Biomedical subjects

A M Fernandez

Publications and source records attributed to A M Fernandez.

At least 19 recordsLinked to original sources

PubMed2005

Insulin-like growth factor I treatment for cerebellar ataxia: addressing a common pathway in the pathological cascade?

In the present work we review evidence supporting the use of insulin-like growth factor I (IGF-I) for treatment of cerebellar ataxia, a heterogeneous group of neurodegenerative diseases of low incidence but high societal impact. Most types of ataxia display not only motor discoordination, but also additional neurological problems including peripheral nerve dysfunctions. Therefore, a feasible therapy should combine different strategies aimed to correct the various disturbances specific for each type of ataxia. For cerebellar deficits, and most probably also for other types of brain deficits, the use of a wide-spectrum neuroprotective factor such as IGF-I may prove beneficial. Intriguingly, both ataxic animals as well as human patients show altered serum IGF-I levels. While the pathogenic significance of IGF-I, if any, in this varied group of diseases is difficult to envisage, disrupted IGF-I neuroprotective signaling may constitute a common stage in the pathological cascade associated to neuronal death. Indeed, treatment with IGF-I has proven effective in animal models of ataxia. Based on this pre-clinical evidence we propose that IGF-I should be tested in clinical trials of cerebellar ataxia in those cases where either serum IGF-I deficiency (as in primary cerebellar atrophy) or loss of sensitivity to IGF-I (as in ataxia telangiectasia) has been reported. Taking advantage of the widely protective and anabolic actions of IGF-I on peripheral tissues, this neurotrophic factor may provide additional therapeutic advantages for many of the disturbances commonly associated to ataxia such as cardiopathy, muscle wasting, or immune dysfunction.

Animals↗

PubMed2001

N-Succinyl-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin: an extracellularly tumor-activated prodrug devoid of intravenous acute toxicity.

Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.

Animals↗

PubMed2001

Insulin-like growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor.

In this study we investigated the molecular mechanisms whereby insulin-like growth factor 1 (IGF-1) induced Twist gene expression and the role of Twist in the anti-apoptotic actions of the IGF-1 receptor. In NIH-3T3 fibroblasts overexpressing the human IGF-1 receptor (NWTb3), treatment with IGF-1 (10(-8) m) for 1 and 4 h increased the level of Twist mRNA as well as protein by 3-fold. In contrast, insulin at physiological concentrations did not stimulate Twist expression in NIH-3T3 fibroblasts overexpressing the human insulin receptor. The IGF-1 effect was specific for the IGF-1 receptor since, in cells overexpressing a dominant negative IGF-1 receptor, IGF-1 failed to increase Twist expression. Pre-incubation with the ERK1/2 inhibitor U0126 or expression of a dominant negative MEK-1 abolished the effect of IGF-1 on Twist mRNA expression in NWTb3 cells, suggesting that Twist induction by IGF-1 occurs via the mitogen-activated protein kinase signaling pathway. In vivo, IGF-1 injection increased the mRNA level of Twist in mouse skeletal muscle, the major site of Twist expression. Finally, using an antisense strategy, we demonstrated that a reduction of 40% in Twist expression decreased significantly the ability of IGF-1 to rescue NWTb3 cells from etoposide-induced apoptosis. Taken together, these results define Twist as an important factor involved in the anti-apoptotic actions of the IGF-1 receptor.

3T3 Cells↗

PubMed2001

Extracellularly tumor-activated prodrugs for the selective chemotherapy of cancer: application to doxorubicin and preliminary in vitro and in vivo studies.

Oligopeptidic derivatives of anthracyclines unable to penetrate cells were prepared and screened for their stability in human blood and their reactivation by peptidases secreted by cancer cells. N-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin was selected as a new candidate prodrug. The NH2-terminal beta-alanine allows a very good blood stability. A two-step activation by peptidases found in conditioned media of cancer cells ultimately yields N-L-leucyl-doxorubicin. In vitro, when MCF-7/6 cancer cells are exposed to the prodrug, they accumulate about 14 times more doxorubicin than MRC-5 normal fibroblasts, whereas when exposed to doxorubicin the uptake is slightly higher in fibroblasts than in MCF-7/6 cells. This increased specificity of the prodrug over doxorubicin was confirmed in cytotoxicity assays using the same cell types. In vivo, the prodrug proved about nine times less toxic than doxorubicin in the normal mouse and also much more efficient in two different experimental chemotherapy models of human breast tumors.

Animals↗

PubMed2001

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death.

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIalpha promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [(3)H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68-79% of pyramidal neurons in the CA1-3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

ATP-Binding Cassette Transporters↗

PubMed2001

Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8) of triosephosphate isomerase: toward a new substrate specificity.

Loop 8 (residues 232-242) in triosephosphate isomerase (TIM) is a highly conserved loop that forms a tight binding pocket for the phosphate moiety of the substrate. Its sequence includes the fully conserved, solvent-exposed Leu238. The tight phosphate-binding pocket explains the high substrate specificity of TIM being limited to the in vivo substrates dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. Here we use the monomeric variant of trypanosomal TIM for exploring the structural consequences of shortening this loop. The mutagenesis, guided by extensive modeling calculations and followed up by crystallographic characterization, is aimed at widening the phosphate-binding pocket and, consequently, changing the substrate specificity. Two new variants were characterized. The crystal structures of these variants indicate that in monomeric forms of TIM, the Leu238 side-chain is nicely buried in a hydrophobic cluster. Monomeric forms of wild-type dimeric TIM are known to exist transiently as folding intermediates; our structural analysis suggests that in this monomeric form, Leu238 of loop 8 also adopts this completely buried conformation, which explains its full conservation across the evolution. The much wider phosphate-binding pocket of the new variant allows for the development of a new TIM variant with a different substrate specificity.

Amino Acid Sequence↗

PubMed2001

Treatment of early seropositive rheumatoid arthritis: a two-year, double-blind comparison of minocycline and hydroxychloroquine.

OBJECTIVE: To compare the efficacy of minocycline with that of a conventional disease-modifying antirheumatic drug (DMARD), hydroxychloroquine, in patients with early seropositive rheumatoid arthritis (RA). METHODS: Sixty patients with seropositive RA of <1 year's duration who had not been previously treated with DMARDs were randomized to receive minocycline, 100 mg twice per day, or hydroxychloroquine, 200 mg twice per day, in a 2-year, double-blind protocol. All patients also received low-dose prednisone. The primary end points of the study were 1) the percentage of patients with an American College of Rheumatology (ACR) 50% improvement (ACR50) response at 2 years, and 2) the dosage of prednisone at 2 years. RESULTS: Minocycline-treated patients were more likely to achieve an ACR50 response at 2 years compared with hydroxychloroquine-treated patients (60% compared with 33%, respectively; P = 0.04). Minocycline-treated patients were also receiving less prednisone at 2 years compared with the hydroxychloroquine group (mean 0.81 mg/day compared with 3.21 mg/day, respectively; P < 0.01). In addition, patients treated with minocycline were more likely to have been completely tapered off prednisone (P = 0.03). Trends favoring the minocycline treatment group were seen when outcomes were assessed according to components of the ACR core criteria set, with the differences reaching statistical significance for patient's global assessment of disease activity (P = 0.004). CONCLUSION: Minocycline is an effective DMARD in patients with early seropositive RA. Patients treated with minocycline were more likely to achieve an ACR50 response and did so while receiving less prednisone. In addition, minocycline-treated patients were more likely to have discontinued treatment with prednisone at 2 years.

Adult↗

PubMed2001

Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity.

Insulin and insulin-like growth factors (IGFs) mediate a variety of signals involved in mammalian development and metabolism. To study the metabolic consequences of IGF-I deficiency, we used the liver IGF-I-deficient (LID) mouse model. The LID mice show a marked reduction (approximately 75%) in circulating IGF-I and elevated growth hormone (GH) levels. Interestingly, LID mice show a fourfold increase in serum insulin levels (2.2 vs. 0.6 ng/ml in control mice) and abnormal glucose clearance after insulin injection. Fasting blood glucose levels and those after a glucose tolerance test were similar between the LID mice and their control littermates. Thus, the high levels of circulating insulin enable the LID mice to maintain normoglycemia in the presence of apparent insulin insensitivity. Insulin-induced autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate (IRS)-1 were absent in muscle, but were normal in liver and white adipose tissue of the LID mice. In contrast, IGF-I-induced autophosphorylation of its cognate receptor and phosphorylation of IRS-1 were normal in muscle of LID mice. Thus, the insulin insensitivity seen in the LID mice is muscle specific. Recombinant human IGF-I treatment of the LID mice caused a reduction in insulin levels and an increase in insulin sensitivity. Treatment of the LID mice with GH-releasing hormone antagonist, which reduces GH levels, also increased insulin sensitivity. These data provide evidence of the role of circulating IGF-I as an important component of overall insulin action in peripheral tissues.

Animals↗

PubMed2000

Estrogen receptors and insulin-like growth factor-I receptors mediate estrogen-dependent synaptic plasticity.

Previous studies have shown that estradiol induces a transient disconnection of axo-somatic inhibitory synapses in the hypothalamic arcuate nucleus of adult ovariectomized rats. The synaptic disconnection is accompanied by an increase in the levels of insulin-like growth factor-I (IGF-I) in the arcuate nucleus, suggesting that IGF-I signaling may be involved in the estrogen-induced synaptic plasticity. The role of estrogen receptors and IGF-I receptors in the synaptic changes has been studied by assessing the number of axo-somatic synapses in ovariectomized rats treated with intracerebroventricular administration of the estrogen receptor antagonist ICI 182,780 and the IGF-I receptor antagonist JBI to ovariectomized rats. Estradiol administration resulted in a significant decrease in the number of axo-somatic synapses on arcuate neurons in control ovariectomized rats. Both the estrogen receptor antagonist and the IGF-I receptor antagonist blocked the estrogen-induced synaptic decrease. This finding suggest that estrogen-induced synaptic plasticity in the arcuate nucleus is dependent on the activation of both estrogen receptors and IGF-I receptors.

Animals↗

PubMed2000

Neurodegeneration is associated to changes in serum insulin-like growth factors.

Serum levels of insulin and insulin-like growth factors and their binding proteins (IGFs and IGFBPs, respectively) are changed in human neurodegenerative diseases of very different etiology, such as Alzheimer's disease, amyotrophic lateral sclerosis, or cerebellar ataxia. However, the significance of these endocrine disturbances is not clear. We now report that in two very different inherited neurodegenerative conditions, ataxia-telangiectasia (AT) and Charcot-Marie-Tooth 1A (CMT-1A) disease, serum levels of IGFs are also altered. Both types of patients have increased serum IGF-I and IGFBP-2 levels, and decreased serum IGFBP-1 levels, while only AT patients have high serum insulin levels. Furthermore, serum IGFs are also changed in three different animal models of neurodegeneration: neurotoxin-induced motor discoordination, diabetic neuropathy, and hereditary cerebellar ataxia. In these three models, serum insulin levels are significantly decreased, serum IGF-I and IGFBP-1, -2, and -3 are decreased in diabetic and neurotoxin-injected rats, while serum IGFBP-1 is increased in hereditary ataxic rats. Altogether, these observations indicate that a great variety of neurodegenerative diseases show endocrine perturbations, resulting in changes in serum IGFs levels. These perturbations are disease-specific and are probably due to metabolic and endocrine derangements, nerve cell death, and sickness-related disturbances associated to the neurodegenerative process. Our observations strongly support the need to evaluate serum IGFs in other neurodegenerative conditions.

Adolescent↗

PubMed2000

Antenatal use of ambroxol for the prevention of infant respiratory distress syndrome.

OBJECTIVE: Our purpose was to evaluate the efficacy and safety of ambroxol for the prenatal prophylaxis of infant respiratory distress syndrome (IRDS). STUDY DESIGN: This was a prospective study with 2 groups of pregnant patients with premature labor or with premature rupture of membranes at an estimated gestation between 27 to 34 completed weeks. Ambroxol treatment group consisted of 39 subjects in whom 1,000 mg of ambroxol diluted in 500 ml of 5% glucose solution was given intravenously for 4 hours once a day for 3 days, while the control group consisted of 41 subjects in whom ambroxol was not administered. Main measures included Apgar scores, clinical signs of one or more of the following: respiratory rate of > 60/min, intercostal retraction, alar flaring, expiratory grunting, cyanosis on room air and radiological evidence of IRDS. Chi-square test was used to determine the statistical significance of the results. RESULTS: Tolerable maternal side effects were noted. Profile of newborns delivered were similar in both groups. Incidence of IRDS was significantly less in the treatment group (p < 0.01). CONCLUSIONS: Antenatal administration of ambroxol resulted in a significant decrease in the incidence of IRDS as well as perinatal morbidity and mortality. Due to the efficacy and safety of this drug, it might be useful for the prevention of IRDS.

Adult↗

PubMed1999

Neuroprotective actions of peripherally administered insulin-like growth factor I in the injured olivo-cerebellar pathway.

Exogenous administration of insulin-like growth factor I (IGF-I) restores motor function in rats with neurotoxin-induced cerebellar deafferentation. We first determined that endogenous IGFs are directly involved in the recovery process because infusion of an IGF-I receptor antagonist into the lateral ventricle blocks gradual recovery of limb coordination that spontaneously occurs after partial deafferentation of the olivo-cerebellar circuitry. We then analysed mechanisms whereby exogenous IGF-I restores motor function in rats with complete damage of the olivo-cerebellar pathway. Treatment with IGF-I normalized several markers of cell function in the cerebellum, including calbindin, glutamate receptor 1 (GluR1), gamma-aminobutyric acid (GABA) and glutamate, which are all depressed after 3-acetylpyridine (3AP)-induced deafferentation. IGF-I also promoted functional reinnervation of the cerebellar cortex by inferior olive (IO) axons. In the IO, increased expression of bax in neurons and bcl-X in astrocytes after 3AP was significantly reduced by IGF-I treatment. On the contrary, IGF-I prevented the decrease in poly-sialic-acid neural cell adhesion molecule (PSA-NCAM) and GAP-43 expression induced by 3AP in IO cells. IGF-I also significantly increased the number of neurons expressing bcl-2 in brainstem areas surrounding the IO. Altogether, these results indicate that subcutaneous IGF-I therapy promotes functional recovery of the olivo-cerebellar pathway by acting at two sites within this circuitry: (i) by modulating death- and plasticity-related proteins in IO neurons; and (ii) by impinging on homeostatic mechanisms leading to normalization of cell function in the cerebellum. These results provide insight into the neuroprotective actions of IGF-I and may be of practical consequence in the design of new therapeutic approaches for neurodegenerative diseases.

Animals↗

PubMed1998

Phospholipid interactions of a peptide from the fusion-related domain of the glycoprotein of VHSV, a fish rhabdovirus.

Previous studies mapped a p2 domain (aa 82-109) which binds phosphatidylserine (PS) (Estepa and Coll, 1996a) and contains three contiguous hydrophobic amino acid heptad repeats followed by a positively charged stretch (Coll, 1995b) in the glycoprotein G of the viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. Anti-p2 antibodies inhibited low-pH VHSV-induced fusion (Estepa and Coll, 1997) and low-pH PS binding to VHSV (Estepa and Coll, 1996a). We report here further studies on the interaction of the synthetic peptide p2 with phospholipid vesicles. The synthetic p2 peptide was able to mediate aggregation, lipid mixing, and leakage of contents only with negatively charged phospholipid vesicles and in a concentration-dependent manner. As shown by its effect on lipid phase transitions deduced from data with fluorescence polarization and differential scanning calorimetry, the p2 peptide becomes inserted into the hydrophobic negatively charged phospholipid vesicle bilayers. In addition, data based on circular dichroism showed that the p2 peptide folds as a structure with a high content of beta-sheets stabilized by interaction with anionic phospholipids. These studies are potentially relevant to viral fusion in VHSV.

Amino Acid Sequence↗

PubMed1998

Insulin-like growth factor I restores motor coordination in a rat model of cerebellar ataxia.

We tested the potential of insulin-like growth factor I (IGF-I) to induce functional recovery in an animal model of cerebellar ataxia because this motor impairment is accompanied in humans and rodents by distinct changes in several components of the IGF-I trophic system. Rats rendered ataxic by deafferentation of the cerebellar cortex with 3-acetylpyridine recovered motor function after IGF-I was administered, as determined by behavioral and electrophysiological tests. When treated with IGF-I, inferior olive neurons, the targets of the neurotoxin, were rescued to various degrees (from 92 to 27% of surviving neurons), depending on the time that treatment with IGF-I was initiated. Furthermore, full recovery was obtained regardless of the route by which the trophic factor was administered (intraventricular or subcutaneous) even in rats with severe neuronal loss. These results suggest that human ataxia could be treated with IGF-I by a simple procedure.

Afferent Pathways↗

PubMed1997

Insulin-like growth factor I modulates c-Fos induction and astrocytosis in response to neurotoxic insult.

Insulin-like growth factor I participates in the cellular response to brain insult by increasing its messenger RNA expression and/or protein levels in the affected area. Although it has been suggested that insulin-like growth factor I is involved in a variety of cellular responses leading to homeostasis, mechanisms involved in its possible trophic effects are largely unknown. Since activation of c-Fos in postmitotic neurons takes place both in response to insulin-like growth factor I and after brain injury, we have investigated whether this early response gene may be involved in the actions of insulin-like growth factor I after brain insult. Partial deafferentation of the cerebellar cortex by 3-acetylpyridine injection elicited c-Fos protein expression on both Purkinje and granule cells of the cerebellar cortex. This neurotoxic insult also triggered gliosis, as determined by an increased number of glial fibrillary acidic protein-positive cells (reactive astrocytes) in the cerebellar cortex. When 3-acetylpyridine-injected animals received a continuous intracerebellar infusion of either a peptidic insulin-like growth factor I receptor antagonist or an insulin-like growth factor I antisense oligonucleotide for two weeks through an osmotic minipump, c-Fos expression was obliterated while reactive gliosis was greatly increased. On the contrary, continuous infusion of insulin-like growth factor I significantly decreased reactive gliosis without affecting the increase in c-Fos expression. These results indicate that insulin-like growth factor I is involved in both the neuronal (c-Fos) and the astrocytic (glial fibrillary acidic protein) activation in response to injury.

Animals↗

PubMed1996

Synthesis of a photoaffinity labeling analogue of the inactivating peptide of the Shaker B potassium channel.

A photoactivatable derivative of the inactivating peptide of the Shaker B potassium channel (ShB peptide) has been synthesized from ShB peptide containing an added cysteine residue at the peptide carboxy-terminus and 1-(p-azidosalicylamido)-4-(iodoacetamido)butane. The peptide derivative restores rapid inactivation in the deletion mutant Shaker Bdelta6-46 potassium channel in a manner indistinguishable from that of the wild-type ShB peptide. Also, both peptides display similar conformational behavior when challenged in vitro by an artificial model target that partly imitates the properties of the putative receptor site for the inactivating peptide in the Shaker B potassium channel. Therefore, we conclude that both functionally and conformationally the photoreactive peptide derivative is an adequate analogue of the wild-type ShB peptide, suitable for photoaffinity labeling of its binding site in the Shaker B potassium channel. Moreover, because the ShB peptide also serves as an efficient inactivating peptide for a large variety of other potassium channels, it appears that the photoreactive analogue may be useful to explore homologous sites in many different channel proteins.

Affinity Labels↗

PubMed1996

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation "entrance" of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.

Amino Acid Sequence↗

PubMed1996

Lipoprotein (a) levels in patients undergoing continuous ambulatory peritoneal dialysis.

Most researchers have found increases of lipoprotein (a) [Lp(a)] in uremic patients, as well as in those undergoing hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). The mechanisms for this increase remain unclear. We studied 71 patients undergoing CAPD, 48 men and 23 women. According to the time spent on CAPD, the patients were divided into three groups: group 0: 29 patients at the starting off point of dialysis treatment; group I: 22 patients with an average stay of 15.2 months; group II: 20 patients with an average stay of 69.3 months on CAPD. We have only observed significant increases of Lp(a) levels in those patients initiating the dialysis, but no significant differences are found in the other groups undergoing CAPD for longer periods when compared to the control group. We found no significant relation between Lp(a) levels and peritoneal protein loss, and not with absorption of glucose from the dialysate either. We have found a positive and significant correlation between Lp(a) levels and urinary protein loss (r = 0.41; p < 0.001). It is possible that an element associated with proteinuria might have an effect on the metabolism of Lp(a) in CAPD patients.

Adult↗