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Biomedical subjects

P Ferrer

Publications and source records attributed to P Ferrer.

At least 19 recordsLinked to original sources
PubMed2005

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst.

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5'-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of L-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at -20 degrees C and 4 degrees C. It was observed that the Km for L-allo-threonine was 38-fold higher than that for L-threonine, suggesting this enzyme can be classified as a specific L-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6-7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible beta-hydroxy-alpha-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of beta-hydroxy-alpha-amino acids.

Enzyme Stability↗
PubMed2004

MR-myelography as an adjunct to the MR examination of the degenerative spine.

Our objective was to analyse the usefulness of single-slice MR-myelography images as a complementary test to conventional MR of the spine in patients with clinically suspected degenerative spine disease. A prospective analysis of 1022 consecutive patients studied with conventional MR and MR-myelography (four acquisitions: one coronal plane, one sagittal plane, and two oblique lateral, right and left planes) examinations was performed. Conventional MR and MR-myelography studies were classified as positive when morphological or structural alterations were noted. Positive MR-myelography information was also categorized as relevant if findings were considered important and radiologically significative to the final diagnosis (either by increasing the radiologists confidence in the diagnosis or because the findings were not observed in the conventional study). Other positive findings were considered non- relevant. There were 62% cases considered positive in the MR-myelography examination. Of them, 3% revealed no alterations on the conventional MR examination. Of the negative MR-myelography studies, 75% had a positive conventional MR examination. Considered as diagnostic techniques, their Kappa concordance was poor. However, MR-myelography complemented the information obtained with the conventional MR examination of the spine, this information being relevant to the final MR diagnosis in 22% of the total number of cases. The contribution of MR-myelography was not different in the three spinal levels. In conclusion, MR-myelography complements the information obtained with conventional MR in a large number of cases.

Aging↗
PubMed2001

Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris.

The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level. Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant. Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.

Bioreactors↗
PubMed2001

Long-term efficacy and safety of oral almotriptan: interim analysis of a 1-year open study.

OBJECTIVE: To assess the long-term tolerability and safety of a single, oral 12.5-mg dose of almotriptan and its efficacy in alleviating pain associated with consecutive migraine episodes occurring during a 12-month period. BACKGROUND: While sumatriptan appears to be effective for treatment of migraine, the drug has several properties that limit its use. Almotriptan, a new selective 5-HT1B/1D agonist, may be better tolerated over the long term. METHODS: This 1-year study was conducted on 806 adults between the ages of 18 and 65 years suffering from migraine, either with or without aura. The patients met the criteria for study as outlined by the International Headache Society and were instructed on drug use. Oral almotriptan 12.5 mg was used to treat the attack of any pain severity and a second dose was permitted in case of relapse of pain during the first 24 hours. Rescue medication was provided. Efficacy and tolerability were assessed by a combination of patient reporting and clinical visit evaluations. RESULTS: Overall, almotriptan was well tolerated. At 2 hours, 81% of attacks were relieved; 56% of the subjects were entirely free of pain. These efficacy data are similar for the first as well as the last attack studied. At analysis, 534 patients had treated their migraines for at least 6 months. About half of all patients experienced at least one adverse event, with 87% of the events being mild or moderate in nature. The most frequent adverse events were back pain (7.23% of patients), bronchitis (5.76%), and influenzalike symptoms (5.62%). Seventy-one percent of the adverse events were not related to almotriptan use. CONCLUSIONS: Almotriptan at an oral dose of 12.5 mg is safe antimigraine treatment. The safety profile results are similar to those obtained in other controlled triptan clinical studies. Almotriptan is efficacious in moderate-to-severe migraine pain and can be used repeatedly in recurrent episodes. The long-term safety data will be reanalyzed when full data become available.

Adult↗
PubMed2001

Tolerability and efficacy of almotriptan in the long-term treatment of migraine.

BACKGROUND: Almotriptan is a highly specific 5-HT(1B/1D) receptor agonist, which acts selectively on blood vessels of the brain. Short-term studies have demonstrated that almotriptan provides rapid, effective and reliable relief of migraine attacks, while offering excellent tolerability. PURPOSE: To assess the long-term tolerability and efficacy of oral almotriptan 12.5 mg administered for every migraine attack over a 1-year period. METHODS: A total of 762 patients treated 13,751 attacks (1-97 per patient); 61.5% of attacks were treated with one 12.5-mg dose, while for 38.5% of attacks, patients took a second dose within 24 h. RESULTS: Three hundred and ninety-one patients (51.3%) experienced a total of 1,617 adverse events (AEs). The majority (88.6%) of AEs were of mild-to-moderate intensity, and only 28.8% of AEs were considered to be related to the study drug. Only 2 patients experienced serious AEs possibly related to almotriptan, syncope and chest pain; both recovered without any sequelae. Patients reported at least 1 AE in 11% of attacks treated. The incidence of AEs decreased during the study. Only 6 (0.8%) study withdrawals were due to AEs considered to be related to almotriptan. Tolerability was not compromised in patients taking 2 doses of almotriptan or in those using migraine prophylactics. Patient age or sex did not influence the incidence of AEs. There was no evidence of tachyphylaxis in those patients completing the study. Pain relief at 2 h after the initial dose was achieved in 84.2% of moderate/severe attacks. Patients were pain free at 2 h after dose in 58.2% of all attacks. Older patients (> 40 years) tended to respond better than younger ones (< 40 years). Efficacy was not modified by use of migraine prophylactics or hormonal contraceptives. Efficacy measurements were consistent on treating repeated moderate/severe migraine attacks. CONCLUSION: This large, open study indicates that the new, specific 5-HT(1B/1D) agonist almotriptan, at a dose of 12.5 mg, is a well tolerated and effective treatment for migraine pain when used over a period of up to 1 year.

Administration, Oral↗
PubMed2001

Production of native and recombinant lipases by Candida rugosa: a review.

The yeast Candida rugosa produces multiple lipase isoenzymes sharing high sequence homology but with some differences in their catalytic properties. The regulation of C. rugosa lipase (CRL) synthesis and secretion in C. rugosa obeys a complex pattern. Fermentation processes for both wild-type and mutant C. rugosa strains are available for lipase production. Native CRL preparations have been extensively used for biotransformations. However, their inherent mixture of isoforms with variable profiles complicates interpretation and brings into question the reproducibility achieved between preparations. Although heterologous CRLs gene expression had been hampered owing to a nonuniversal codon usage, recent advances have made heterologous CRLs available. This will expand and improve the industrial utility of CRLs even further. The purpose of this review is to provide a summary of the recent advances on the production of native and recombinant lipases by C. rugosa.

Animals↗
PubMed2000

[Value of MR myelography in the diagnosis of the spine disorders].

To evaluate the utility of myelography obtained with MR imaging (MR-myelography) as a complementary tool in patients studied with a conventional MR examination of the spine. 275 consecutive patients were included. All of them were studied with MR-myelography in 2 planes, coronal and sagittal, with a turbo spin-echo single-shot technique, as a complement to a conventional MR study of the spine; 130 were males and 145 women, with ages ranging from 20 to 71 years (mean, 45 years). The analyzed variables were age, sex, vertebral segment studied, alteration of the dural sac, intradural nerve roots, emergent roots, and presence of intradural lesions, meningeal cysts, and spinal stenosis. The added value of MR-myelography regarding conventional MR was categorized. MR-myelography obtained new information in 88 cases (32%), being considered irrelevant information in 42 cases and relevant in 46 cases (16.7% of all cases) (amputations of the emergent roots and alterations of the intradural roots). MR-myelography did not contribute to any type of additional information to the conventional MR study in 187 cases (68% of all studies). MR-myelography is a rapid acquisition technique that supplements the conventional MR study of the spine, contributing with relevant new information in the analysis of the spine diseases 16.7% of cases.

Adult↗
PubMed2000

MR imaging of Baker cysts --prevalence and relation to internal derangements of the knee.

RATIONALE AND OBJECTIVES: To evaluate the prevalence of popliteal cysts in patients studied with magnetic resonance (MR) imaging. Cyst presence and volume will be related to other internal derangement of the knee. MATERIALS AND METHODS: Three hundred and eighty-two consecutive patients with a MR study of the knee were included. Images were obtained in the three spatial orthogonal planes and evaluated through consensus. Four categories were established for the Baker cyst and synovial fluid (absence, minimum, moderate and massive), and the presence of meniscal, cruciate ligaments and cartilage lesions were recorded. Statistical analysis was carried out with bivariate analysis (chi(2) of Pearson and Gamma tests). RESULTS: From the 382 patients, 145 had Baker cysts (38.0%). Cyst content was minimum in 99, moderate in 34 and massive in 12. Joint effusion was observed in 269 patients (70.4%), being minimal in 140 patients, moderate in 119 and massive in ten. Meniscal lesions were observed in 195 patients (51%), while 58 patients (15%) had a cruciate ligament lesion. Baker cyst had a statistically significant direct relationship with the presence and quantity of synovial fluid (P=0.002) and with the presence and type of meniscal lesion (P=0.01) but not with cruciate ligaments or cartilage lesions. CONCLUSIONS: The prevalence of Baker cysts in MR studies of the knee is high. Its presence and volume are related to the quantity of synovial fluid, and to the presence and severity of meniscal lesions.

Adolescent↗
PubMed2000

Consistent efficacy and tolerability of almotriptan in the acute treatment of multiple migraine attacks: results of a large, randomized, double-blind, placebo-controlled study.

In this double-blind study, the efficacy and tolerability of a single dose of almotriptan (6.25 or 12.5 mg) was compared with placebo in the treatment of three consecutive migraine attacks of moderate or severe intensity. Of 1013 randomized patients, 722 evaluable patients completed the study. The total number of attacks relieved (severe or moderate pain reduced to mild or no pain) at 2 h post-dose was significantly higher (P < 0.001) after treatment with almotriptan 6.25 or 12.5 mg compared with placebo (60% and 70% vs. 38%, respectively). Moreover, a consistent response was achieved across and within patients for almotriptan 6.25 or 12.5 mg compared with placebo (pain relief in at least two out of three attacks within 2 h for 64% and 75% vs. 36%, respectively) and less than one-third of the patients relapsed within 24 h. Almotriptan was well tolerated with no significant differences between the almotriptan and placebo treatment groups in the percentage of patients reporting adverse events. Overall, the 12.5-mg dose was associated with the most favourable efficacy/tolerability ratio and is, therefore, the recommended dose.

Acute Disease↗
PubMed1999

Epiregulin is a potent vascular smooth muscle cell-derived mitogen induced by angiotensin II, endothelin-1, and thrombin.

Vasoactive GTP-binding protein-coupled receptor agonists such as angiotensin II (AII), endothelin-1 (ET-1), and alpha-thrombin (alpha-Thr) have been reported to indirectly stimulate vascular smooth muscle cell (VSMC) proliferation by regulating the expression of one or more autocrine growth factors. Using ion-exchange, gel-filtration, and reverse-phase chromatographic purification methods, we isolated a major mitogenic protein present in AII-stimulated rat aortic smooth muscle (RASM) cell conditioned medium. Twenty N-terminal amino acids of the purified peptide were identified, and they had 75% amino acid sequence identity with mouse epiregulin, an epidermal growth factor (EGF)-related growth factor. We cloned the cDNA for rat epiregulin to determine its pattern of expression in G-protein-coupled receptor agonist-stimulated cells and confirm its activity as a mitogen. After treatment of RASM cells with AII, ET-1, or alpha-Thr for 1 h, induction of two epiregulin transcripts was observed, including a 4.8-kb transcript and a novel transcript of approximately 1.2 kb. Recombinant rat epiregulin was strongly mitogenic for RASM cells, stimulating DNA synthesis to levels similar to those induced by serum or platelet-derived growth factor and approximately 3-fold above that observed with saturating concentrations of EGF. In addition, epiregulin caused rapid EGF receptor activation in RASM cells. However, relative levels of EGF receptor tyrosine phosphorylation stimulated by epiregulin were less than those induced by EGF or betacellulin. Taken together, these results indicate that epiregulin is a potent VSMC-secreted mitogen, induced in common by AII, ET-1, and alpha-Thr, that may contribute to VSMC proliferation and vascular remodeling stimulated by vasoactive agonists.

3T3 Cells↗
PubMed1998

Conservation of read-through transcription of the Drosophila serendipity genes during evolution is gratuitous.

The Drosophila melanogaster serendipity (sry) genes sry alpha and sry beta, which code for unrelated proteins and functions, are transcribed both as monocistronic (sry alpha, srv beta) and dicistronic (sry beta-sry alpha) polyadenylated transcripts, the three types of transcripts being differentially expressed throughout development. We show here that, while the sry gene cluster is conserved in two other distantly related Drosophila species, sry beta-sry alpha dicistronic transcription is observed in one of them, Drosophila pseudoobscura but not in the other, Drosophila virilis, indicating that this mode of transcription is not subject to selection during evolution. Sequence comparison of the intergenic sry beta-sry alpha region suggests that sry, beta-sry alpha read-through transcription results from interference between initiation of transcription at the sry alpha promoter and 3' processing of the upstream sry beta transcript. From these and previous analyses, we propose that read-through transcription at the sry locus reflects its clustered gene organisation but is functionally gratuitous. This conclusion is supported by the autonomous rate of evolutionary sequence divergence shown by each individual sry gene.

Animals↗
PubMed1997

Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo.

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.

Activins↗
PubMed1997

Body position effects on sternocleidomastoid and masseter EMG pattern activity in patients undergoing occlusal splint therapy.

This study was conducted in order to determine the effects of body position on electromyographic (EMG) activity of sternocleidomastoid and masseter muscles, in 15 patients with myogenic cranio-cervical-mandibular dysfunction undergoing occlusal splint therapy. EMG activity was recorded by placing surface electrodes on the sternocleidomastoid and masseter muscles (contralateral to the habitual sleeping side of each patient). EMG activity at rest and during swallowing of saliva and maximal voluntary clenching was recorded in the following body positions: standing, supine and lateral decubitus. In the sternocleidomastoid muscle significant higher EMG activities at rest and during swallowing were recorded in the lateral decubitus position, whereas during maximal voluntary clenching EMG activity did not change. In the masseter muscle significant higher EMG activity during maximal voluntary clenching in a standing position was observed, whereas EMG activity at rest and during swallowing did not change. The opposite pattern of EMG activity supports the idea that there may exist a differential modulation of the motor neuron pools of the sternocleidomastoid and masseter muscles, of peripheral and/or central origin. This suggests that the presence of parafunctional habits and body position could be closely correlated with the clinical symptomatology in these muscles in patients with myogenic craniomandibular dysfunction.

Adult↗
PubMed1996

Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109. A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall.

Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B. subtilis. This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S. cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast. Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O. xanthineolytica LLG109. The amplified fragment has been cloned and sequenced. The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O. xanthineolytica LLG109. By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O. xanthineolytica LLG109. All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions. The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids. The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced. The cloning of the lytic beta-1,3-glucanase from O. xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned. The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation. In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S. cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.

Amino Acid Sequence↗
PubMed1996

Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Actinomycetales↗
PubMed1995

Separate cis-regulatory sequences control expression of serendipity beta and janus A, two immediately adjacent Drosophila genes.

The genes janus (jan) A and B, and serendipity (sry) beta and delta are two pairs of duplicated genes that are adjacent to each other on the third chromosome of Drosophila melanogaster. The jan A and sry beta genes are expressed throughout development in both males and females. They are transcribed in opposite orientations from start sites separated by only 173 bp of DNA. We report here the complete sequence of the jan A and B genes in Drosophila pseudoobscura, a species distantly related to D. melanogaster in which the overall organization of the sry beta, jan A and jan B genes is identical to that in D. melanogaster. Sequence comparison of the jan A-sry beta intergenic region and 5'-transcribed domain of each gene between D. melanogaster and D. pseudoobscura reveals short stretches of conserved sequences that may correspond to cis-acting regulator elements. In order to test the possibility that some cis-acting regulatory sequences are shared by the two genes, we carried out a deletion analysis of the jan A/sry beta intergenic region in D. melanogaster using transgenic lacZ fusion genes. Our results show that sry beta cis-acting sequences are located in the (-117; +137) 5'-region of the gene and that jan A cis-regulatory sequences are included in the (-56; +151) 5'-domain of this gene. Together these data indicate that in spite of the physical proximity of the jan A and sry beta genes, their transcription is regulated by separate cis-acting sequences.

Amino Acid Sequence↗
PubMed1995

Orally active endothelin receptor antagonist BMS-182874 suppresses neointimal development in balloon-injured rat carotid arteries.

Vascular smooth muscle cell (SMC) proliferation is an important component in the development of restenosis. Because endothelin (ET) has been reported to act as an SMC mitogen, we postulated that the orally active ETA receptor antagonist BMS-182874 would suppress the development of the intimal lesion that develops in rat carotid arteries after balloon injury. Using cultured rat aortic SMC, we noted that ET-1-stimulated increases in [3H]thymidine incorporation were blocked by BMS-182874. To determine the effect of the drug on intimal lesion formation, we treated rats with BMS-182874 (100 mg/kg orally, p.o.) or vehicle once daily for 3 weeks, beginning 1 week before balloon injury. Two weeks after injury, drug-treated rats had a 35% decrease in lesion area and a 34% decrease in the lesion/media ratio as compared with the vehicle-treated rats. In situ hybridization (ISH) analysis of balloon-injured rat carotid arteries showed an increase in ETA receptor mRNA. These data support the concept that ETA receptor activation contributes to intimal lesion formation by promotion of SMC proliferation and suggest a potential use for ETA receptor antagonists in the amelioration of hyperproliferative vascular diseases, including restenosis.

Administration, Oral↗