Diagnostic improvement of prostate cancer using an extensive biopsy protocol.
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Journal Article↗
Biomedical subjects
E Blanc
Publications and source records attributed to E Blanc.
Real-space molecular-dynamics structure refinement.
Real-space targets and molecular-dynamics search protocols have been combined to improve the convergence of macromolecular atomic refinement. This was accomplished by providing a local real-space target function for the molecular-dynamics program X-PLOR. With poor isomorphous replacement experimental phases, molecular dynamics does not improve real-space refinement. However, with high-quality anomalous diffraction phases convergence is improved at the start of refinement, and torsion-angle real-space molecular dynamics performs better than other available least-squares or maximum-likelihood methods in real or reciprocal space. It is shown that the improvements result from an optimization method that can escape local minima and from a reduction of overfitting through the implicit use of phases and through use of a local refinement in which errors in remote parts of the structure cannot be mutually compensating.
Hydroxymethylglutaryl CoA Reductases↗
Critical initial real-space refinement in the structure determination of arginine kinase.
Arginine kinase (AK), a homologue of creatine kinase, catalyses the reversible transfer of a phosphoryl group between a guanidino phosphate and ADP. The family of phosphagen kinases eluded structure determination for over 25 years until an inactive form creatine kinase (CK) structure was determined [Fritz-Wolf et al. (1996). Nature (London), 381, 341-345]. The structure determination of the active-form transition-state complex was non-trivial, owing to the distant relatedness and domain reorientation of AK compared with CK. Phases from a molecular-replacement solution of the large domain, supplemented by single isomorphous replacement and inter-crystal averaging, did not reveal interpretable electron density for the small domain. Reciprocal-space refinement of the initial model (Rfree = 0.54) by any of the commonly used methods, including post facto application of maximum-likelihood methods, led to overfitting without significant improvement of the partial initial model. By contrast, in the local real-space refinements which proved successful, the interdependence of atoms is limited to immediate neighbors, and atomic positions are not influenced by errors or omissions in remote parts of the structure. Modest improvement was possible without overfitting, and this was critical to the calculation of improved phases. Phases were refined and extended from 4.0 to 2.5 A resolution by Fourier inversion of omit maps, combination with isomorphous replacement phases and averaging between crystal forms, after several batches of real- and reciprocal-space atomic refinement. The final structure refinement, against a 1.86 A cryo data set yielded a high-quality model with R = 0.196 and Rfree = 0.224.
Arginine Kinase↗
Diagnostic value of ten systematic TRUS-guided prostate biopsies.
OBJECTIVE: To evaluate the improvement in the rate of detection of prostate cancer using an extensive protocol involving ten transrectal biopsies. METHODS: A total of 162 patients submitted to transrectal ultrasound-guided biopsy for elevated prostate-specific antigen (PSA) and/or abnormality on digital rectal examination were studied consecutively and prospectively. Five biopsies were performed in each lobe: between the three standard biopsies on each side, two additional biopsy specimens were taken in the same plane and at the same 45 degrees angle. RESULTS: The complication rate with the ten-biopsy protocol was 1.85%. Prostate cancer was detected in 40.1% of the patients. In the overall series, the percentage of diagnostic improvement brought about by this ten-biopsy protocol was +3.1%. The percentage improvement was greatest (+4.9%) in patients with PSA </=10 ng/ml. CONCLUSION: Increasing the number of biopsy cores without altering the angle of biopsy and/or the zone sampled does not lead to a significant improvement in the detection of prostate cancer.
Adult↗
[Value of the use of a questionnaire in the evaluation of incontinence surgery].
OBJECTIVE: To assess the contribution of a questionnaire in the evaluation of patients operated for urinary stress incontinence. PATIENTS AND METHODS: From 1988 to 1996, 42 patients underwent bladder neck suspension for urinary stress incontinence. Only 7 patients had no history of pelvic surgery, 24 patients presented pure urinary stress incontinence and 19 presented mixed incontinence, 37 patients were evaluable with a mean follow-up of 2 years. The evaluation consisted of two aspects: clinical assessment, based on review of the case files, showing that 77% of patients were continent, and a questionnaire (non-validated translation of "Q7" and "UD16"). This questionnaire evaluated quality of life (7 items) and symptoms. Each item was scored from 0 to 3. RESULTS: To interpret the results we divided the three main scores into three intervals, the first third corresponding to good results. The percentage of good results for the overall score, quality of life score and symptom score was 62%, 73% and 46% respectively. The most discriminative questions of the questionnaire can be used to analyse urine leaks related to effort, urgency episodes of incontinence. A score of 0 or 1 was considered to be a good result. The percentage of good results for these three questions was therefore 72%, 60% and 64%, respectively. CONCLUSION: The cure rate therefore varies as a function of the criteria selected: the questionnaire allows the urologist to more accurately assess the patient's postoperative state.
Adult↗
Solution structure of two new toxins from the venom of the Chinese scorpion Buthus martensi Karsch blockers of potassium channels.
The solution structure of BmTX2 purified from the venom of the Chinese Buthid Buthus martensi has been determined by 2D NMR spectroscopy techniques which led to the description of its 3D conformation. The structure consists of a triple-stranded beta-sheet connected to a helical structure. This helix encompasses 10 residues, from 11 to 20, begins with a turn of 310 helix, and ends with an alpha helix. The three strands of beta sheet comprise residues 2-6, with a bulge covering residues 4 and 5, 26-29, and 32-35, with a type I' beta turn centered on residues 30-31. We also characterized the solution structure of BmTX1. The two toxins which are potent blockers of both large-conductance calcium-activated potassium channels (BKCa channels) and voltage-gated potassium channels (Kv1. 3) are highly superimposable and possess the same structural characteristics. Analysis of these structures allows us to hypothesize that, besides the main surface of interaction described by the functional map of charybdotoxin, one can expect that the binding of scorpion toxins on BKCa channels may involve residues on the edge of this surface.
Amino Acid Sequence↗
Transition state structure of arginine kinase: implications for catalysis of bimolecular reactions.
Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with fluctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-A resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transition-state analog complex studied here, a nitrate mimics the planar gamma-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.
Animals↗
Determination of the relative precision of atoms in a macromolecular structure.
Several real-space indices and temperature factors are compared with respect to their correlation with atomic positional error and their ability to indicate atoms and residues with the worst of subtle errors. The best index, rED, is a correlation coefficient between model and map electron densities, similar to one proposed earlier, but incorporating two improvements. Firstly, resolution is accounted for explicitly by calculating the model electron density by Fourier transformation of resolution-truncated scattering factors. Secondly, the deviation between model and map electron densities is assigned to neighboring atoms according to their contribution to the electron density of each grid point. With maps of various qualities, rED is the single index with best correlation to atomic error with grouped or individual atoms, and it is the most reliable indicator of poor residues. With poorer omit maps, imprecision of individual atoms is best diagnosed by a combination of low rED or high B factor. With the improved methods, 60-70% of the least precise atoms can detected in a fully refined structure. Similarly, 40-80% of the least precise atoms of an unrefined model can be detected by comparison with an isomorphous replacement map. This is useful in assessing and improving the quality of a model, but not sufficient to confidently validate all atoms of a structure at sub-atomic resolution.
Electrons↗
[Preliminary results of the treatment of 44 patients with localized cancer of the prostate using transrectal focused ultrasound].
OBJECTIVE: To evaluate the efficacy and morbidity of treatment of localized prostatic cancer using transrectal high-intensity focused ultrasound. MATERIAL AND METHODS: 44 patients (mean age: 72 years) with clinical stage T1 (20) or T2 (24) prostatic cancer, not eligible for radical prostatectomy, were treated by two different prototypes of the Ablatherm (Technomed Medical System). The second prototype includes several safety devices designed to reduce morbidity. 99 sessions were performed, i.e. an average of 2.25 sessions per patient: the prostate was treated in one session (5 patients), 2 sessions (26 patients), 3 sessions (10 patients), or 4 sessions (3 patients), usually under spinal anaesthesia. The mean PSA was 9.92 ng/ml and the mean prostatic weight was 37 g. RESULTS: Complications occurred in 10 (50%) of the first 20 patients treated (1993-1995): 2 recto-urethral fistulas, 2 asymptomatic rectal burns, 2 cases of stable urinary retention, 1 case of severe incontinence, 3 cases of bladder neck sclerosis, and 1 febrile urinary tract infection. In the 24 patients treated subsequently (1995-1997), complications occurred in only 4 patients (16%): 1 case of stable urinary retention, 1 febrile urinary tract infection, 1 case of bladder neck sclerosis, 1 case of stress incontinence. A complete response (repeated negative follow-up biopsies) was obtained in 27 patients (61%). The mean post-treatment PSA level in patients of this group was 1.09 +/- 1.06 and remained stable (mean follow-up: 346 days; range: 60-1250). A residual cancer was detected in 17 patients (39%). 8 patients were considered to be failures (mean post-treatment PSA: 4.8 ng/ml) and received adjuvant treatment (hormonal: 3; external radiotherapy: 5). Complementary treatment by focused ultrasound will be performed in another 9 patients, if their PSA rises above 3 ng/ml (patients of this group are symptomatic with a mean post-treatment PSA of 1.6 ng/ml). CONCLUSION: The morbidity of treatment by transrectal focused ultrasound has now been reduced. These results suggest that this new treatment can constitute a useful option for certain patients with localized cancer, not suitable for radical prostatectomy.
Adenocarcinoma↗
[Diagnostic value of 10 systematized and ultrasound-guided transrectal prostatic biopsies].
OBJECTIVE: To study the improvement of prostatic cancer detection provided by ten strictly systematized transrectal prostatic biopsies. MATERIAL AND METHODS: This prospective study was conducted in 162 patients submitted to a series of 10 ultrasound-guided transrectal prostatic biopsies due to the presence of elevated PSA and/or an abnormality on digital rectal examination. Five biopsies were taken from different sites in each lobe: 2 biopsies were inserted between the 3 biopsies usually performed in the standard protocol, while maintaining the angle of entry of the needle recommended in this protocol. RESULTS: The complication rate was 1.85% with the 10-biopsy technique. Prostatic cancer was detected in 40.1% of the 162 patients. The percentage diagnostic improvement provided by the 10-biopsy protocol in the overall patient population was +3.1% compared to the standard protocol. The highest percentage diagnostic improvement was observed in the group of patients with PSA < or = 10 ng/ml (+4.9%) and in the group of patients with an ultrasound prostatic volume < or = 40 cc (+4%). CONCLUSION: The most marked diagnostic improvement appears to be related more to sampling of prostatic zones presenting a statistically increased risk of cancer (peripheral zone) than to the increased number of prostatic biopsies performed.
Adenocarcinoma↗
1H-NMR-derived secondary structure and overall fold of a natural anatoxin from the scorpion Androctonus australis hector.
The venom of the scorpion Androctonus australis hector contains several protein neurotoxins of which structure and structure/activity relationships have been extensively studied. It also contains polypeptides such as Aah STR1, which are not toxic, while having highly similar sequences to fully active toxins. We have determined the solution structure of Aah STR1 by use of conventional two-dimensional NMR techniques followed by distance-geometry and energy minimization. We have demonstrated that, despite its lack of toxicity, Aah STR1 is structurally highly related to anti-mammal scorpion toxins specific for Na+ channels. The calculated structure is composed of a short alpha-helix (residues 26-33) connected by a tight turn to a three-stranded antiparallel beta-sheet (sequences 3-6, 38-41 and 44-48). This beta-sheet is right-handed twisted as usual for such secondary structures. The beta-turn connecting the strands 38-41 and 44-48 belongs to type II'. The overall fold of Aah STR1 is typical of beta-type scorpion toxins. This is, however, the first example of such a fold in Old World scorpion toxins. Either the absence of a basic residue in position 63 or the high mobility of loops, compared to active beta-type neurotoxins, may explain the lack of activity of this protein.
Amino Acid Sequence↗
Potential use of real-space refinement in protein structure determination.
Through testing refinement protocols using free R-factor estimates of model quality, it is shown that real-space refinement can be a useful addition to conventional reciprocal-space refinement, even for protein structures with poor electron-density maps derived from multiple isomorphous replacement. By alternating real- and reciprocal-space refinements, starting with an experimental map, then calculating 2F(o) - F(c) maps, it is demonstrated with the structure of HMG-CoA reductase, that quick automatic refinement can yield a model with a free R factor 1.5% better than exhaustive reciprocal-space refinement, and within 1% of a model that was interactively rebuilt and refined repeatedly.
Journal Article↗
Solution structure of TsKapa, a charybdotoxin-like scorpion toxin from Tityus serrulatus with high affinity for apamin-sensitive Ca(2+)-activated K+ channels.
TsKapa (TsK), purified from the Buthidae Tityus serrulatus is a very high potent ligand for small-conductance apamin-sensitive calcium-activated potassium channels (SK). It is able to efficiently compete with apamin for binding on this channel (K0.5 = 0.3 nM) [Legros, C. et al., FEBS Lett. 390:81-84, 1996]. The solution structure of TsK has been determined by 2D-NMR techniques, which led to the full description of its 3D conformation: a short alpha helix from residues 14 to 20 and a three-stranded antiparallel beta sheet (residues 2-3, 27-29, and 32-34). The interaction of TsK with the SK potassium channel has been modeled according to the charge anisotropy of the ligand. The resulting dipole moment orientates TsK so that it presents toward the receptor, a surface, mainly basic, encompassing residues K18 and K19 on one side and R9 and Y8 on the other. Despite its three-dimensional structure that is related with scorpion toxins active on voltage-gated potassium channels such as charybdotoxin, the pharmacological activity and specificity of TsK is related with shorter scorpion toxins (i.e., possessing an only two-stranded beta sheet) such as scyllatoxin (also named leiurotoxin I) or P05.
Amino Acid Sequence↗
4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.
Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4-hydroxynonenal potentiates oxidative cascades. Collectively, these findings suggest that 4-hydroxynonenal plays important roles in oxidative impairment of synaptic functions that would be expected to promote excitotoxic cascades.
ATP-Binding Cassette Transporters↗
Differential involvement of disulfide bridges on the folding of a scorpion toxin.
Leiurotoxin I is a neurotoxin, blocker of Ca(2+)-activated apamin-sensitive K+ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys3-Cys21, Cys8-Cys26 and Cys12-Cys28. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were placed by alpha-aminobutyrate residues to suppress one disulfide bridge, were analyzed by 1H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys3-Cys21 and a loss of organized structure for another analog lacking disulfide bridge Cys12-Cys28. These analogs are, respectively, fully active and weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggest that disulfide bridge Cys12-Cys28 is essential for the folding process. In contrast, the lack of disulfide bridge Cys3-Cys21 does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.
Amino Acid Sequence↗
Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltage-gated potassium channels.
Maurotoxin (MTX), purified from the scorpionid Scorpio maurus is a potent ligand for potassium channels. It shows a broad specificity as being active on Kv1.1 (Kd = 37 nM), Kv1.2 (Kd = 0.8 nM), Kv1.3 (Kd = 150 nM) voltage-gated potassium channels, as well as on small-conductance calcium-activated potassium channels. It has a unique disulfide pairing among the scorpion toxins family. The solution structure of MTX has been determined by 2D-NMR techniques, which led to the full description of its 3D conformation: a bended helix from residues 6 to 16 connected by a loop to a two-stranded antiparallel beta sheet (residues 23 to 26 and 28 to 31). The interaction of MTX with the pore region of the Kv1.2 potassium channel has been modeled according to their charge anisotropy. The structure of MTX is similar to other short scorpion toxins despite its peculiar disulfide pairing. Its interaction with the Kv1.2 channel involves a dipole moment, which guides and orients the toxin onto the pore, toward the binding site, and which thus is responsible for the specificity.
Amino Acid Sequence↗
Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel.
The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an alpha-helix stretch (residues 5-12). This alpha-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an alpha-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional 1H nuclear magnetic resonance and show that 1) the proline residue does not disturb the alpha-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an alpha-helix running from residues 5 to 12, two antiparallel stretches of beta-sheet (residues 15-20 and 23-27) connected by a type I' beta-turn, and three disulfide bridges connecting the alpha-helix to the beta-sheet.
Amino Acid Sequence↗
Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown.
The dependence on Ca2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca2+ concentrations were buffered by bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca2+ buffering on intracellular and extracellular Ca2+ concentration ([Ca2+]i and [Ca2+]e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca2+]i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca2+]i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca2+ ions with BAPTA produced a reduction of both [Ca2+]e and [Ca2+]i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 microM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca2+]i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca2+]e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca(2+)-insensitive way.
Animals↗