of death. A significant direct correlation between VEGF and CD34 implicates the process of angiogenesis. Topo-II is a proliferative marker and it correlated directly with COX-2, indicating that expression of COX-2 may be greater in more proliferative tumors. Increased expression of EGFR, VEGF, and COX-2 has identified patients with a worse prognosis in cancer of the cervix. These data support the investigation of therapeutics that target these proteins in carcinoma of the cervix.

Adenocarcinoma↗
PubMed2001

Maternal selectivity suppression through peripheral anosmia affects neither overall nursing frequency and duration, nor lactation performance in ewes.

The effects of prepartum peripheral anosmia on nursing activity, milk production and growth of the lambs, were assessed by comparing intact (n=10) and anosmic (n=10) multiparous Columbia and Rambouillet ewes and their single lamb during the first 2 months of lactation. Intact mothers only nursed their own lamb (98%) while most of the nursing activity in anosmic mothers concerned alien lambs (78%). On the other hand, the total duration and the frequencies of nursing did not differ significantly between groups (P>0.05). Nevertheless, the total percentage of nursing of own lamb by anosmic mothers (22%) was higher than expected at random (10%). Milk production or lambs' weights did not differ between groups. We conclude that prepartum anosmia resulted in the failure of ewes to develop true selective nursing up to the 8th week of lactation, although some preferential mother-young relationship yet developed. On the other hand, it did not affect significantly overall nursing activity.

Journal Article↗
PubMed2003

Prognostic value of quality of life scores for time to progression (TTP) and overall survival time (OS) in advanced breast cancer.

The purpose of the study was to investigate whether baseline quality of life (QoL) and changes in QoL scores from baseline are prognostic for time to progression (TTP) and/or overall survival (OS) in patients with advanced breast cancer receiving docetaxel (T) or sequential methotrexate and 5-fluorouracil (MF). QoL was assessed at baseline and before each treatment using the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30). Survival curves and probabilities were estimated using the Kaplan-Meier technique. The Cox proportional hazards regression model was used for both the univariate and multivariate analyses to explore relationships between baseline QoL variables and TTP, as well as OS. In the univariate analysis, more severe pain and fatigue at baseline were predictive for a shorter OS; global QoL, physical functioning and appetite loss had a borderline significance (P=0.0130 for global QoL; P=0.0256 for physical functioning: P=0.0149 for appetite loss). World Health Organization (WHO) performance status was significantly predictive for OS. In the multivariate analysis, more severe pain at baseline was predictive for a shorter OS. In contrast, baseline QoL had no prognostic value for the duration of TTP. QoL change scores from baseline QoL predicted neither OS nor TTP. Our findings suggest that while QoL measurements are important in evaluating patients' QoL, they have no great importance in predicting primary clinical endpoints such as TTP or OS in advanced breast cancer patients.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Copper-promoted overall transformation of 4-tert-butylphenol to its para-hydroxyquinonic derivative, 2-hydroxy-5-tert-butyl-1,4-benzoquinone. Biomimetic studies on the generation of topaquinone in copper amine oxidases.

Topaquinone (TPQ) is a cofactor present at the active site of copper amine oxidases, derived from a Tyr residue inserted in the polypeptide chain through a copper-dependent but otherwise largely unknown mechanism. A simple model system was developed that permits to obtain the overall transformation of 4-tert-butylphenol, chosen as a model for Tyr, into a TPQ-like, para-hydroxyquinonic structure in the presence of Cu(II)-imidazole mononuclear complexes.

Amine Oxidase (Copper-Containing)↗
PubMedDate not supplied

An approach to the assessment of overall uncertainty of determination of dose using an ESR technique.

An approach to the assessment of overall uncertainty of dose reconstruction by means of an ESR with additive dose technique is proposed. This approach takes account of uncertainties caused by different sources giving a quantitative measure of uncertainty of the determined dose. Dose is determined as an interval assessment rather than a deterministic value, allowing for the analytical estimation of both the mean value and the 95% confidence intervals. The effects of the number of additional irradiations and the value of dose increment on the uncertainty of dose determination are analyzed.

Dental Enamel↗
PubMed2000

Single centre experience on primary correction of common arterial trunk: overall survival and freedom from reoperation after more than 15 years.

OBJECTIVE: To identify predictors of early and late outcomes of common arterial trunk (CAT) after primary surgical correction, such as clinical state prior to surgery, age and weight at presentation, implications of truncal valve abnormalities and associated anomalies of this complex congenital malformation. METHODS: A consecutive series of 106 patients, operated between 1976 and 1998, were reviewed retrospectively. Using the 'Van Praagh' classification, 59 patients presented as A1, 33 as A2, six as A3, and eight as A4. The mean age at operation was 8.6 months (range, 14 days-8.9 years; median, 4.4 months), and the mean weight was 5.2 kg (range, 2.5-30.8 kg; median, 4.4 kg). At the time of operation, 32 patients had congestive heart failure, five were on ventilator support for less than 1 week, and 21 for more than 1 week. RESULTS: Between 1976 and 1989, the early mortality was 21%, and between 1990 and 1998, it dropped to 13%. After 1, 10 and 15 years, the overall survival was 66, 61 and 59%. The 1, 5, 10 and 15 year freedom from reoperation was 82, 60, 22 and 10%, respectively. Clinical condition prior to intervention, truncal valvar dysfunction, and coronary anomalies were significantly associated with poor surgical outcome, whereas weight and age at presentation in our series were not. CONCLUSIONS: In the current era of paediatric cardiac surgery, primary surgical repair of CAT can be carried out with reasonable early and late mortality. However, our data suggest that a high incidence of reoperation, mainly due to the outgrowth and failure of the conduit, has to be expected. The patient's clinical state after diagnosis is decisive for the timing of intervention.

Child↗
PubMed1997

Predictors of early and overall outcome in coronary artery bypass without cardiopulmonary bypass.

OBJECTIVE: Cardiopulmonary bypass in coronary artery bypass graft operations may adversely affect the outcome especially in high-risk patients. The purpose of this study is to evaluate results of coronary artery bypass performed without cardiopulmonary bypass, in a relatively high-risk cohort, and to identify predictors of unfavorable outcome. METHOD: Three hundred and thirteen (313) patients, 246 (79%) of whom had high-risk conditions, who have a coronary anatomy suitable for coronary artery bypass surgery without cardiopulmonary bypass, underwent this procedure between December 1991 and July 1995. Mean number of grafts/patient was 1.8 (1-5), and only 71 patients (23%) received a graft to the circumflex coronary system. RESULTS: Early unfavorable outcome events included operative mortality (12 patients, 3.8%), nonfatal perioperative myocardial infarction (eight patients, 2.6%), emergency reoperation (three patients, 0.9%), sternal infection (five patients, 1.6%), and nonfatal stroke (two patients, 0.6%). Multivariate analysis revealed angina pectoris class IV (odds ratio 5.4) and age > or = 70 years (odds ratio 5.0) as independent predictors of early mortality. Preoperative risk factors such as repeat coronary artery bypass grafting (50 patients, 16%), ejection fraction < or = 0.35 (85 patients, 27%), acute myocardial infarction (86 patients, 28%), cardiogenic shock (ten patients, 3.2%), chronic renal failure (25 patients, 8%), chronic obstructive pulmonary disease (20 patients, 6%), and peripheral vascular disease (51 patients, 16%) did not increase early mortality. During 33 months of follow-up (range 1-57 months), there were 42 deaths, at least 16 cardiac-related (one and four years actuarial survival of 90% and 76% respectively), and 39 patients (12.5%) in whom angina returned. Calcified aorta (odds ratio 2.6) and old myocardial infarction (odds ratio 1.8) were independent predictors of overall unfavorable events. CONCLUSIONS: Coronary artery bypass grafting without cardiopulmonary bypass can be performed with relatively low operative mortality in certain high-risk subgroups of patients; however, an increased risk of graft occlusion is a potential disadvantage. This procedure should therefore be considered only for patients with suitable coronary anatomy, in whom cardiopulmonary bypass poses a high risk. Although the risk of stroke is relatively low, the procedure is still hazardous for patients aged 70 years and over.

Adult↗
PubMedDate not supplied

Pilot test of a behavioral skill building intervention to improve overall diet quality.

OBJECTIVE: To determine the effect of a cognitive and behavioral skills building intervention delivered via a small group or correspondence on improvement in total diet quality. DESIGN: Randomized, controlled trial comparing 2 intervention groups with a usual care (UC) group. PARTICIPANTS: Generally healthy men (n = 35) and women (n = 63); mean age = 49.6 years (range = 29 to 71 years). INTERVENTION: 20-session behavioral and cognitive skills curriculum to train participants to improve personal dietary habits that were inconsistent with public health guidelines. One group (weekly meeting [WM]) met in small groups with 2 cofacilitators. A correspondent (CR) group received the curriculum via mail and an interactive study Web site. The UC group received a copy of a consumer nutrition book. MAIN OUTCOME MEASURE: Modified Healthy Eating Index (MHEI) score derived from 9 components of the US Department of Agriculture's Healthy Eating Index. RESULTS: The WM group significantly improved their MHEI score compared with the CR (P =.04) and UC (P =.002) groups. The CR group's improvement in MHEI score was not significantly different from that of the UC group (P =.19). CONCLUSIONS AND IMPLICATIONS: A behaviorally focused intervention can improve overall diet quality, especially if delivered through small-group meetings.

Adult↗
PubMed2002

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking.

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.ADP.P(i) states accumulate in the steady state [Lionne, C., et al. (1995) FEBS Lett. 364, 59]. We have now studied the kinetics of P(i) release with chemically cross-linked myofibrils that, when adequately cross-linked, appear to be a good model for isometric contraction. By using a method that is specific for free P(i) and rapid quench flow that measures the amount of (A)M.ADP.P(i) states and free P(i), we show that (A)M.ADP.P(i) states predominate which suggests that the overall ATPase is limited by P(i) release kinetics. Therefore, under our experimental conditions with myofibrils prevented from shortening, the concentration of (A)M.ADP states is low, as with rapidly shortening and relaxed myofibrils. This result is difficult to reconcile with the sensitivity of force development in fibers and myofibrils to P(i) which implies interaction of P(i) with an (A)M.ADP state. We discuss two models for accommodating the mechanical and chemical kinetics with reference to the duty cycle in skeletal muscle.

Adenosine Diphosphate↗
PubMed2003

The closely related estrogen-regulated trefoil proteins TFF1 and TFF3 have markedly different hydrodynamic properties, overall charge, and distribution of surface charge.

The human trefoil proteins TFF1 and TFF3 are expressed predominantly in the gastrointestinal tract. They are also expressed and regulated by estrogens in malignant breast epithelial cells. TFF1 and TFF3 are small cysteine-rich acidic secreted proteins of 60 and 59 amino acids with similar isoelectric points of 4.75 and 3.94, respectively. Each contains one trefoil domain that is characterized by several conserved features including six cysteine residues with conserved spacing. TFF1 and TFF3 form intermolecular disulfide bonds via an extra-trefoil domain cysteine residue and are present in vivo as monomers and homodimers and as complexes with other proteins. The TFF1 dimer is more active than the TFF1 monomer. In the present study the hydrodynamic and charge properties of TFF1 and TFF3 monomers and homodimers have been compared and shown to differ markedly. Notably, TFF1 is significantly more asymmetric than TFF3 (frictional coefficients 1.25 and 1.12, respectively, p < 0.001), and homodimerization of TFF1 results in a greater increase in asymmetry than for TFF3. The overall charges of TFF1 and TFF3 are very different at neutral pH. Titration curves predicted significant differences in charge across a wide pH range that agreed well with experimental data. The locations of charged amino acids in the primary sequences and in the tertiary structures of TFF1 and TFF3 were examined. This revealed interesting divergence in both the distribution and local topology of charged amino acid side chains. The significant differences between the shape, size, and surface charge of these two closely related molecules may account for their divergent biological activities.

Amino Acid Sequence↗
PubMed2006

The alkali molten globule state of ferrocytochrome c: extraordinary stability, persistent structure, and constrained overall dynamics.

This paper describes the structural and dynamic properties of a hitherto uncovered alkali molten globule (MG) state of horse "ferrocytochrome c" (ferrocyt c). Several experimental difficulties mainly because of heme autoxidation and extraordinary stability of ferrocyt c have been overcome by working with the carbonmonoxide-bound molecule under extremely basic condition (pH 13) in a strictly anaerobic atmosphere. Structural and molecular properties extracted from basic spectroscopic experiments suggest that cations drive the base-denatured CO-liganded protein to the MG state. The stability of this state is approximately 5.2 kcal mol(-)(1), and the guanidinium-induced unfolding transition is sharp (m(g) approximately 2.3 kcal mol(-)(1) M(-)(1)), suggesting contents of rigid tertiary structure. Strategic experiments involving the measurement of the CO association rate to the base-denatured protein and intrachain diffusion rates measured by laser photolysis of CO indicate a substantially restricted overall motion and stiffness of the polypeptide chain in the MG state. Possible placement of the state in the folding coordinate of ferrocyt c is discussed.

Alkalies↗
PubMed2006

Multiple steps determine the overall rate of the reduction of 5alpha-dihydrotestosterone catalyzed by human type 3 3alpha-hydroxysteroid dehydrogenase: implications for the elimination of androgens.

Human type 3 3alpha-hydroxysteroid dehydrogenase, or aldo-keto reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5alpha-dihydrotestosterone (DHT, potent androgen) to form 3alpha-androstanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The k(cat) for the NADPH-dependent reduction of DHT catalyzed by AKR1C2 is 0.033 s(-1). We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low k(cat) value. Stopped-flow experiments to measure the formation of the AKR1C2.NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on k(cat) (1.5) and a slightly larger effect on k(cat)/K(m) (2.1) were observed in the steady state. In the transient state, the maximum rate constant for the single turnover of DHT (k(trans)) was determined to be 0.11 s(-1) for the NADPH-dependent reaction, which was approximately 4-fold greater than the corresponding k(cat) x k(trans) was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent (D)k(trans) of 3.5. Thus, the effects of isotopic substitution on the hydride transfer step were masked by slow events that follow or precede the chemical transformation. Transient multiple-turnover reactions generated curvilinear reaction traces, consistent with the product formation and release occurring at comparable rates. Global fitting analysis of the transient kinetic data enabled the estimate of the rate constants for the three-step cofactor binding/release model and for the minimal ordered bi-bi turnover mechanism. Results were consistent with a kinetic mechanism in which a series of slow events, including the chemical step (0.12 s(-1)), the release of the steroid product (0.081 s(-1)), and the release of the cofactor product (0.21 s(-1)), combine to yield the overall observed low turnover number.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗
PubMed2006

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae.

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.

Binding Sites↗
PubMed1996

Chemical modification of recombinant HIV-1 capsid protein p24 leads to the release of a hidden epitope prior to changes of the overall folding of the protein.

It was found that the affinity of a monoclonal antibody directed against a recombinantly expressed HIV-1 capsid protein p24 (rp24) strongly increased after chemical modification of the Iysine residues of rp24 with different amounts of maleic anhydride. The extent and the sites of modification were analyzed by MALDI-TOF mass spectrometry. Unmodified rp24 and the differently modified rp24 samples were tested for binding the murine monoclonal antibody CB4-1 which recognizes the epitope GATPQDLNTML comprising residues 46-56 of rp24. An increase in the number of modified lysine residues led to enhanced binding affinity of CB4-1. Most pronounced effects were observed after substitution of the first amino groups: an average number of three modified residues per protein molecule increases the binding affinity by a factor of 23, but the substitution of the remaining nine residues increases the binding affinity only by a factor of 11. Fully modified rp24 variant proteins were bound by CB4-1 with Kd values comparable to that of the peptide epitope. Conformation and stability of the unmodified rp24, highly (rp24F, 9 residues; rp24G, 11 residues) modified, and fully modified protein (rp24I, 11 lysine residues and N-terminus) were analyzed by circular dichroism (CD) and fluorescence spectroscopy under different solvent conditions. Little difference in conformation and unfolding behavior was observed between the unmodified and highly modified rp24, which differ drastically in the antibody binding behavior. The fully modified sample, however, displayed a significant decrease in alpha-helical content. Thus, the epitope seems to be hidden (cryptotope) in the unmodified rp24 in a low-affinity binding conformation and becomes displayed at low levels of chemical modification which obviously induce subtle structural changes prior to changes of the overall folding observable by spectroscopic means.

Amino Acid Sequence↗
PubMed1996

Alterations of rings B and C of colchicine are cumulative in overall binding to tubulin but modify each kinetic step.

The role of the elimination of ring B and/or the modification of ring C of colchicine in tubulin binding kinetics and thermodynamics has been characterized, using four different molecules. These ligands are colchicine (COL); 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC), in which the central ring B has been reduced to one bond; allocolchicine (ALLO), in which ring C has been replaced by a six-membered ring; and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), where the same two modifications are made simultaneously. This paper describes the kinetics of association of ALLO with tubulin. The binding is accompanied by a fluorescence increase with slow biphasic kinetics, indicating binding to fast and slow tubulin isotypes. Binding to each of these isotypes occurs in two steps: a fast initial binding followed by a slower isomerization step. The K1 and k2 values for ALLO at 25 degrees C are 14,000 +/- 2,000 and 25,000 +/- 6,000 M-1 (fast and slow isotypes) and 0.055 +/- 0.003 s-1 and 0.013 +/- 0.001 s-1 (fast and slow isotype), respectively. For ALLO the reaction standard enthalpy change of the initial binding is 68 +/- 5 kJ.mol-1 (fast isotype) and 45 +/- 33 kJ.mol-1 (slow isotype) and the activation energy for the second forward step is 58 +/- 14 kJ.mol-1 (fast isotype) and 81 +/- 17 kJ.mol-1 (slow isotype). Displacement kinetics of bound ALLO by podophyllotoxin was monoexponential. The activation energy for the isomerization in the off direction is 107 +/- 7 kJ.mol-1. Comparison of the thermodynamic parameters for all four compounds shows that the modifications of both rings are cumulative with respect to overall binding. For the intermediate state there is a mutual influence of both modifications, suggesting an alteration of the reaction pathway.

Biphenyl Compounds↗