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Biomedical subjects

A Puig

Publications and source records attributed to A Puig.

At least 19 recordsLinked to original sources
PubMed2001

Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis.

A method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/100ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a significant increase (more than 100%) in the number of phages detected. No other changes in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the culture conditions and media described, taking into consideration that bile renders the media cloudy making it difficult to follow the host growth and that most laboratories do not have the facilities to work under strict anaerobic conditions. Nalidixic acid (100 microg/ml) and kanamycin (100 microg/ml) in the assay medium significantly reduce the background flora from polluted samples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at (-70+/-10) degrees C with BSA-sucrose as cryoprotector, storing of 1-2 ml in glass vials at (-70+/-10) degrees C and using them directly to inoculate fresh broth allows the obtention of cultures ready for phage enumeration in about 2.5 h. All these developments have been incorporated into a procedure that makes the method for detecting phages infecting B. fragilis as workable as the standardised methods available for the detection of coliphages.

Anaerobiosis↗
PubMed2000

Nonepileptic seizures in pregnancy.

The authors report five patients with recurrent psychogenic seizures (PS) during pregnancy, with multiple emergency room visits and continued intake of antiepileptic drugs obtained from various sources, despite awareness of the psychogenic nature of their attacks and the risks of antiepileptic drug use in pregnancy. These patients demonstrate that preexisting PS may persist during pregnancy, and there will be patients who continue to take antiepileptic drugs despite awareness of the risks inherent to these treatments. New-onset or persisting PS with pregnancy can be indicative of serious emotional conflicts, and the child should be considered at risk.

Adolescent↗
PubMed1999

Diversity of bacteroides fragilis strains in their capacity to recover phages from human and animal wastes and from fecally polluted wastewater.

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10(1) to approximately 10(3) phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.

Animals↗
PubMed1999

Mirtazapine in major depression with comorbid generalized anxiety disorder.

BACKGROUND: A high proportion of patients with generalized anxiety disorder (GAD) have comorbid depressive illness. The presence of anxiety in depression has significant prognostic implications. Because of mirtazapine's early anxiolytic effects, the present study was undertaken as a preliminary investigation in patients with a diagnosis of major depression with comorbid GAD. METHOD: Mirtazapine was administered to 10 patients with DSM-IV major depressive disorder and comorbid GAD in an 8-week open-label study. Mirtazapine was increased from an initial daily dose of 15 mg to a maximum daily dose of 45 mg. RESULTS: Patients were found to have significant reductions in Hamilton Rating Scale for Depression scores, Hamilton Rating Scale for Anxiety scores, and Beck Depression Inventory scores, with improvement noted after the first week of therapy and continuing improvement over the 8 weeks of study. CONCLUSION: These positive preliminary findings support the further investigation of mirtazapine's potential value as a treatment for generalized anxiety disorder in addition to its established efficacy as an antidepressant drug.

Adult↗
PubMed1997

A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities.

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.

Amino Acid Sequence↗
PubMed1996

Wastewater from the manufacture of rubber vulcanization accelerators: characterization, downstream monitoring and chemical treatment.

The content of wastewater resulting from the manufacture of rubber antioxidants and accelerators by a factory situated in the Ebro basin (Spain) has been determined using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The change in the pollutants was studied in the riverbed via two modules which continuously gathered pollutants on various solid supports (activated carbon and XAD-2 resins). These modules were located in Bocal Station, lying a further 100 km downstream from the factory, and from the Zaragoza water supply. Forty-six different compounds were identified at Bocal Station, the majority resulting from the production of rubber additives. Due to the immunity of different waste substances, and to the toxic nature of some, we studied their reaction when subjected to techniques of chemical oxidation using ozone.

Antioxidants↗
PubMed1995

Simple concentration method for bacteriophages of Bacteroides fragilis in drinking water.

A membrane of inorganic material with a honeycomb pore structure was used to concentrate phages infecting Bacteroides fragilis from drinking water. Phages were removed from the membrane with 0.25 M glycine buffer pH 9.5. Phages were not inactivated by storage in this buffer neutralized to pH 7.0 for at least 9 days at 4 degrees C. The method allows recovery of around 50% in drinking water. When the turbidity of the water increased, the efficiency of the concentration decreased markedly. The efficiency of concentration was evaluated versus a presence/absence test in 317 water samples with turbidity level below the threshold of drinking water. Results obtained by concentration of 11 provided data which were significantly more informative than the presence/absence tests carried out on 100 ml. A number of additional tests carried out with both somatic and F-specific coliphages indicated that these conclusions can be extended to these groups of bacteriophages.

Bacteriophages↗
PubMed1995

[Patients with chronic airflow limitation: effects of the inspiratory muscle training with threshold load valve, built with appropriate technology, associated to nutritional support].

AIM: To assess prospectively the effects of a controlled program of inspiratory muscle training program and nutritional support in patients with chronic obstructive lung disease (COPD). PATIENTS AND METHODS: Twenty-three patients with COPD were randomly assigned into four groups. Group I received a 1000 kcal/day nutritional supplement, given as a casein based enteral nutritional formula; group III was subjected to inspiratory muscle training, using an inexpensive pressure threshold load valve constructed according to the Appropriate Technology principles of the WHO, adjusted at 30% of Maximal Inspiratory Mouth Pressure and received also the nutritional supplement; group IV was trained but did not receive the nutritional supplement and group II was not trained nor supplemented. Patients were studied during three months and monthly, inspiratory muscle function, exercise capacity and anthropometry were measured. RESULTS: A significant improvement in exercise capacity, maximal inspiratory pressure and inspiratory muscle endurance was observed in the four groups throughout the study. Trained subjects had greater improvement in their inspiratory muscle endurance, compared to untrained subjects. Nutritional support had no effect in inspiratory muscle function or exercise capacity. No changes in anthropometric measures were observed. CONCLUSIONS: The pressure threshold load valve used in this study, improved inspiratory muscle endurance and nutritional support had no effect in patients with COPD.

Aged↗
PubMed1994

Anti-chaperone behavior of BiP during the protein disulfide isomerase-catalyzed refolding of reduced denatured lysozyme.

Folding catalysts of the endoplasmic reticulum (ER), such as protein disulfide isomerase (PDI), accelerate the slow chemical steps, such as disulfide bond formation, that accompany protein folding. Molecular chaperones of the ER, notably the heavy chain-binding protein, BiP (grp78), bind and release unfolded proteins in an ATP-dependent fashion. In vitro, the fate of reduced, denatured lysozyme is dependent on whether the substrate interacts first with BiP or PDI. Depending on the ratio of PDI to substrate and order in which the components of the reaction are mixed, PDI can exhibit a foldase/chaperone activity, which increases the rate and extent of lysozyme refolding, or it can function as an anti-chaperone that promotes the formation of inactive, disulfide-linked lysozyme aggregates (Puig, A., and Gilbert, H.F. (1994) J. Biol. Chem. 269, 7764-7771). Reduced, denatured lysozyme, but not the native protein, interacts with BiP and efficiently stimulates its peptide-dependent ATPase activity. When present at substoichiometric amounts, BiP, like PDI, facilitates the formation of large, inactive lysozyme aggregates that are non-covalently associated with BiP. BiP and PDI compete for a limited number of sites in these insoluble aggregates. If BiP is present at a high molar excess, the chaperone binds unfolded lysozyme and inhibits its aggregation by maintaining it in a soluble, yet inactive, conformation, both in the presence or absence of ATP. Increasing concentrations of BiP decrease the extent, but not the initial rate, of refolding, suggesting that BiP and PDI compete for unfolded lysozyme and that the BiP-lysozyme complex is not a very good substrate for PDI either in the presence or absence of ATP. Depending on the BiP and PDI concentrations, unfolded lysozyme may either be efficiently refolded into the native conformation in a PDI-catalyzed reaction, or it may form both soluble and insoluble BiP-lysozyme complexes. In vitro, PDI- and BiP-facilitated aggregation, as well as the competition of the two proteins for substrate, reproduces many of the features of the quality control system of the ER.

Adenosine Triphosphate↗
PubMed1994

The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase.

The complexity of protein folding is often aggravated by the low solubility of the denatured state. The inefficiency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and pathways leading to the native structure. Protein disulfide isomerase (PDI), a resident foldase of the endoplasmic reticulum, catalyzes the in vitro oxidative refolding of reduced, disulfide-containing proteins, including denatured lysozyme. Depending on the concentrations of foldase and denatured substrate and the order in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem 269, 7764-7771). PDI's chaperone activity leads to quantitative recovery of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorporate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperone activity and behaves as an anti-chaperone at all substrate and PDI concentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but they are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennarz, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfide isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme-PDI aggregates are disulfide cross-linked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PDI into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti-chaperone activity. PDI's anti-chaperone activity could serve as a quality control device by providing an efficient mechanism to retain misfolded proteins in the endoplasmic reticulum (Marquardt, T., and Helenius, A. (1992) J. Cell. Biol. 117, 505-513).

Animals↗
PubMed1994

Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.

Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit "anti-chaperone" activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).

Animals↗
PubMed1994

New alleles at microsatellite loci in CEPH families mainly arise from somatic mutations in the lymphoblastoid cell lines.

In the analysis of 40 CEPH families, under the EUROGEM project, with a total of 29 microsatellites (26 CA-repeats, a TCTA-repeat within the vWFII-3 gene, a TTA-repeat within the PLA-2 gene, and an AAAT-repeat intragenic to the NF1 gene) from human chromosomes 12, 17, and 21, we have detected 21 cases of abnormal segregation of alleles in 16 pedigrees for a total of 14 markers (48%). In 11 cases, the abnormal transmissions were of somatic origin, 10 of which (91%) occurred in the lymphoblastoid cell lines. In 9 other cases, it was not possible to determine if the origin of the new alleles was somatic or germline, and in one case hemizygosity in several family members was observed, so its origin was germline. The 20 new mutations detected in the 22,852 meioses analysed represent a mutation frequency of 8.7 x 10(-4) per locus per allele. The germline mutation rate could be as high as 3.9 x 10(-4) per locus per gamete (from 0 to 3.9 x 10(-4)), but the rate of somatic mutations detected in the study was much higher (4.8 x 10(-4) to 8.7 x 10(-4) per locus per allele). Individual mutation rates ranged from 0 to 3.8 x 10(-3). Among the markers analysed, all three that were tri- or tetranucleotide repeats showed one or two new alleles, compared to only 10 of the 26 (38%) CA-repeats showing mutations. Three CEPH families (102, 45 and 1333) each had several mutational events, and one individual (10210) had somatic mutations for two microsatellites from different chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

[Hepatitis C virus infection in patients with porphyria].

Hepatitis C virus antibodies were studied in sera coming from 39 patients with porphyria (cutanea tarda in 17, variegate in 8, intermittent acute in 4, coproporphyria in 2 and protoporphyria in 8). Nine of 17 patients with porphyria cutanea tarda had positive antibodies, but none of the patients with other types of porphyria. All subjects with porphyria cutanea tarda had histological or laboratory liver abnormalities. There was no relationship between the presence of antibodies and frequency of alcoholism, diabetes, or carbohydrate intolerance. Family background of porphyria was significantly less frequent among patients with positive hepatitis C virus antibodies. In 13 patients, a liver biopsy was performed, always showing signs of chronic hepatitis, whose magnitude was higher in those with positive antibodies. It is concluded that, as reported previously, hepatitis C virus may be an activating factor for porphyria cutanea tarda or may potentiate its accompanying liver disease.

Enzyme-Linked Immunosorbent Assay↗