Biomedical subjects
Publications and source records attributed to A Nieto.
Adding glucose to Saccharomyces cerevisiae cells growing among nonfermentable carbon sources leads to glucose repression. This process may be resolved into several steps. An early repression response requires any one of the three glucose kinases present in S. cerevisiae (HXK1, HXK2, or GLK1). A late response is only achieved when Hxk2p is present.
We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.
The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.
We assessed the potential of 99mTc labelled specific polyclonal antibodies (99mTc-PoAb) for the diagnosis of hydatid disease by immunoscintigraphy. Experimentally infected mice and rabbits were used for this purpose. A specific rabbit antibody recognizing total somatic antigen from hydatid membranes (HCMA) was obtained. PoAb biological activity before labelling was checked according to Barbieri et al. 99mTc-PoAb labelling was performed according to Thakur et al.; the radiochemical purity was higher than 90%. The following studies of 99mTc-PoAb were made: post-labelling biological activity; in vitro stability; blood and renal kinetics in normal mice up to 24 hours after intravenous (i.v.) and intraperitoneal (i.p.) administration; biodistribution in normal and infected mice after i.p. or i.v. injection, and in rabbits after i.v. administration. Biodistribution studies in normal mice, after both administration routes, showed considerable hepatic uptake of activity. An important uptake in cysts after i.p. administration in mice, indicating successful targeting, was also confirmed by autoradiography images. Intravenously administered 99mTc PoAb was not significantly targeted to peritoneal cysts in either animal species, due to inherent limitations to these animal models. Results obtained with i.p. administration suggest that specific hydatid imaging may be possible. Both the mice and rabbit models revealed hepatic uptake which, combined with the short isotope half-life, prevent the drawing of any final conclusions regarding the usefulness of 99mTc-labelling in hydatid disease.
OBJECTIVE: To compare the efficacy, tolerance and recurrence rate of endometriosis after 5-year follow-up of treatment with Gestrinone and Buserelin, respectively. STUDY DESIGN: A prospective study with randomized follow-up of 5 years duration (minimum) for each patient was done. We included 43 cases of endometriosis diagnosed by laparoscopy or laparotomy and treated them with Gestrinone (Group G, n = 25 cases) or Buserelin intranasal spray (Group B, n = 18) for 6 months. RESULTS: General data: Age, height, weight of patients and AFS score of endometriosis were without significant differences in either group. Specific data: A) Global clinical efficacy was good or excellent in 74% (16/25) of group G and in 78% (14/18) of group B without significant differences. B) Global clinical tolerance was good in 50% of the patients in group G and in 0% in group B (p < 0.001). C) Global evaluation after 5-year follow-up showed "success" only for 36% of patients in group G and in 33% in group B (no significant differences), with "failure" in 40% and 33%, respectively (no significant differences). CONCLUSIONS: 1) Gestrinone and Buserelin intranasal spray are valid treatments for the remission of endometriosis, with "success", "failure" and "clinical recurrence" rates similar after a follow-up of 5 years of initial treatment. 2) The most significant androgenic effect of Gestrinone was the presence of acne. Vascular effects were also considered as very undesirable effects according to the comments of patients. On the contrary, the effects of analogs are generally better tolerated.
The objective of our study was to analyse the fluctuation of cardiotocographic scores during labor in fetal growth retardation (FGR). The study took place at the University hospital "Principe de Asturias", Alcalá de Henares, Madrid, Spain. 170 at term FGR fetuses and 170 at term fetuses without FGR as control group were compared using modified Fischer scores, which were blindly performed at 3, 5 and 10 cm of cervical dilatation. As results we found out that the mean value of the Fischer score was significantly lower in FGR at 3 cm of cervical dilatation (8.1 +/- 1 vs 8.7 +/- 0.6), as well as at 5 cm (7.6 +/- 0.9 vs 8.1 +/- 0.7) and at 10 cm (6.7 +/- 0.8 vs 7.4 +/- 0.8). Poor prognosis cardiotocograms were also more common in FGR than in the control group in the three cut-offs points studied (7.5% vs 0.6% at 3 cm, 9.8 vs 1.8 at 5 cm and 37.9 vs 11.6 at complete cervical dilatation). The afore mentioned differences were more remarkable as labor advanced. Conclusions are that poor fetal heart rate recordings were detected in FGR at the beginning of labor when compared with control group fetuses and the differences between both groups increased while the labor prolonging.
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Cisplatin-based induction chemotherapy has been extensively tested in nasopharyngeal carcinoma for the improvement of local and systemic control and survival of this disease. In this study, we report the results of the treatment with induction chemotherapy in 40 patients with locally advanced carcinoma of the nasopharynx (LANPC) with four courses of cisplatin (P) 25 mg/m2 per day and 5-fluorouracil (F) 1000 mg/m2 per day both in a 4-days continuous infusion, with or without leucovorin (L) 250 mg/m2 per day in 2-hour infusion at the beginning of daily administration of PF, followed by sequential radiotherapy. All except one were in stage IV. The overall response after induction chemotherapy was 93%, with 55% CR and 38% PR. Definitive overall response after radiotherapy was 98%, with 80% CR and 18% PR. At a maximum follow up of 11 years, the overall survival rate is 55%. Induction chemotherapy with continuous infusion of PF with or without leucovorin followed by radiotherapy is a highly active regimen for the treatment of locally advanced nasopharyngeal carcinoma with response and survival rates comparable to other combinations of sequential or simultaneous chemotherapy and radiotherapy.
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Microsomes and plasmatic membranes from rat liver bind radioactive uteroglobin (UG) in vitro with high affinity (Kd = 1.7 x 10(-10) M. The binding is saturable and specific and dependent on previous reduction of UG with dithiothreitol. Microsomes from rat spleen or lung or from rabbit endometrium also possess a similar ability. Binding capacity is not affected by previous treatment of microsomes with phospholipase A2 or peptide-N-glycosidase F but is lost after brief treatment with trypsin. The complex formed between UG and the binding component can be solubilized from microsomes with 5 mM CHAPS and it elutes with an apparent Mr of 90,000 in a Sephacryl 200 column. The complex is resistant to 8 M urea but is completely dissociated by Triton X-100. The UG-binding protein(s) has been partially purified from solubilized microsomes and membranes by affinity chromatography. The results are discussed in relation to a possible physiological effect of UG on cellular membranes.
Complement has been shown to lyse protoscoleces of Echinococcus granulosus, but products from this parasite are able to consume complement, and this has been proposed as an evasion mechanism. The murine secondary hydatidosis model, with intraperitoneal inoculation, is used in this work to assess the occurrence in vivo of complement consumption by the parasite as well as the role of complement during the establishment of infection. Although the measurement of systemic levels of C3 activation, total C3, and hemolytic complement in challenged mice yielded no evidence of complement consumption, the relevance of local consumption at the site of infection cannot be ruled out. The role of complement during establishment of infection was assessed by comparing parasite burdens in normal and complement-depleted mice. Complement depletion by treatment with cobra venom factor caused a 79% reduction in cyst numbers (P < 0.05). Possible explanations of this unexpected result are discussed. The results presented suggest that lysis or opsonization by host complement are not effective against the establishing parasite in this model. They also indicate the significance of complement activation by the parasite needs to be studied at a local level.
Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively. Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids. All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1-113 of the PB2 protein. Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates. The application of these antibodies as research and diagnostic tools is discussed.
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With the aim of studying whether a general vaccination against invasive disease caused by Haemophilus influenzae type b (Hib) is economically profitable bearing in mind the efficacy and safety of the vaccine, its price and the global cost that this disease has in our area, a review is conducted of patients admitted due to invasive disease caused by Hib in the Children's Hospital La Fe, Valencia, born between 1984 and 1993. They total 100, 63 who have meningitis. In the 81 cases (56 with meningitis) born between 1984 and 1990 (years that can be regarded as "closed" since all the patients were younger than 5 years of age) the total cost has been calculated for hospitalization, care during the acute phase, care for the sequelae (6 severe and 7 mild) and death (5 cases). The mean annual cost of care can be calculated at 62 million pesetas, without making an economic valuation of the loss of life, and at 205 million pesetas taking this factor into account. The annual cost of vaccinating the 7000 babies under one year of age and falling within the Hospital's catchment area, on the basis of a vaccination pattern of three doses (at 2, 4 and 6 months) or four doses (at 2, 4, 6 and 15 months) would amount to 63 or 84 million pesetas, normal price to public (not covered by National Health Service), and 40 or 51 million pesetas if acquired by National Health Service. As a conclusion we can state that, even from the economic point of view, without quantifying the cost of the loss of life, a public general anti-Hib vaccination would be profitable in our area since it would mean an administration cost lower than that of the care required by patients. This is without taking into account the fact that emotional, family and social serious disturbances would also be avoided due to hospitalization, sequelae and deaths caused by a disease which is today perfectly preventable.
Specific anti-Toxoplasma gondii antibodies were analyzed by latex agglutination during the 5 months of pregnancy in the serum of sheep from two flocks in the Uruguayan north-west: one flock (79 sheep) kept under an intensive management system and the other (494 sheep) kept under an extensive management system. Titers obtained using the latex test correlated with those obtained by indirect immunofluorescence. Pregnancy was confirmed by laparoscopy in all sheep from the smaller flock and by determination of serum progesterone levels in the seroconverted sheep from the larger flock. The percentage of sheep originally exhibiting significant serum levels of anti-T. gondii antibodies (13.9% in the smaller flock and 28.5% in the larger one) as well as the observed levels of seroconversion (22.8% and 7.7%, respectively), indicated a high prevalence of this infection in the north-west of Uruguay. In addition, birth rates of seroconverted sheep in the larger flock were significantly different from those of non-seroconverted animals. These results, although preliminary, suggest that Toxoplasma infection would produce considerable economic losses in extensive ovine production in Uruguay.
In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit.
In this work we analysed the modulation of the antibody response to Echinococcus granulosus antigens via anti-idiotype (Ab2) administration. In a first set of experiments, we determined the antibody response to hydatid cyst fluid antigen (HCFA) of mice immunized with Ab2. Results showed that Ab2 elicited anti-HCFA antibodies in mice that had never been exposed to HCFA before. Moreover, that response was characterized by booster effect, avidity maturation and an inverse correlation between avidity and Ab2 dose. We infected these mice and measured the titre and avidity of anti-HCFA antibodies during 250 days of infection. Lack of avidity maturation was the most important feature observed, suggesting that the parasite (either protoscolex or cyst) could modulate the antibody response of its host. In a second set of experiments, we investigated the presence of regulatory anti-idiotypes in the Ab2 preparation. In this context we treated neonates with different doses of Ab2, immunized them with HCFA 12 weeks later and determined their anti-HCFA titres. A specific and Ab2 dose-dependent suppression of the response to HCFA was observed, suggesting the existence of regulatory anti-idiotypes in the Ab2 preparation. Although this Ab2 could be involved in the regulation of the anti-HCFA response, other anti-idiotypes could also be involved. Our results show that it may be possible to improve the avidity of the anti-HCFA response via the administration of the anti-idiotype Ab. However, the live parasite could successfully revert this effect by mechanisms not yet characterized.