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Biomedical subjects

M Royo

Publications and source records attributed to M Royo.

At least 19 recordsLinked to original sources
PubMed2002

Changes in molecular forms of prostate-specific antigen during treatment with finasteride.

OBJECTIVE: To study the influence of finasteride treatment on the molecular forms of prostate-specific antigen (PSA) in patients with benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: Total PSA, free PSA and PSA complexed to alpha1-antichymotrypsin (PSA-alpha1ACT) were measured in plasma and serum from 40 men with BPH and a total PSA of < 20 ng/mL, using in-house and commercial immunoassays, before and during treatment with finasteride (30 men) or placebo (10 men). RESULTS: The baseline values were not significantly different between the groups, with mean (sd) total plasma PSA levels of 3.6 (4.3) and 4.8 (5.9) ng/mL in the finasteride and placebo groups, respectively. Finasteride, but not placebo, induced a significant reduction in total PSA, free PSA and PSA-alpha1ACT levels in plasma and serum (P < 0.001). However, complexed-to-total (c/t) and free-to-total (f/t) PSA ratios remained constant in both groups, both in plasma and serum, during the follow-up. CONCLUSION: The decrease in total PSA after finasteride treatment results from a proportional reduction in its two major molecular forms, free PSA and PSA-alpha1ACT, which explains why the c/t and f/tPSA ratios do not change significantly despite treatment. This suggests that routine analysis of molecular forms of PSA could improve the utility of the change in total PSA associated with finasteride for the early diagnosis of prostate cancer. It also suggests that any subsequent change in both ratios, particularly an increase in c/tPSA or a decrease in f/tPSA ratio, could be considered an early sign of neoplastic degeneration rather than a therapeutic consequence.

Aged↗
PubMed2001

Synthesis and structure determination of kahalalide F (1,2).

Kahalalide F, the only member of the family of peptides called kahalalides, isolated from the sacoglossan mollusc Elysia rufescens and the green alga Bryopsis sp., with important bioactivity, is in clinical trials for treatment of prostate cancer. An efficient solid-phase synthetic approach is reported. Kahalalide F presents several synthetic difficulties: (i) an ester bond between two beta-branched and sterically hindered amino acids; (ii) a didehydroamino acid; and (iii) a rather hydrophobic sequence with two fragments containing several beta-branched amino acids in a row, one of them terminated with a saturated aliphatic acid. The cornerstones of our strategy were (i) a quasiorthogonal protecting system with allyl, tert-butyl, fluorenyl, and trityl-based groups, (ii) azabenzotriazole coupling reagents, (iii) formation of the didehydroamino acid residue on the solid phase, and (iv) cyclization and final purification in solution. HPLC, high-field NMR, and biological activity studies showed that the correct stereochemistry of the natural product is that proposed by Rinehart et al. whereas the stereochemistry proposed by Scheuer et al. is that of a biologically less active diastereoisomer.

Amino Acid Sequence↗
PubMed2001

[PSA:alpha 1-antichymotrypsin ratio/total PSA in the diagnosis of prostatic cancer in the range of total PSA from 4 to 10 ng/ml].

OBJECTIVE: To evaluate the usefulness of the ratio PSA:alpha-1-antichymotrypsin/total PSA (C/T) in the diagnosis of prostate cancer in the range of total PSA between 4 and 10 ng/mL. MATERIAL AND METHODS: By using home-made ELISAs we have determined plasmatic concentrations of total PSA and complex PSA:alpha-1-ACT in 300 patients with total PSA between 4-10 ng/mL. All samples were obtained before any manipulation that could interfere the PSA levels. RESULTS: By prostatic biopsy 85 patients (28.3%) were diagnosed of prostate cancer (CaP) and 215 (71.6%) of benign prostatic hyperplasia (BPH). The mean values of the complex PSA:alpha-1-ACT (4.2 ng/mL in the BPH patients vs 5.0 ng/mL in the CaP patients) and of the C/T ratio (0.70 vs 0.82, respectively) showed significant differences between both groups (p = < 0.0001). The total PSA did not show differences (6.1 ng/mL vs 6.0 ng/mL; p = 0.79). From all three parameter evaluated, the ratio C/T had the biggest area under the ROC (0.884) and statistically significant differences in comparison with total PSA (0.490; p = < 0.001) and the complex PSA:alpha-1-ACT (0.696: p = < 0.001). Therefore, by using a ratio C/T > 0.62 to decide the performance of a biopsy, 27% of the patients with BPH could have avoided this procedure with a 100% sensitivity. Increasing the ratio to 0.68 the specificity is 47% and the sensitivity is 95.2%. Rectal examination did not have influence on the cut-off, sensitivity, specificity and area under the ROC of the ratio C/T. CONCLUSIONS: Our results confirm that the ratio C/T improve the diagnostic capacity of the total PSA between 4-10 ng/ml. Moreover, the rectal examination does not influence the selection of ratio C/T cut-off suggestives of CaP neither the diagnostic efficacy.

Aged↗
PubMed2001

[Factors affecting the PSA:alpha 1 antichymotrypsin/total PSA ratio].

OBJECTIVES: To evaluate the influence of several factors, including age, prostate volume, total PSA (PSA-T), clinical stage and Gleason on the PSA:alpha 1ACT/PSA-T (C/T) ratio. MATERIAL AND METHODS: Using in-house assays, we measured plasma levels of PSA-T and PSA:alpha 1ACT complex in 622 patients with benign prostate hyperplasia (BPH) (455 with hystological confirmation and 167 with clinical evidence of absence of malignance) and in 255 patients with prostate cancer (CaP), and determined the correlation between different parameters. RESULTS: In BPH patients, PSA-T and PSA:alpha 1ACT significantly increased with age. There was a positive correlation between age and PSA-T (r = 0.161, p < 0.0001) and PSA:alpha 1ACT (r = 0.141, p = 0.001). In contrast, the C/T ratio remained constant and below 70% in all decades. Similar results were obtained in CaP patients. In BPH patients, there was a positive correlation between prostate volume and PSA-T and PSA:alpha 1ACT, but not with the C/T ratio. In CaP patients, however, there was a negative correlation between prostate volume and the C/T ratio. An excellent correlation was found between PSA-T and PSA:alpha 1ACT, and a good correlation between PSA-T and the C/T ratio and between PSA:alpha 1ACT and C/T ratio. A multiple regression analysis showed that, in HBP and CaP patients, PSA-T and PSA:alpha 1ACT complex were the only parameters that significantly and independently influenced the C/T ratio. CONCLUSIONS: The C/T ratio is independent of age, prostate volume, Gleason and clinical stage. Therefore, these factors need not to be considered when using the C/T ratio.

Adult↗
PubMed2000

Substituted guanidines: introducing diversity in combinatorial chemistry.

The guanidine moiety is an important motif present in many biologically active compounds. Fully substituted guanidines are of key importance for the development of bioactive molecules. The present paper reports on an efficient procedure for the direct solid-phase conversion of amines to fully substituted guanidines under very mild conditions.

Chromatography, High Pressure Liquid↗
PubMed2000

Prostate-specific antigen complexed to alpha(1)-antichymotrypsin in the early detection of prostate cancer.

OBJECTIVES: To study the usefulness of the complexed-to-total (C:T) prostate-specific antigen (PSA) ratio in the early detection of prostate cancer in patients with a total PSA value <4.0 ng/ml. PATIENTS AND METHODS: Total PSA and PSA complexed to alpha(1)-antichymotrypsin were measured in plasma from 193 men with benign prostatic hyperplasia (BPH) and 34 with prostate cancer. The diagnosis was confirmed in 28 BPH and 16 prostate cancer patients by biopsy and in 165 BPH and 18 prostate cancer patients by histological study following transurethral prostatectomy or open prostatectomy. RESULTS: The area under the receiver operating characteristic (ROC) curve was significantly greater for the C:T PSA ratio (0.908) than for total PSA (0.692) (p<0.001). Using a cut-off point of 0.83 for the C:T PSA ratio and regardless of the digital rectal examination (DRE) finding, 20 of the 34 prostate cancer patients would have been given a correct diagnosis (59% sensitivity) and in only 8 of the 193 BPH patients would a biopsy have been necessary (96% specificity). With a cut-off of 0.79, the sensitivity increased to 85% with a specificity of 92%. When the analysis was restricted to the 44 patients with abnormal DRE, the area under the ROC curve for the C:T PSA ratio was 0.919, and a cut-off point of 0. 78 gave a sensitivity of 87% and a specificity of 93%. Using a cut-off of 0.63, all prostate cancers were detected (100% sensitivity) and 54% of the negative biopsies would have been eliminated. For the 183 patients diagnosed following surgery, a cut-off of 0.82 gave a sensitivity of 72% and a specificity of 94%. CONCLUSION: Our results show that the C:T PSA ratio significantly improves the clinical utility of the PSA assay for detecting prostate cancer in patients with total PSA < 4 ng/ml, increasing the sensitivity without significantly increasing the number of biopsies.

Aged↗
PubMed1999

Inhibition of human sperm-zona-free hamster oocyte binding and penetration by protein C inhibitor.

Protein C inhibitor is a heparin-dependent serine protease inhibitor present in plasma at about 0.08 mumol l-1. Protein C inhibitor inhibits activated protein C and other coagulation factors. Previously, we described the presence of high protein C inhibitor levels in human semen (3.1 mumol l-1) and showed potential roles of the inhibitor in human reproduction. Here, we show that protein C inhibitor is present in an active form in follicular fluid at about 0.1 mumol l-1 and that purified, functionally active human plasma-derived and inactive, semen-derived protein C inhibitor and a synthetic peptide derived from its sequence inhibited both binding and penetration of zona-free hamster oocytes by human sperm. The binding inhibition by protein C inhibitor was dose dependent, with 50% inhibition at 0.037 mumol l-1 inhibitor (45 +/- 17 sperm per egg versus 90 +/- 23 in control experiments). The inhibitor also blocked in a dose-dependent manner the penetration of zona-free hamster eggs by human sperm (20 +/- 7% fertilized eggs at 0.1 mumol l-1 protein C inhibitor versus 55 +/- 10% in control experiments). Polyclonal antiprotein C inhibitor or antipeptide antibodies partially abolished the effect of protein C inhibitor and peptide on the inhibition of the binding and penetration of zona-free hamster oocytes by human sperm. The effect of the protein C inhibitor was not dependent on its antiprotease activity since purified semen-derived protein C inhibitor which did not have antiprotease activity gave comparable results. We conclude that protein C inhibitor may be involved in human reproduction at several steps, including the fertilization process.

Amino Acid Sequence↗
PubMed1999

Factor V Leiden and antibodies against phospholipids and protein S in a young woman with recurrent thromboses and abortion.

We describe the case of a 39-year-old woman who suffered two iliofemoral venous thromboses, a cerebral ischemic infarct and recurrent fetal loss. Initial studies showed high levels of antiphospholipid antibodies (APAs) and a moderate thrombocytopenia. After her second miscarriage, laboratory diagnosis revealed that the woman was heterozygous for the factor V Leiden mutation and had a functional protein S deficiency as well as anti-protein S and anti-beta 2-glycoprotein I antibodies. The impairment of the protein C pathway at various points could well explain the recurrent thromboses in the patient and supports the role of a disturbed protein C system in the pathophysiology of thrombosis in patients with APAs.

Abortion, Habitual↗
PubMed1998

Addressing the tertiary structure of human parathyroid hormone-(1-34).

Parathyroid hormone (PTH) regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renal tubular cells and is fully functional as the N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped" conformation with N- and C-terminal helices brought in close proximity by a turn has been postulated. The general acceptance of this hypothesis, despite limited experimental evidence, has altered the direction of the design of PTH-analogs. Examining the structure of human PTH-(1-34) under conditions that encompass the different environments the hormone may experience in the approach to and interaction with the G-protein-coupled receptor (including benign aqueous and saline solutions and in the presence of dodecylphosphocholine), we observe no evidence for a U-shape conformation or any tertiary structure. Instead, the N- and C-terminal helical domains, which vary in length and stability depending on the conditions, are separated by a highly flexible region of undefined conformation. These observations are in complete accord with recent conformational studies of PTH-related protein analogs containing lactams (Mierke, D. F., Maretto, S., Schievano, E. , DeLuca, D., Bisello, A., Mammi, S., Rosenblatt, M., Peggion, E., and Chorev, M. (1997) Biochemistry 36, 10372-10383) or a model amphiphilic alpha-helix (Pellegrini, M., Bisello, A., Rosenblatt, M., Chorev, M., and Mierke, D. F. (1997) J. Med. Chem. 40, 3025-3031). Reliable structural data from different environmental conditions are absolutely requisite for the next step in the design of non-peptide PTH analogs.

Circular Dichroism↗
PubMed1998

Longitudinal evaluation of the complexed-to-total prostate specific antigen ratio in men with prostate disease. Effect of treatment.

The longitudinal changes in the complexed-to-total prostate specific antigen (PSA) ratio were evaluated in 90 men with benign prostatic hyperplasia (BPH) and 50 men with prostate cancer. The influence of treatment on this ratio was studied in 45 BPH patients and 50 patients with prostate cancer. Using a cut-off of 0.80 for the complexed-to-total PSA ratio, the large majority of prostate cancer patients had a ratio above the cut-off before treatment in serial determinations, whereas most BPH patients had a ratio consistently below that value. However, the few prostate cancer patients who had a ratio < or = 0.80 showed this low ratio in serial determinations, as did BPH patients who had a ratio > or = 0.80. During treatment, the ratio significantly decreased in 43 of the 50 patients with prostate cancer in parallel with the decrease in total PSA, and 34 of the 41 patients that had a pretreatment ratio > 0.80 showed a ratio < or = 0.80 during hormonal therapy. Our results show that neither the physiological changes in total and complexed PSA nor the treatment of BPH patients change the diagnostic efficacy of the complexed-to-total PSA ratio, whereas in prostate cancer patients under hormonal therapy, the ratio decreased in parallel with the decrease in total PSA. This suggests that, apart from improving the diagnostic efficacy of total PSA, the complexed-to-total PSA ratio could also be used to monitor BPH patients for newly developed tumours or to monitor therapy in patients with prostate cancer.

Antineoplastic Agents, Hormonal↗
PubMed1998

Free and complexed prostate specific antigen in the differentiation of benign prostatic hyperplasia and prostate cancer: studies in serum and plasma samples.

PURPOSE: We prospectively evaluated serum and plasma concentrations of total and free prostate specific antigen (PSA), and PSA complexed to alpha1-antichymotrypsin in 170 patients who underwent biopsy, including 59 with prostate cancer and 111 with benign prostatic hyperplasia. We compared the usefulness of the ratios of free-to-total and complexed-to-total PSA for distinguishing between prostate cancer and benign prostatic hyperplasia, and studied the influence of blood clotting on the ratios. MATERIALS AND METHODS: Blood samples were processed to generate serum and citrated plasma. To calculate complexed-to-total and free-to-total PSA we assayed plasma and serum samples for total and complexed PSA using homemade immunoassays, and total and free PSA using the Immulite assay. The 2 total PSA assays were compared using the Tandem-E PSA assay. Receiver operating characteristics curves were constructed for the total population, and for 2 to 20, 4 to 20, 2 to 10 and 4 to 10 ng./ml. total PSA. RESULTS: In all groups complexed-to-total PSA had higher specificity than free-to-total and total PSA, especially at 90 to 100% sensitivity. Generally citrated plasma samples provided higher specificity than serum samples for all sensitivity values. The best performance for complexed-to-total and free-to-total PSA was obtained in the subset of patients in whom total PSA was 2 to 10 ng./ml. CONCLUSIONS: Our results indicate that the complexed-to-total PSA ratio performed better for classifying disease status than the free-to-total PSA ratio in the whole patient population and in the diagnostic gray zone of 2 to 10 ng./ml. In addition, plasma samples should be used to calculate the complexed-to-total and free-to-total PSA ratio.

Aged↗
PubMed1998

New insights into interactions between the human PTH/PTHrP receptor and agonist/antagonist binding.

We prepared a polyclonal antiserum [Ab-(88-97)] against residues 8-97 of the NH2-terminal tail of the human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor. Ab-(88-97) bound specifically to the receptor, as assessed by fluorescence-activated cell sorter analysis of HEK C21 cells, which stably express approximately 400,000 hPTH/PTHrP receptors per cell. Unlike PTH, Ab-(88-97) binding did not elicit either adenosine 3',5'-cyclic monophosphate or intracellular calcium concentration signaling responses in these cells. Incubation of C21 cells for 90 min at 4 degrees C with hPTH-(1-34) plus antiserum reduced the Ab-(88-97) binding to the cells by up to 40-50% of control values in a PTH concentration-dependent fashion with a half-maximal effective concentration of approximately 5 nM. The decrease in Ab-(88-97) binding caused by hPTH-(1-34) was completely reversed by coincubation with hPTHrP-(7-34). We conclude that residues 88-97 of the hPTH/PTHrPR are involved, either directly or indirectly, in agonist but not antagonist binding to the receptor.

Animals↗
PubMed1997

Conformational consequences of i, i + 3 cystine linkages: nucleation for alpha-helicity?

Methods to introduce specific secondary structural elements into peptides and proteins are vital for the rational design of peptide and non-peptide drug candidates as well as in the de novo design of proteins. Here the incorporation of a disulfide linkage between cysteine residues spaced three amino acids apart (i, i + 3) as a method to induce helicity is examined. Two dodecamer peptides, A-V-S-E-C-Q-L-C-H-D-K-G-NH2, differing in the chirality of the cysteine at the fifth position (the i position), have been synthesized and conformationally studied both in the linear and cyclized form. This peptide sequence, derived from the N-terminal sequence of parathyroid hormone related protein, does not form helices, even as part of the 1-34 fully active domain of the protein. The four analogs (two cyclic and two linear) were analyzed both in aqueous solution and in the presence of sodium dodecyl sulfate micelles. In aqueous solution the linear peptides display no evidence for secondary structure, while the cyclization induces a turn centered about the cysteine residues. In the presence of micelles the linear form of the peptides adopts bent conformations, containing turns, but results from both NMR and CD provide no evidence of helices. The oxidized L,L-peptide in the micellar solution does not present a well defined conformation, although the presence of one helical turn is evident. The cyclic D,L analog adopts a helical structure (not an alpha-helix) extending from residue 2 to 9, with non-standard phi, psi values caused by the presence of the D-amino acid. These results clearly illustrate that the ability of D-Cys(i), Cys(i + 3) cyclization to initiate helix formation depends greatly on the solvent used. Therefore, any drug-design principle utilizing this modification for helix nucleation must keep the environment in which the peptide is biologically active in mind.

Amino Acid Sequence↗
PubMed1997

Reference ranges for the concentrations of total and complexed plasma prostate-specific antigen and their ratio in patients with benign prostate hyperplasia.

OBJECTIVE: To establish the normal distribution and reference ranges of complexed and total prostate-specific antigen (PSA) and the complexed-to-total PSA ratio according to the age of the patients, so that PSA can be used to distinguish between prostate cancer and benign prostate hyperplasia (BPH). MATERIAL AND METHODS: Using specific ELISAs, total PSA and PSA complexed to alpha 1-antichymotrypsin (complexed PSA) were determined in 237 BPH patients, 160 with histologically confirmed BPH and 77 in whom prostate cancer was excluded by digital rectal examination, transrectal ultrasound and total PSA measurement. RESULTS: Both total and complexed PSA correlated with patient age (r = 0.424 and r = 0.379, p < 0.0001, respectively). However, no correlation was found between the complexed-to-total PSA ratio and age (r = 0.026, p > 0.2). The mean complexed-to-total PSA ratio for the 237 BPH patients was 0.69 +/- 0.11, and only 23 had a ratio > 0.8. CONCLUSIONS: These results show that the cut-off point of 0.8 established for the complexed-to-total PSA ratio is the same for men of all ages, and that the use of this ratio may avoid many negative prostate biopsies, confirming that PSA: alpha 1ACT is a potential marker for differential diagnosis of prostate carcinoma and BPH.

Adult↗
PubMed1996

Conformational characterization of a peptide mimetic of the third cytoplasmic loop of the G-protein coupled parathyroid hormone/parathyroid hormone related protein receptor.

The third-cytoplasmic loop of the G-protein coupled receptor responsible for the activity of parathyroid hormone and parathyroid hormone-related protein has been structurally characterized in aqueous solution in the presence of sodium dodecylsulfate and dodecylphosphocholine micelles. The high-resolution conformation of the 29-amino acid peptide containing the sequence of the cytoplasmic loop was obtained by CD and nmr. The structure was refined using a two-step distance geometry based method that first includes the removal of all side chains to quickly locate the globular fold of the peptide. After a simulated annealing protocol, the side chains are added in a random fashion. The resulting conformation was further refined with nuclear Overhauser enhancement restrained molecular dynamics using a two-phase simulation cell consisting of carbon tetrachloride and water as a mimetic of the biphasic, hydrophobic/hydrophilic character of the micelles in which the experimental measurements were carried out. The topological orientation of the cycloplasmic loop within the micelle was determined by addition of 5-doxylstearate and monitoring the decrease of nmr signal intensities from the radical-induced relaxation. The conformation and relative orientation of the peptide provided insight into the mechanism by which the cytoplasmic portion of the receptor activates the heterotrimeric, guanine nucleotide-binding regulatory protein, one of the first steps in signal transduction.

Amino Acid Sequence↗
PubMed1996

Heparin stimulation of the inhibition of activated protein C and other enzymes by human protein C inhibitor--influence of the molecular weightof heparin and ionic strength.

The ability of unfractionated (UF) heparin and low-molecular-weight heparin (LMWH) to potentiate the inhibition of fibrinolytic and coagulation factors by protein C inhibitor (PCI) was studied. Inhibition of activated protein C (APC), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), thrombin, factor Xa (Xa), factor XIa (XIa) and plasma kallikrein (KK) by PCI was found to be dependent on the size of the polysaccharide. In general, maximal stimulation was reached with UF heparin, except in the case of KK. Differences in heparin stimulation were more pronounced for thrombin, APC, uPA, tPA and XIa, whereas inactivation of Xa by PCI was less dependent on the presence of heparin, and kallikrein showed higher potentiation with LMWH than with UF heparin. The second-order rate constants for enzyme inhibition by PCI were strongly dependent on the ionic strength, and, in general, with an ionic strength higher than 0.15 the heparin stimulation of the inhibition reactions was drastically reduced. These results may explain the large discrepancies in the literature on the effect of heparin on the stimulation of enzyme inhibition by PCI. They also show that LMWH is less efficient in stimulating the PCI inhibition of APC, uPA and tPA, which could contribute to the antithrombotic effect of these enzymes.

Animals↗