Biomedical subjects

N Jiménez

Publications and source records attributed to N Jiménez.

At least 19 recordsLinked to original sources

PubMed2006

Effects of steatosis on drug-metabolizing capability of primary human hepatocytes.

The suitability of liver grafts discarded for transplantation because of macrosteatosis for preparing human hepatocyte cultures for in vitro drug metabolism studies has been examined. Lower cell viability and yield of isolation procedure were obtained from fatty livers (>40% steatosis) with respect to normal tissue. Significant reductions in 7-ethoxycoumarin O-deethylation (ECOD) and testosterone oxidations were found in hepatocytes prepared from steatotic livers. The potential impact of lipid accumulation on P450 enzymes was studied in vitro by incubation of cultured hepatocytes with long chain free fatty acids (FFA). Treatment of cells with 0.25-3mM FFA induced dose-dependent accumulation of lipids in the cytosol. Decreased ECOD and testosterone oxidation were found after 14h of exposure to 1mM or 2mM FFA (about 60-70% and 30-60% of control, respectively). The effects of fat-overloading on individual P450s were analyzed both at activity and mRNA level. CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities were reduced after hepatocyte incubation with 1mM (to 45-65% of control) or 2mM (to 20-50%) FFA for 14h. Reductions in P450 transcripts were also found in hepatocytes treated with 1mM FFA. Our findings showed a general down-regulation of P450s involved in drug metabolism in fat-overloaded hepatocytes. The results suggest that, despite their reduced P450 function, human hepatocytes obtained from donors with steatosis are metabolically competent and could be used for drug metabolism studies.

Cells, Cultured↗

PubMed2006

Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing.

Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell viability is the preculture of hepatocytes before cryopreservation. By the use of this procedure, high-quality cells, estimated in terms of post-thaw recovery, viability, adaptation of hepatocytes to culture, drug-metabolizing capability and cytochrome P450 induction, are obtained. Therefore, cryopreserved hepatocytes can provide a regular source of metabolically competent cells for in vitro investigations of the metabolic profile of new drugs and drug-drug interactions in pharmaco-toxicological research.

Animals↗

PubMed2006

Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers.

High-pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze-substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high-pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high-pressure freeze, freeze-substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high-pressure freezing, freeze-substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.

Animals↗

PubMed2005

Identification of carcass and meat quality quantitative trait loci in a Landrace pig population selected for growth and leanness.

The identification of QTL related to production traits that are relevant for the pig industry has been mostly performed by using divergent crosses. The main objective of the current study was to investigate whether these growth, fatness, and meat quality QTL, previously described in diverse experimental populations, were segregating in a Landrace commercial population selected for litter size, backfat thickness, and growth performance. We have found QTL for carcass weight (posterior P > 0.75), cutlet weight (posterior P > 0.99), weight of ham (posterior P > 0.75), shoulders weight (posterior probability > 0.99), and shear firm-ness (posterior P > 0.99) on pig Chromosome 2. Moreover, QTL with posterior P > 0.75 for fat thickness between the 3rd and 4th ribs (Chromosome 7), rib weights (Chromosome 8), backfat thickness (Chromosomes 8, 9, and 10), and b Minolta color component (Chromosome 7) were identified. These results indicate that commercial purebred populations retain a significant amount of genetic variation, even for traits that have been selected for many generations.

Adipose Tissue↗

PubMed2004

The immunosuppressant drug FK506 prevents Fas-induced apoptosis in human hepatocytes.

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.

Adult↗

PubMed2004

Low diversity in the major histocompatibility complex class II DRB1 gene of the Spanish ibex, Capra pyrenaica.

During the last two centuries, the Spanish ibex (Capra pyrenaica) has shown a significant demographic decline as a result of the progressive destruction of its natural habitat, disease epidemics, and uncontrolled hunting. Partial sequencing of the class II MHC DRB1 gene revealed that the Spanish ibex has remarkably low levels of genetic variation at this locus, with only six different DRB1 alleles and an observed heterozygosity of 0.429-0.579. The rates of nonsynonymous vs synonymous substitutions were significantly different in the peptide-binding region (dN/dS=5.347, P=0.002), a feature that indicates that the DRB1 gene is under positive selection. A phylogenetic analysis of the Spanish ibex and a set of domestic goat DRB1 alleles revealed that the reported sequences represent four major allelic lineages. The limited allelic repertoire of the DRB1 gene in the Spanish ibex is likely the direct result of the recent history of population bottlenecks and marked demographic decline of this species. A genetic survey of 13 microsatellite loci was consistent with this idea. The Spanish ibex subspecies C. p. hispanica and C. p. victoriae consistently showed considerably lower levels of microsatellite heterozygosity (Ho=0.184-0.231) and allelic diversity (mean number of alleles per locus=2-2.4) than those reported in other wild ruminants. This study demonstrates the significance of both natural selection and the demographic history of populations in determining patterns of genetic variation at MHC loci. In addition, our results emphasize the importance of locally adapted populations for the preservation of genetic diversity.

Amino Acid Sequence↗

PubMed2003

Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits.

The chicken insulin-like growth factor (IGF)1 and IGF2 genes have been partially sequenced in six individuals of each of two chicken strains of the Black Penedesenca breed (PN and MN). These two strains are genetically diverse for growth traits. Sequence alignment revealed the existence of three single nucleotide polymorphisms (SNP) (IGF1-SNP1, IGF2-SNP2, and IGF2-SNP3). These three SNP and a fourth IGF1 polymorphism (IGF1-SNP4) were typed in 60 individuals from each strain by using PCR-RFLP or primer extension analysis. No significant associations among these four SNP, growth traits, and plasma IGF1 concentration were identified. In contrast, suggestive associations (P < or = 0.05) were found between IGF1-SNP1 and average daily gain at 107 d and feed efficiency at 44, 73, and 107 d. However, these associations were not simultaneously found in both strains suggesting that they might have been produced by linkage disequilibrium with another mutation located in the IGF1 locus or another linked gene. Since the PN and MN strains differ very markedly on their feed intake, the chicken leptin gene was included in the sequence analysis. Unfortunately, attempts to amplify several regions of this gene were unsuccessful. Even when primers complementary to highly conserved regions were used, the PCR consistently failed. Other authors have reported similar problems when trying to amplify avian leptin sequences.

Animals↗

PubMed2001

Peptidylglycine alpha-amidating monooxygenase- and proadrenomedullin-derived peptide-associated neuroendocrine differentiation are induced by androgen deprivation in the neoplastic prostate.

Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy.

Adrenomedullin↗

PubMed2001

Modifications of intracellular Ca2+ signalling during nerve growth factor-induced neuronal differentiation of rat adrenal chromaffin cells.

Postnatal sympathetic neurons (SNs) and chromaffin cells (CCs) derive from neural crest precursors. CCs can differentiate in vitro into SN-like cells after nerve growth factor (NGF) exposure. This study examines changes of intracellular Ca2+ homeostasis and dynamics of CCs under conditions that promote a neuronal phenotype. Spontaneous Ca2+ fluctuations, a frequent observation in early cultures of CCs, diminished after > 10 days in vitro in control cells and ceased in NGF-treated ones. At the same time, Ca2+ rises resulting from entry upon membrane depolarization, gradually increased both their size and peak d[Ca2+]i/dt, resembling those recorded in SNs. Concomitantly, caffeine-induced Ca2+ rises, resulting from Ca2+ release from intracellular stores, increased their size and their peak d[Ca2+]i/dt by > 1000%, and developed transient and sustained release components, similar to those of SNs. The transient component, linked to regenerative Ca2+ release, appeared after > 10 days of NGF treatment, suggesting a delayed steep enhancement of Ca2+-induced Ca2+ release (CICR). Immunostaining showed that proteins coded by the three known isoforms of ryanodine receptors (RyRs) are present in CCs, but that only RyR2 increased significantly after NGF treatment. Since the transient release component increased more steeply than RyR2 immunostaining, we suggest that the development of robust CICR requires both an increased expression of RyRs and more efficient functional coupling among them. NGF-induced transdifferentiation of chromaffin cells involves the enhancement of both voltage-gated Ca2+ influx and Ca2+ release from intracellular stores. These modifications are likely to complement the extensive morphological and functional reorganization required for the replacement of the endocrine phenotype with the neuronal one.

Adrenal Glands↗

PubMed2000

Prephenate dehydratase from the aphid endosymbiont (Buchnera) displays changes in the regulatory domain that suggest its desensitization to inhibition by phenylalanine.

Buchnera aphidicola, the prokaryotic endosymbiont of aphids, complements dietary deficiencies with the synthesis and provision of several essential amino acids. We have cloned and sequenced a region of the genome of B. aphidicola isolated from Acyrthosiphon pisum which includes the two-domain aroQ/pheA gene. This gene encodes the bifunctional chorismate mutase-prephenate dehydratase protein, which plays a central role in L-phenylalanine biosynthesis. Two changes involved in the overproduction of this amino acid have been detected. First, the absence of an attenuator region suggests a constitutive expression of this gene. Second, the regulatory domain of the Buchnera prephenate dehydratase shows changes in the ESRP sequence, which is involved in the allosteric binding of phenylalanine and is strongly conserved in prephenate dehydratase proteins from practically all known organisms. These changes suggest the desensitization of the enzyme to inhibition by phenylalanine and would permit the bacterial endosymbiont to overproduce phenylalanine.

Amino Acid Sequence↗

PubMed1999

Expression of adrenomedullin and proadrenomedullin N-terminal 20 peptide in human and rat prostate.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two recently discovered hypotensive peptides translated from the same message transcript (preproAM mRNA). In this article we report the presence of AM, PAMP, and their mRNA in human and rat prostate and of AM receptor mRNA in rat prostate. PreproAM mRNA was found in the epithelium of normal human and rat prostate glands by in situ hybridization. In humans, it was mainly expressed in the basal cells. In rat, its expression was higher in the ducts than in the acini of all the prostate lobes. Immunocytochemistry identified a similar distribution pattern for AM compared with its mRNA but showed different locations for AM and PAMP immunoreactivity. The former was widespread in the epithelia, whereas the latter was almost exclusively found in neuroendocrine cells. In rat, Western blot analysis confirmed the presence of high levels of AM peptide in the ventral lobe and of its precursor in the ventral and dorsolateral lobes. Immunoreactivity for serotonin, chromogranin A, PAMP, and AM defined four subpopulations of prostate neuroendocrine-like cells in rat, a cell type that has not been previously described.

Adolescent↗

PubMed1998

Description of the characteristics of cases with noncontiguous neural tube defects identified in a series of consecutive births.

Van Allen et al. [(1973) Am. J. Med. Genet. 47:723-743] provided evidence for multisite closure of the neural tube in humans. Reynolds et al. [(1995) Proceedings of the Greewood Genetic Center 14:70-71] and Seller [(1995) J. Med. Genet. 32:205-207] described 13 and seven cases of noncontiguous neural tube defects (NTDs) respectively and concluded that the presence of noncontiguous NTDs cannot be explained on the basis of the model of a single initiation site with bidirectional closure. Here we present a series of 14 consecutive infants with noncontiguous NTDs, describing their characteristics. These show that noncontiguous NTDs are clinically heterogeneous, may have differences in sex ratio, and could have causal heterogeneity. The different combinations of closure failure defects have shown proportions in our population that are different from those in the populations studied by Reynolds et al. and Seller.

Birth Weight↗

PubMed1998

[Brachmann-de-Lange syndrome in our population: clinical and epidemiological characteristics].

INTRODUCTION: We present the study of the clinical and epidemiological characteristics of Brachmann-de Lange syndrome in our population. PATIENTS AND METHODS: In this study we present the analysis of 13 cases of Brachmann-de Lange syndrome identified among 24,696 infants with congenital defects registered by the Spanish Collaborative Study of Congenital Malformations (ECEMC) between April 1976 and June 1996. RESULTS: The minimum estimation of the prevalence in our population is 0.97 per 100,000 live births. We have epidemiologically confirmed the presence of intrauterine growth retardation and have observed that parental ages tend to be relatively young. We have observed a wide range of clinical expression of this syndrome. One hundred percent of our cases have limb reduction defects, followed in frequency by craniofacial alterations (84.62%), abnormal hair distribution (76.92%) and genital defects (69.23%). Upper limbs are predominantly affected and one case of diaphragmatic hernia is worth mentioning. We underline the importance of the differential diagnosis with Fryns'syndrome. CONCLUSIONS: The cases studied correspond to the most severe form of the syndrome, reason for which the prevalence is a minimal estimate. However, the mild forms of the syndrome are more frequent and it is important to consider that the face, especially the form of the eyebrow, could be a good guide for the diagnosis of mild forms of the syndrome.

De Lange Syndrome↗

PubMed1997

Ca(2+)-induced Ca2+ release phenomena in mammalian sympathetic neurons are critically dependent on the rate of rise of trigger Ca2+.

The role of ryanodine-sensitive intracellular Ca2+ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca2+ release in individual fura 2-loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR components shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations > or = 10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca(2+)-mediated feedback loops, suggesting that it constitutes a Ca(2+)-induced Ca(2+)-release phenomenon. The dynamics of release is markedly affected when Sr2+ substitutes for Ca2+, indicating that Sr2+ release may operate with lower feedback gain than Ca2+ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca2+ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca2+ and Ca(2+)-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca2+ reservoir is full, the rapid initial Ca2+ rise determines a faster occupation of the ryanodine receptor Ca2+ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca2+ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store's Ca2+ content. This study suggests that transmembrane Ca2+ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca2+] near the Ca2+ release channels sufficiently fast to overcome their Ca(2+)-dependent inactivation. Conversely, caffeine-induced Ca2+ release can undergo considerable amplification especially when Ca2+ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca2+ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca2+ stores.

Anesthetics, Local↗

PubMed1994

Short rib-polydactyly syndrome and pericentric inversion of chromosome 4.

We report on a newborn infant with clinical and radiological manifestations of some type of short rib-polydactyly syndrome who died soon after birth. Chromosomal studies on peripheral blood lymphocytes and chondrocytes demonstrated an apparently balanced pericentric inversion of chromosome 4 (present in the mother also). This association may have occurred by chance but, if not, the chromosomal breakpoints could interrupt the gene responsible for short rib-polydactyly syndromes, or else be related to the mechanism of short rib-polydactyly syndromes.

Abortion, Habitual↗

PubMedDate not supplied

Nerve growth factor with insular cortical grafts induces recovery of learning and reestablishes graft choline acetyltransferase activity.

Rats showing disrupted taste aversion due to insular cortex (IC)-lesions received either IC-grafts with NGF, grafts without NGF, or NGF alone. An additional group served as lesioned controls. Only those animals that received IC-grafts with NGF recovered the ability to learn the conditioned taste aversion task, at 15 days post-graft. Choline acetyltransferase (ChAT) activity in the IC-grafts with, but not without NGF, was similar to the IC activity of unoperated controls. In contrast, glutamate decarboxylase activity was similar in all the groups. These findings suggest that IC-grafts associated with NGF induce recovery of learning abilities in IC-lesioned rats, which correlates with reestablishment of ChAT activity in the grafts at 15 days post-implantation.

Animals↗

PubMedDate not supplied

Graft-induced recovery of inhibitory avoidance conditioning in striatal lesioned rats is related to choline acetyltransferase activity.

Four groups of male Wistar rats showing disrupted inhibitory avoidance conditioning due to striatal lesions received either striatal or ventral mesencephalic brain grafts. Two additional non-lesioned groups were used as controls. Half of the groups was retrained in an inhibitory avoidance task at fifteen days postgraft and the other half at sixty days postgraft. Those animals receiving striatal grafts significantly improved their ability to acquire the inhibitory avoidance task at fifteen and sixty days postgraft, as opposed to those receiving mesencephalic grafts, which did not show behavioral recovery. Choline acetyltransferase and glutamate decarboxylase activities, as well as dopamine content, were measured in the grafted tissue. Striatal grafts showed levels of choline acetyltransferase activity similar to the control group. Moreover, a positive correlation was found between the choline acetyltransferase activity and the behavioral recovery. In contrast, both glutamate decarboxylase activity and dopamine levels were significantly lower in striatal and in mesencephalic grafts, as compared to the controls. These results show that striatal but not mesencephalic grafts can promote the restoration of the ability to acquire an inhibitory avoidance task even at early stages (15 days) of the development of the grafts. The results also suggest that acetylcholine plays an important role in behavioral recovery.

Animals↗

PubMedDate not supplied

[Genitourinary changes in multiple sclerosis: the need for a urodynamic study].

OBJECTIVE: In this study we have evaluated the urinary and sexual alterations of patients with multiple sclerosis (MS) by means of neurourological and neuroandrological studies. PATIENTS AND METHODS: In 41 patients with MS we took a clinical history and made a neurourological physical examination. We also did a full urodynamic study including measurement of flow and postmictional residue, cystomanometry and test of the detrusor pressure/mictional flow. Selective electromyography of the periurethral sphincter and a cystourethrographic study were also done. In patients complaining of erection dysfunction this was also studied by means of an erection test with intracavernosal vasoactive drugs and special neurophysiological techniques including the obtention of S2-S4 evoked potentials in the bulbo-cavernosal muscle and the somatosensorial potentials of the pudendal nerve. RESULTS: In patients with MS there is a predominance of mixed and irritative urinary symptoms rather than obstructive symptoms. The vesico-urethral dysfunction of upper motor neurone type was the most frequent urodynamic diagnosis. There were no statistically significant differences between the urinary symptoms of the different urodynamic diagnoses. Ninety percent of the patients with erectile dysfunction had associated vesico-urethral dysfunction of upper motor neurone type and 100% of the patient with erectile dysfunction had dysfunction of the sphincter of the urinary bladder. CONCLUSIONS: In patients with MS there is no correlation between the clinical symptoms and urodynamic diagnosis. Therefore urodynamic diagnosis is essential not only for diagnosis but also for development of suitable treatment. The urodynamic diagnosis of dysfunction of the bladder sphincter is a risk factor in patients with MS. In our series 100% of the cases with erectile dysfunction also had dysfunction of the bladder sphincter.

Adult↗