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Biomedical subjects

M Prieto

Publications and source records attributed to M Prieto.

At least 37 records · Page 2Linked to original sources
PubMed2003

Genetic heterogeneity of bovine viral diarrhoea viruses from Spain.

Forty-four bovine viral diarrhoea virus (BVDV) isolates collected from the north of Spain between 1989 and 2000 were characterised at the molecular level. For 24 of these isolates the entire N(pro) gene sequence was determined. Phylogenetic analysis revealed that 21 isolates belong to subgroup BVDV-1b, while two BVDV-1c isolates and one BVDV-1h isolate were found. Furthermore, 20 additional virus isolates were analysed by differential reverse transcription-polymerase chain reaction (RT-PCR) and classified as BVDV-1. The results from our study demonstrate that BVDV-1b is the most prevalent subgroup present in bovine dairy herds of Spain, while there is no evidence for the presence of BVDV-2.

Amino Acid Sequence↗
PubMed2003

Axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes.

The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional disabilities. The inability of neurons to regenerate their axon is appreciably due to an inhospitable environment made of an astrocytic scar. We generated mice knock-out for glial fibrillary acidic protein and vimentin, the major proteins of the astrocyte cytoskeleton, which are upregulated in reactive astrocytes. These animals, after a hemisection of the spinal cord, presented reduced astroglial reactivity associated with increased plastic sprouting of supraspinal axons, including the reconstruction of circuits leading to functional restoration. Therefore, improved anatomical and functional recovery in the absence of both proteins highlights the pivotal role of reactive astrocytes in axonal regenerative failure in adult CNS and could lead to new therapies of spinal cord lesions.

Animals↗
PubMed2003

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies.

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.

Antibiotics, Antitubercular↗
PubMed2003

Kidney transplantation without calcineurin inhibitors using sirolimus.

INTRODUCTION: With the introduction of new immunosuppressive medicines, it has become possible to determine the extent to which nephrotoxic medicines contribute to CAN. The aim of this study is to compare the safety and efficacy of calcineurin inhibitor (CI) free immunosuppression in a prospective, randomized trial comparing sirolimus-mycophenolate mofetil (MMF)-prednisone to tacrolimus- MMF-prednisone. METHODS: Patients are randomized at the time of transplant to receive either tacrolimus (target level 12 to 15 ng/mL in the first month) or sirolimus (target level 12 to 18 ng/mL in the first month). All patients also receive MMF (750 mg bid) and prednisone tapered to 10 mg/d by 3 months and thymoglobulin induction (1.5 mg/kg/d on days 0, 1, 2, 4 and 6). RESULTS: At this point we have 4-month follow-up in 85 patients. The acute rejection rate is 7.5% (3/40) in the tacrolimus group and 6.7% (3/45) in the sirolimus group. We have discontinued sirolimus in eight patients so far, with wound complications being the most common indication. Renal function appears to be better in the sirolimus group at 1 month after transplantation, but the difference is not statistically significant. CONCLUSIONS: While longer follow-up is needed, these results demonstrate that total avoidance of CI can be achieved with extremely low acute cellular rejection rates using sirolimus-based immunosuppression in combination with thymoglobulin, MMF, and prednisone.

Calcineurin Inhibitors↗
PubMed2003

Use of the Advia 120 hematology analyzer in the differential cytologic analysis of biological fluids (cerebrospinal, peritoneal, pleural, pericardial, synovial, and others).

The centralization of our laboratories and the demand for new parameters to measure have led to an increase in the number of biological fluid samples, which are generally sent for urgent analysis. Due to this they cannot be processed by manual methods. Meeting this increased demand for assistance is a challenge for the laboratory, and the challenge has been met by the automated hematology area. A study of the reliability of the Advia 120 hematology analyzer has been carried out through leukocyte and red blood cell counting of 179 biological fluids: cerebrospinal, peritoneal or ascitic, pleural, pericardial, synovial, and others. The automated leukocyte counts of cerebrospinal fluid samples containing up to 0.150 x 10(9) leukocytes/L are correlated with counts obtained with the manual reference method in a Neubauer counting chamber (r = 0.958; P = .0001). Applying Passing-Bablok regression analysis to these results indicates a slope p of 1.155 (95% confidence interval [CI], 0.915-1.347) and an ordinate intercept b of 0.0076 (95% CI, 0.012-0.034), showing the results to be perfectly interchangeable. In the comparison of the manual analysis of the leukocyte differential using the May-Grünwald-Giemsa staining method with the analysis using the automated method, the percentage of polymorphonuclear granulocytes of the Advia 120 basophil/lobularity method is significantly correlated (r = 0.844; P = .0001) with that obtained with the manual count. The results of Passing-Bablok regression analysis (p = 0.859 [95% CI, 0.58-1.190]; b = 8.8 [95% CI, -12.09-24.2]) indicate that these two counting methods are also perfectly interchangeable. Automated leukocyte and differential counts of peritoneal or ascitic fluids also show good correlations with the manual method, and the results are not statistically different. Pretreating synovial fluid samples with hyaluronidase enzyme allows their processing on the Advia 120; no significant differences were found between manual and automated methods with respect to leukocyte counts and differentials. Finally, results with pleural fluid samples indicated that leukocyte and differential counts obtained with the Advia 120 showed significant differences from results obtained with manual methods because of the high incidence of mesothelial, lymphoid, and other tumoral cells in this kind of fluid sample. This result shows that use of hematology analyzers is questionable for these kinds of samples, especially from oncology patients with tumors. A procedure is proposed for the processing of these pleural fluids.

Ascitic Fluid↗
PubMed2002

Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1.

Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.

Biodegradation, Environmental↗
PubMed2002

Desensitization of alpha(2)-adrenoceptors which regulate noradrenaline synthesis and release after chronic treatment with clorgyline in the rat brain.

The sensitivity of alpha(2)-adrenoceptors which regulate synthesis and release of noradrenaline was investigated in hippocampus, parietal cortex, and hypothalamus of rats treated with clorgyline. After administering a DOPA decarboxylase inhibitor, the in vivo tyrosine hydroxylase activity and the noradrenaline content were evaluated. Acute and chronic treatment with clorgyline led to both increases of noradrenaline levels and decreases of tyrosine hydroxylase activity, determined as the accumulation of DOPA. Whereas the alpha(2)-adrenoceptor agonist clonidine induced a similar reduction in tyrosine hydroxylase activity in the group subjected to the acute treatment and in the control group, it failed to do so after chronic clorgyline treatment. In hippocampal and cortical synaptosomes, a reduction in the sensitivity of alpha(2)-adrenoceptors which regulate [(3)H]noradrenaline release, reflected by the shift to the right of the concentration-effect curves for oxymetazoline, was also found after the repeated treatment. These results indicate a desensitization of alpha(2)-adrenoceptors after chronic treatment with clorgyline.

Adrenergic alpha-Agonists↗
PubMed2001

Thymoglobulin induction decreases rejection in solitary pancreas transplantation.

BACKGROUND: Solitary pancreas transplants, both pancreas transplant alone (PTA) and pancreas after kidney (PAK), have higher rejection rates and lower graft survivals than simultaneous pancreas-kidney transplants (SPK). The aim of this study is to compare three different antibody induction regimens in solitary pancreas transplant recipients and to assess the role of surveillance pancreas biopsies in the management of these patients. METHODS: Solitary pancreas transplant recipients between 01/98 to 02/00 (n=29) received induction with either daclizumab (1 mg/kg on day 0, 7, 14), OKT 3 (5 mg/day x0-7), or thymoglobulin (1.5 mg/kg/day x0-10). Maintenance immunosuppression was similar for the three groups. All rejections were biopsy-proven either by surveillance/protocol or when clinically indicated. RESULTS: The 1-year graft survival was 89.3% overall and 91.7% in the thymoglobulin group. Thymoglobulin significantly decreased rejection in the first 6 months when compared with OKT3 or daclizumab (7.7 vs. 60 vs. 50%). Acute rejections were seen on surveillance biopsies in the absence of biochemical abnormalities in 40% of patients. CONCLUSIONS: Thymoglobulin induction regimen led to a low incidence of acute rejection and a high rate of graft survival in solitary pancreas transplants. In addition, surveillance biopsies were useful in the detection of early acute rejection in the absence of biochemical abnormalities.

Adult↗
PubMed2001

Ureteric bud derivatives express angiotensinogen and AT1 receptors.

Inactivation of the renin-angiotensin system interferes with the morphogenesis of the renal medulla. Thus ureteric bud (UB) derivatives may be a target for angiotensin production and action. To begin to test this hypothesis, we examined the cellular expression of angiotensinogen (Ao) and AT(1) receptor proteins during rat metanephrogenesis by immunohistochemistry. In addition, we tested whether UB-derived cells in culture express the Ao and AT(1) proteins. On embryonic day E15, Ao and AT(1) are expressed in the UB branches and stromal mesenchyme. S-shaped bodies, including the vascular cleft, express AT(1) but not Ao. The metanephric mesenchyme and pretubular aggregates are Ao negative and AT(1) negative. Expression of Ao and AT(1) in UB branches and ampullae is also observed on E16. However, UB expression of Ao is transient and is no longer detectable in the developing distal nephron beyond E17. On E17, both Ao and AT(1) are expressed in capillary loop glomeruli and proximal tubules, whereas UB branches express AT(1) only. By E18, the majority of Ao immunoreactivity is clustered in terminally differentiated proximal tubules, whereas AT(1) receptors are expressed in both proximal and distal nephron segments. The specificity of Ao and AT(1) staining was documented by the elimination/attenuation of immunoreactivity after preadsorption of the primary antibodies with their respective antigens. Consistent with the in vivo findings, the AT(1) protein is abundantly expressed in cellular lysates of mouse UB (E11.5) and IMCD3 (adult) cells. Moreover, AT(1) receptors in UB and IMCD3 cells are functional, since angiotensin II treatment elicits the tyrosine phosphorylation of the mitogen-activated protein kinases, ERK1/2. To our knowledge, this is the first demonstration of Ao and AT(1) protein expression in the developing distal nephron. Angiotensin II may have a paracrine role in the ontogeny of the collecting system.

Angiotensinogen↗
PubMed2001

Exclusion of a cholesterol analog from the cholesterol-rich phase in model membranes.

Vesicles of phosphatidylcholine/cholesterol mixtures show a wide composition range with coexistence of two fluid phases, the 'liquid disordered' (cholesterol-poor) and 'liquid ordered' (cholesterol-rich) phases. These systems have been widely used as models of membranes exhibiting lateral heterogeneity (membrane domains). The distributions of two fluorescent probes (a fluorescent cholesterol analog, NBD-cholesterol, and a lipophilic rhodamine probe, octadecylrhodamine B) in dimyristoylphosphatidylcholine/cholesterol vesicles were studied, at 30 degrees C and 40 degrees C. The steady-state fluorescence intensity of both probes decreases markedly with increasing cholesterol concentration, unlike the fluorescence lifetimes. The liquid ordered to liquid disordered phase partition coefficients K(p) were measured, and values much less than unity were obtained for both probes, pointing to preference for the cholesterol-poor phase. Globally analyzed time-resolved energy transfer results confirmed these findings. It is concluded that, in particular, NBD-cholesterol is not a suitable cholesterol analog and its distribution behavior in phosphatidylcholine/cholesterol bilayers is in fact opposite to that of cholesterol.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol interaction.

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25 degrees C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE--filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin--filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates).

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.

Alkanes↗
PubMed2001

Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study.

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40 degrees C) inside the coexistence range of liquid-ordered (l(o)) and liquid-disordered (l(d)) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM)-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l(o)/l(d) partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l(d) phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l(o) in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l(d) in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l(o) phase from initially pure l(d), and domain growth being faster in formation of l(d) phase from initially pure l(o).

Biophysical Phenomena↗
PubMed2001

Interaction of alpha-melanocyte stimulating hormone with binary phospholipid membranes: structural changes and relevance of phase behavior.

The interaction of alpha-melanocyte stimulating hormone (alpha-MSH) with negatively charged binary membrane systems composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], (DMPC/DMPG) or DMPC/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA), both at a 3:1 ratio, was studied using complementary techniques (differential scanning calorimetry, infrared and ultraviolet absorption spectroscopy, and steady-state and time-resolved fluorescence). The peptide structure in buffer, at medium to high concentrations, is a mixture of aggregated beta-strands and random coil, and upon increasing the temperature the random coil configuration becomes predominant. At low concentrations (micromolar) there are essentially no aggregates. When in interaction with the lipidic systems this transition is prevented and the peptide is stabilized in a specific conformation different from the one in solution. The incorporation of alpha-MSH into phosphatidic acid-containing systems produced a significant alteration of the calorimetric data. Lateral heterogeneity can be induced by the peptide in the DMPA-containing mixture, at variance with the one of DMPG. In addition, the lipid/water partition coefficient for the peptide in the presence of DMPC/DMPA is greater in the gel phase as compared to the fluid phase. From the high values of limiting anisotropies it can be concluded that the peptide presents a very reduced rotational dynamics when in interaction with the lipids, pointing out to a strong interaction. Overall, these results show that the structure and stability of alpha-MSH in a negatively charged membrane environment are substantially different from those of the peptide in solution, being stabilized in a specific conformation that could be important to eliciting its biological activity.

Biophysical Phenomena↗
PubMed2001

Effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline in the rat brain.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) induces a degeneration of noradrenergic axons originating in the locus coeruleus. The sensitivity of alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline was investigated in three brain regions which receive an unequal innervation from locus coeruleus, 21 days after DSP4 (50 mg/kg) administration. After giving treated rats a dopa decarboxylase inhibitor, the in vivo tyrosine hydroxylase activity and the tissue concentrations of noradrenaline were also evaluated. Relevant reductions of noradrenaline levels were found in hippocampus and parietal cortex (91% and 77.5%, respectively; P<0.001) together with a less pronounced reduction in hypothalamus (32%, P<0.01). The administration of the neurotoxin led to decreases of the basal tyrosine hydroxylase activity, determined as the accumulation of 3,4-dihydroxyphenylalanine, in hippocampus and parietal cortex (75% and 50.5%, respectively; P<0.001), but not in hypothalamus. The inhibitory effect of clonidine on tyrosine hydroxylase activity was markedly reduced in hippocampus of rats treated with DSP4 (10+/-5% vs 57+/-3% in the control group, P<0.001) but was not changed in parietal cortex and hypothalamus. Moreover, in hippocampus, a lack of functionality of the alpha2-adrenoceptors which regulate K(+)-evoked [3H]noradrenaline release was determined. However, in cortical synaptosomes the concentration-effect curve for the oxymetazoline shifted to the right. The administration of the neurotoxin did not modify the inhibitory effects of the agonist in hypothalamus. These results support the previously described selectivity of DSP4 for noradrenergic terminals arising from locus coeruleus and suggest a more severe lesioning of the hippocampus than the parietal cortex.

Animals↗