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Biomedical subjects

P Ramos

Publications and source records attributed to P Ramos.

At least 37 records · Page 2Linked to original sources
PubMed2000

Fibrinogen in systemic lupus erythematosus: more than an acute phase reactant?

OBJECTIVE: To investigate whether plasma fibrinogen (FNG) measured longitudinally in a cohort of patients with systemic lupus erythematosus (SLE) increased over observational time faster than in a control group, and whether its increase might depend upon age, disease duration, disease activity, and medications. METHODS: Hospital based retrospective study with repeated measurements of plasma FNG and C-reactive protein (CRP) for patients and controls and erythrocyte sedimentation rate (ESR) and lupus activity index (LAI) for patients only. Study groups included patients with SLE: n = 96 (95% female), and healthy controls: n = 39 (95% female). Of the patients, 42% had SLE only, 23% had SLE with antiphospholipid antibodies (aPL), and 34% had SLE with aPL related thrombosis. RESULTS: Median baseline FNG was higher in patients (357 mg/dl; 95% CI 339-375) than in controls (271 mg/dl; 95% CI 251-291) by 86 mg/dl (95% CI 56-115, p < 0.001); in older subjects than younger (in patients and in controls); in patients with thrombosis than in other patient groups (by an average of 35 mg/dl; 95% CI 9-61 mg/dl): and in patients with longer disease duration (p = 0.05). Mean FNG increased faster in patients (19 mg/dl/year; 95% CI 12-26 mg/dl) than in controls (2.6 mg/dl/year; 95% CI 2.0-3.2 mg/dl). The increase was faster than the age effect and independent of patient group and disease activity. CONCLUSION: Plasma FNG in patients with SLE increases throughout followup regardless of disease activity, mimicking the age related increment observed in population based studies. The rapidity of the increment may reflect the prematurity of vascular disease typical of SLE.

Acute-Phase Proteins↗
PubMed1999

The cardiac acetylcholine-activated, inwardly rectifying K+-channel subunit GIRK1 gives rise to an inward current induced by free oxygen radicals.

Reactive oxygen species (ROS) play a crucial role in pathophysiology of the cardiovascular system. The present study was designed to analyze the redox sensitivity of G-protein-activated inward rectifier K+ (GIRK) channels, which control cardiac contractility and excitability. GIRK1 subunits were heterologously expressed in Xenopus laevis oocytes and the resulting K+ currents were measured with the two-electrode voltage clamp technique. Oxygen free radicals generated by the hypoxanthine/xanthine oxidase system led to a marked increase in the current through GIRK channels, termed superoxide-induced current (I(SO)). Furthermore, I(SO) did not depend on G-protein-dependent activation of GIRK currents by coexpressed muscarinic m2-receptors, but could also be observed when no agonist was present in the bathing solution. Niflumic acid at a concentration of 0.5 mmol/l did not abolish I(SO), whereas 100 micromol/l Ba2+ attenuated I(SO) completely. Catalase (10(6) i.u./l) failed to suppress I(SO), whereas H2O2 concentration was kept close to zero, as measured by chemiluminescence. Hence, we conclude that O2*- or a closely related species is responsible for I(SO) induction. Our results demonstrate a significant redox sensitivity of GIRK1 channels and suggest redox-activation of G-protein-activated inward rectifier K+ channels as a key mechanism in oxidative stress-associated cardiac dysfunction.

Acetylcholine↗
PubMed1999

Insulin-induced up-regulation of lipoprotein lipase messenger ribonucleic acid and activity in mammary gland.

The effects of insulin on lipoprotein lipase (LPL) messenger RNA (mRNA) were studied in mammary glands from virgin and late-pregnant rats. Virgin and pregnant rats (at 17 days of gestation) were subjected to a continuous infusion (35 ml/day) with 50% glucose for 72 h to produce a prolonged hyperinsulinemic-euglycemic condition. Gestation causes a 4-fold increase in LPL mRNA accompanied by a 3- to 4-fold increase in total LPL activity. Experimental hyperinsulinemia, generated by the 50% glucose infusion, had a similar effect in both pregnant and virgin rats, by enhancing (2- to 3-fold) both their LPL activity and LPL mRNA. Thus, total LPL activity and LPL mRNA significantly correlated with plasma insulin (r = 0.789, P < 0.001; and r = 0.772, P < 0.001, respectively). Furthermore, total LPL activity was correlated with LPL mRNA (r = 0.765, P = 0.001). In conclusion, the present study shows that insulin participates in the control of LPL expression in mammary glands, revealing its role as a modulator of the enzyme at a mRNA level.

Animals↗
PubMed1998

Organization and evolution of olfactory receptor genes on human chromosome 11.

Olfactory receptors (OR) are encoded by a large multigene family including hundreds of members dispersed throughout the human genome. Cloning and mapping studies have determined that a large proportion of the olfactory receptor genes are located on human chromosomes 6, 11, and 17, as well as distributed on other chromosomes. In this paper, we describe and characterize the organization of olfactory receptor genes on human chromosome 11 by using degenerate PCR-based probes to screen chromosome 11-specific and whole genome clone libraries for members of the OR gene family. OR genes were identified by DNA sequencing and then localized to regions of chromosome 11. Physical maps of several gene clusters were constructed to determine the chromosomal relationships between various members of the family. This work identified 25 new OR genes located on chromosome 11 in at least seven distinct regions. Three of these regions contain gene clusters that include additional members of this gene family not yet identified by sequencing. Phylogenetic analysis of the newly described OR genes suggests a mechanism for the generation of genetic diversity.

Amino Acid Sequence↗
PubMed1998

Copper can promote oxidation of LDL by markedly different mechanisms.

Oxidation of LDL (0.1 microM) in PBS with copper concentrations ranging from 0.03 to 10 microM, equal to 0.3-100 Cu2+/LDL, was investigated by monitoring the formation of conjugated dienes at 234 nm. With all 8 LDL samples examined, the kinetics changed strongly at submicromolar Cu2+ concentrations. Based on time-course of the formation of conjugated dienes, cholesteryl linoleate hydroxides and hydroperoxides as well as the antioxidant consumption, two oxidation types were distinguished. Type A oxidations, observed at relatively high Cu2+ concentrations of 10-100 Cu2+/ LDL, represented the conventional kinetics of LDL oxidation with an inhibition period (= lag-time) followed by a propagation phase. In contrast, type C oxidations proceeded after a negligibly short lag time followed by a distinct propagation phase. The rate of this propagation increased rapidly to 0.5 mol diene/mol LDL and then slowed down in the presence of alpha-,gamma-tocopherols and carotenoids, which were consumed faster than tocopherols. The increase in diene absorption was due to the formation of both hydroxides and hydroperoxides suggesting a high initial decomposition of hydroperoxides. At submicromolar concentrations of about 0.1 to 0.5 microM, type C and type A oxidation can be combined resulting in 4 consecutive oxidation phases, i.e. 1st inhibition and 1st propagation (belonging to type C), followed by 2nd inhibition and 2nd propagation (belonging to type A). Increasing copper concentrations lowered the 1st propagation and shortened the 2nd inhibition periods until they melted into one apparent kinetic phase. Decreasing [Cu2+] increased the 1st propagation and 2nd inhibition but lowered the 2nd propagation phase until it completely disappeared. A threshold copper concentration, denoted as Cu(lim), can be calculated as a kinetic constant based on the Cu2+-dependence for the rate of 2nd propagation. Below Cu(lim), LDL oxidation proceeds only via type C kinetics. The Cu2+-dependence of the oxidation kinetics suggests that LDL contains two different Cu2+ biding sites. Cu2+ at the low-affinity binding sites, with half-saturation at 5-50 Cu2+/LDL, initiates and accelerates the 2nd propagation by decomposing lipid hydroperoxides. Cu2+ bound to the high-affinity binding sites, with half-saturation at 0.3-2.0 Cu2+/LDL, is responsible for the 1st propagation. Arguments in favor and against this propagation being due to tocopherol mediated peroxidation (TMP) are discussed. If the lag-time concept is extended to the conjugated diene curves seen for combined oxidation profiles, then a true inhibition phase does not apply to this time interval, but instead represents the time elapsed before the onset of the 2nd propagation phase.

Adult↗
PubMed1998

Breakdown of the inner and outer blood retinal barrier in streptozotocin-induced diabetes.

Using vitreous fluorophotometry and quantitative fluorescence microscopy the authors studied the permeability of the blood-retinal barrier (BRB) to fluorescein in control and in 8 days streptozotocin-induced diabetic rats. Vitreous fluorophotometry showed that fluorescein permeates BRB in control and in diabetic rats. However, in diabetic rats the permeability to fluorescein was significantly increased as compared to control rats. The vitreous penetration ratio (VPR) values for total and free fluorescein at 60 min, were higher for diabetic rats (231.2+/-12.9 min-1 for total fluorescein and 1299.24+/-58.0 min-1 for free fluorescein) than for control rats (95.5+/-3.5 min-1 for total fluorescein and 646.6+/-55. 0 min-1 for free fluorescein) (P<0.05). Quantitative confocal fluorescence microscopy confirmed these findings and identified the site of leakage across the BRB by comparing the relative importance of the fluorescein leakage across the outer and inner BRB. In control rats the fluorescence levels remained relatively low in the photoreceptor layer, next to the outer BRB but in the inner nuclear layer, next to the inner BRB reached values that were almost ten times higher. These results suggest that in retinas of control rats fluorescein penetrates predominantly through the inner BRB. In diabetic rats the fluorescence levels in the photoreceptor and in the inner nuclear layer were significantly increased as compared to the fluorescence levels in controls rats. Nevertheless, in the inner nuclear layer the fluorescence levels were also generally higher than the fluorescence levels at the photoreceptor layer. The rates of fluorescence levels between the inner nuclear layer and the photoreceptor layer were apparently 3:1, 60 min after the single intravenous injection of fluorescein. Also, the fluorescein penetration in the inner nuclear layer of the diabetic rats is higher than that observed in the inner nuclear layer of the control rats (P<0.001). These findings suggest that the permeability to fluorescein of both components of the BRB is increased 8 days after the induction of diabetes by streptozotocin and that the permeability of the retinal vasculature is preferentially affected.

Animals↗
PubMed1998

Changes in the kinase activity of the insulin receptor account for an increased insulin sensitivity of mammary gland in late pregnancy.

Mammary gland is an organ that undergoes cycles of growth, differentiation, and function during pregnancy and lactation. Although it is known that the gland enhances its sensitivity to insulin during lactation, it remains to be investigated whether this increased sensitivity develops during pregnancy and which are the molecular mechanisms underlying such a change. To address this issue, virgin and late-pregnant rats were subjected to a continuous infusion with 50% glucose for 72 h to produce a prolonged hyperinsulinemic-euglycemic condition. Insulin sensitivity in mammary gland was determined as the glucose utilization index by using 2-[3H]-deoxyglucose. Furthermore, binding characteristics and kinase activity were studied by means of both [125I]insulin binding and in vitro phosphorylation studies with insulin receptors partially purified from mammary gland. Whereas the glucose utilization index in mammary gland from nonpregnant rats remained unaffected by hyperinsulinemia, glands from pregnant rats displayed a high insulin-dependent glucose uptake. This effect was not paralleled by changes in the binding characteristics of insulin to the high-affinity receptor, suggesting that the high insulin sensitivity of mammary gland in pregnancy is not accounted for by changes at the level of hormone-receptor interaction. Autophosphorylation studies showed that insulin-stimulated kinase activity of insulin receptors from mammary gland was 6- and 20-fold higher in pregnant than in virgin animals under normo- and hyperinsulinemic conditions, respectively. Moreover, insulin dose-response curves revealed that the efficacy of insulin to stimulate kinase activity of the insulin receptor was markedly higher in pregnant than in virgin rats, whereas its potency (ED50 approximately 15 nM) was not changed. These data, therefore, show that mammary glands develop increased insulin sensitivity during late pregnancy, caused by an augmented kinase activity of the insulin receptor.

Animals↗
PubMed1998

[Clinical and laboratory basis for the diagnosis and understanding of neonatal metabolic diseases].

The authors divide neonatal metabolic diseases into two major groups: intoxication and energy deficiency. The main signs which allow for the suspicion of the diagnosis are indicated for each group. The complementary examinations to be carried out by the Clinical Pathology Service of the Central Hospital and those which must be carried out by the metabolic diseases Reference Centre are reviewed. Based on the clinical framework and on the examination results, the authors establish five syndromatic groups to orientate diagnosis. The authors conclude by presenting differential diagnosis tables based on the original systematic classification by Jean-Marie Saudubray, with up-dated modifications from their own experience.

Humans↗
PubMed1998

Controlled-rate versus uncontrolled-rate cryopreservation of peripheral blood progenitor cells: a prospective multicenter study. Group for Cryobiology and Biology of Bone Marrow Transplantation (CBTMO), Spain.

BACKGROUND AND OBJECTIVE: Cryopreservation of hemopoietic progenitors for transplantation has been traditionally performed by the use of a controlled-rate freezer. Several groups have reported successful cryopreservation of progenitor cells at -80 degrees C without a controlled-rate freezer. In an attempt to elucidate whether both methods are equally efficient, we compared controlled-rate versus uncontrolled cryopreservation of peripheral blood progenitor cells (PBPC) in a prospective, multicenter study. DESIGN AND METHODS: Apheresis products from patients undergoing PBPC mobilization were split into two aliquots, and cryopreserved simultaneously by both methods, in autologous plasma plus 10% dimethylsulfoxide. Controlled-rate samples were placed into a programmable freezer with a cooling rate of 1-2 degrees C/min. Uncontrolled-rate samples were directly introduced into a -80 degrees C mechanical freezer. After thawing, cell counts, assays for viability, clonogenic cultures and CD34+ cell enumeration were performed. RESULTS: A total of 105 cases were included. No significant differences were found in viability (mean 88.8 +/- 13% in the controlled-rate group vs. 89.7 +/- 12% in the uncontrolled-rate group), nucleated cell loss (23.5 +/- 23% vs. 23 +/- 22%), mononuclear cell loss (19 +/- 23% vs. 19.1 +/- 22%), and loss of CD34+ cells (34.3 +/- 33% vs. 28.6 +/- 34%). On the other hand, recovery of granulomonocytic colony-forming units (CFU-GM), was significantly better with the controlled-rate technique, than with the non-controlled-rate method (104.3 +/- 95 vs. 86.5 +/- 80, respectively; p = 0.048). INTERPRETATION AND CONCLUSIONS: Our results indicate that both techniques are suitable for cryopreservation of PBPC, although a better recovery of committed progenitors is achieved by the controlled-rate method. Therefore, the use of controlled-rate freezer should probably be recommended.

Blood Preservation↗
PubMed1997

The interaction of lower alcohols with apoB in spin labeled human plasma low density lipoproteins (LDL).

In this study the interaction of alcohol with the macromolecular lipid-protein assembly represented by human plasma low density lipoproteins (LDL) was investigated. The spin label which covalently binds to the side chain amino group of lysines as well as terminal amino groups was attached to the spin labeled apoprotein (apoB) of native LDL in order to observe the protein component in the electron spin resonance (ESR) spectrum. The interaction of different lower alcohols (methanol, ethanol, propanol and butanol) with the spin labeled LDL was studied for two alcohol concentrations (0.3 and 3.0 M). The ESR spectra indicate a decrease of the hyperfine splitting and narrowing of the linewidth upon the action of alcohol that leads to the conclusion that alcohol provokes a change in the apoB conformation. These findings are explained by following the arguments of the phospholipid mediated mechanism of alcohol action, through the modulation of the lipid packing free volume which results in the protein conformational change.

Alcohols↗
PubMed1997

Chondroitin 4-sulphate exhibits inhibitory effect during Cu2+-mediated LDL oxidation.

Chondroitin 4-sulphate (C4S), a basic component of the extracellular matrix of the artery wall, inhibited copper-induced low density lipoprotein (LDL) oxidation by prolonging the lag time and reducing the rate of propagation. LDL oxidation was assessed by monitoring conjugated dienes and low level chemiluminescence. A possible initial key reaction in LDL oxidation, the reduction of copper(II) to copper(I) by LDL, was decreased in the presence of C4S. Moreover, C4S protected tryptophan residues of Apo-B-100 in the early stage of LDL oxidation and during the subsequent propagation phase. The presence of the sulphate group in position 4 of N-acetylgalactosaminyl residues of C4S is crucial for protective activity. In fact, the structurally related chondroitin 6-sulphate, also present in tissues, had no effect on LDL oxidation. These data suggest that C4S may contribute to protect LDL against oxidation in arterial intima.

Adult↗
PubMed1997

Nitroxide reduction with ascorbic acid in spin labeled human plasma LDL and VLDL.

The LDL and VLDL were spin labeled with Tempo which partitions both in the aqueous and lipid phase. The ESR spectra were measured in the equilibrium state as well as during the reduction of the spin label with ascorbic acid. The kinetics of the concentration decay curves was parametrized with two exponentials. The theoretical simulation of the experimental spectra revealed a drastic linewidth narrowing in the VLDL samples exposed to the ascorbic acid. Since the transport properties of the specific monolayer are reflected in the observed reaction rates, the analysis of the fatty acid composition of phospholipids, triglycerides and cholesterol esters in LDL and VLDL was performed. It is concluded that different lipid packing at the surface of LDL and VLDL might be the consequence of different intermolecular forces between phospholipids and cholesterol. This finding was connected to the experimentally detected different reaction kinetics in LDL and VLDL as well as their different susceptibility to the ESR linebroadening effects during the nonequilibrium conditions of the spin label reduction with ascorbic acid.

Ascorbic Acid↗
PubMed1997

Subcutaneous versus intravenous low-dose IL-2 therapy after autologous transplantation: results of a prospective, non-randomized study.

Use of IL-2 therapy after autologous transplantation is currently being explored to reduce relapse rate. Low doses of the cytokine induce significant immunomodulation avoiding the severe side-effects associated with high-dose IL-2 therapy. However, low-dose IL-2 is usually given by continuous infusion through central venous lines with the consequent risks of thrombosis and infections. Twenty-six consecutive patients who received autologous transplants received low-dose IL-2 after stable engraftment had been achieved. The first 13 patients (group A) were scheduled to receive 400,000/IU/m2/day for 3 months by continuous intravenous infusion. Ten of these patients suffered infectious episodes, mainly bacteriemias that often necessitated delaying IL-2 therapy (median delivered dose: 32% of planned). The next 13 patients were then assigned to receive IL-2 (800,000-1,000,000 IU/m2/day for 3 months) subcutaneously (group B). For group B patients, median dose intensity was 84% (P = 0.01 when compared with group A patients). Only one severe infectious episode was observed in these patients. Clinical toxicity in group B patients consisted mainly of s.c. nodules. Immunomodulation, measured as an increase in the absolute number of CD56+ cells and CD56+(bright) cells, was higher in patients who received the cytokine by the subcutaneous route (median peak increase of CD56+ cells: 160 and 220% for group A and B patients respectively; median peak increase of CD56+(bright) cells: 210% and 310% for group A and B respectively, P < 0.05 between groups A and B). No statistically significant increment of T lymphocytes was observed in any group. No hematologic toxicity was observed apart from eosinophilia, which was very marked in group B (P < 0.01). Our results show that low-dose s.c. IL-2 therapy is associated with low clinical and hematologic toxicity after autologous transplantation. The immunomodulation achieved is no less than that achieved with the i.v. approach.

Acute Kidney Injury↗
PubMed1997

Immunohistochemical analysis of murine CD95/Fas/Apo-1 receptor and its ultrastructural distribution in the thymus.

CD95/Fas/Apo-1 is a cell surface receptor that, upon contact with its ligand, induces cells to die by apoptosis. In view of the importance of Fas receptor (FasR) in immunologic tolerance, an immunohistochemical analysis of FasR expression was performed in the lymphoid and certain parenchymal tissues of normal and mutant MRL/lpr mice using a rabbit polyclonal anti-Fas receptor antibody. FasR was expressed by immunoperoxidase (IP) in the cortex and at the corticomedullary junction of the thymus of normal mice. By immunoelectron microscopy FasR was detected on the cell membrane of normal thymocytes. In MRL/lpr mice, FasR protein expression could not be clearly detected. FasR protein expression was not detected in the heart, liver or ovary by IP, presumably reflecting the low number of receptors in these tissues.

Animals↗
PubMed1997

High-dose granulocyte-colony stimulating factor (G-CSF) in vitro induces the growth of high proliferative potential colony forming cells (HPP-CFC) in patients undergoing blood stem cell mobilization.

We evaluated the role of high-dose granulocyte colony stimulating factor (G-CSF) in vitro, in inducing the generation of high-proliferative potential colony forming cells (HPP-CFC), from either mononuclear cells or purified CD34+ cells. Both normal controls and patients undergoing peripheral blood stem cell (PBSC) mobilization and transplantation were studied. In serum-driven agar cultures, G-CSF stimulated the proliferation of HPP-CFC in a dose dependent manner (r = 0.92). The number of HPP-CFC was four-fold greater in mobilized patients than in normal controls. Purified CD34+ cells yielded 11-fold more colonies than mononuclear cells. HPP-CFC from mobilized patients showed replating capacity, giving rise to secondary colonies of more mature appearance. In serum-free cultures, the effect of G-CSF appeared to be mediated by synergistic interaction with stem cell factor. Our results suggest that G-CSF stimulates primitive hematopoietic cells that are detectable in increased amounts in patients receiving mobilization therapy. Therefore, determination of G-CSF induced HPP-CFC could be a useful tool in the evaluation of mobilization strategies.

Antigens, CD34↗
PubMed1997

RRR-alpha-tocopherol can be substituted for by Trolox in determination of kinetic parameters of LDL oxidizability by copper.

LDL resistance to oxidation is characterized by the lag time that precedes the rapid generation of conjugated dienes in copper-induced oxidation. Lag time (y) is described by the equation y = kx + a, where k is the efficacy constant of alpha-tocopherol (alpha-TOH), x the alpha-TOH content of LDL, and a the alpha-TOH-independent variable, all of which show considerable between-subject variability. To answer the question of whether loading of LDL with Trolox can substitute for the more timeconsuming loading with RRR-alpha-TOH in large scale studies focusing on determination of k and a values, LDL from 15 healthy subjects was loaded in vitro with 0-50 mol Trolox/mol LDL or 0-250 mM RRR-alpha-TOH. In addition, five of these subjects were supplemented in vivo with 800-1200 IU/d RRR-alpha-TOH for 17 days and k and a were determined. The ratios k(Trolox):k(alpha-TOH) and alpha(Trolox):a(alpha-TOH) were calculated. The k(Trolox) was only slightly higher than k(alpha-TOH), in contrast to alpha(Trolox), which was almost twice as high as a(alpha-TOH), indicating that, in the case of Trolox, a includes the contribution of alpha-TOH to the lag time. In the case of alpha-TOH, a includes only the additional antioxidants that occur naturally in LDL. The ratios k(Trolox):k(alpha-TOH) and alpha(Trolox):a(alpha-TOH) showed little variability, both between subjects and between in vitro and in vivo experiments. A close correlation was found between k(alpha-TOH) and a(alpha0TOH), calculated using this ratio, and k(alpha-TOH) and a(alpha-TOH), obtained in our experiments. We conclude that from this ratio, k(alpha-TOH) and a(alpha-TOH) can be reliably calculated when k(Trolox) and a(Trolox) are determined experimentally, thus allowing us to obtain k and a values in a more convenient way.

Adult↗