Biomedical subjects

Francisco Parra

Publications and source records attributed to Francisco Parra.

16 recordsLinked to original sources

PubMed2006

NMR analysis of carbohydrate-protein interactions.

Carbohydrate-protein interactions are frequently characterized by dissociation constants in the microM to mM range. This is normally associated with fast dissociation rates of the corresponding complexes, in turn leading to fast exchange on the nuclear magnetic resonance (NMR) chemical shift time scale and on the NMR relaxation time scale. Therefore, NMR experiments that take advantage of fast exchange are well suited to study carbohydrate-protein interactions. In general, it is possible to analyze ligand binding by observing either protein signals or ligand resonances. Because most receptor proteins to which carbohydrates bind are rather large with molecular weights significantly exceeding 30 kDa, the analysis of the corresponding protein spectra is not trivial, and only very few studies have been addressing this issue so far. We, therefore, focus on NMR experiments that employ observation of free ligand, that is, carbohydrate signals to analyze the bound state. Two types of NMR experiments have been extremely valuable to analyze carbohydrate-protein interactions at atomic resolution. Whereas transferred nuclear Overhauser effect (NOE) experiments deliver bioactive conformations of carbohydrates binding to proteins, saturation transfer difference (STD) NMR spectra provide binding epitopes and valuable information about the binding thermodynamics and kinetics. We demonstrate the power of a combined transfer NOE/STD NMR approach for the analysis of carbohydrate-protein complexes using selected examples.

Carbohydrate Conformation↗

PubMed2006

Structure and function of RNA replication.

Contrary to their host cells, many viruses contain RNA as genetic material and hence encode an RNA-dependent RNA polymerase to replicate their genomes. This review discusses the present status of our knowledge on the structure of these enzymes and the mechanisms of RNA replication. The simplest viruses encode only the catalytic subunit of the replication complex, but other viruses also contribute a variable number of ancillary factors. These and other factors provided by the host cell play roles in the specificity and affinity of template recognition and the assembly of the replication complex. Usually, these host factors are involved in protein synthesis or RNA modification in the host cell, but they play roles in remodeling RNA-RNA, RNA-protein, and protein-protein interactions during virus RNA replication. Furthermore, viruses take advantage of and modify previous cell structural elements, frequently membrane vesicles, for the formation of RNA replication complexes.

Genome, Viral↗

PubMed2005

The recombinant rabbit hemorrhagic disease virus VP60 protein obtained from Pichia pastoris induces a strong humoral and cell-mediated immune response following intranasal immunization in mice.

Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.

Administration, Intranasal↗

PubMed2005

Isolation and characterization of a new Vesivirus from rabbits.

This report describes the isolation, cDNA cloning, complete genome nucleotide sequence, and partial characterization of a new cultivable calicivirus isolated from juvenile feeder European rabbits (Oryctolagus cuniculus) showing symptoms of diarrhea. Absence of neutralization by type-specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons of the open reading frame 1-encoded polyprotein with those of other caliciviruses demonstrate that this new calicivirus is a putative novel member of the Vesivirus genus which is closely related to the marine calicivirus subgroup. According to its putative classification, this new virus has been named rabbit vesivirus.

Animals↗

PubMed2005

High-level expression and immunogenic properties of the recombinant rabbit hemorrhagic disease virus VP60 capsid protein obtained in Pichia pastoris.

The VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) (Spanish isolate AST/89) was cloned and expressed in Pichia pastoris. The transformed yeast was grown at high cell density and an expression level of about 1.5 g VP60L(-1) culture was obtained. The protein was detected associated with the cell debris fraction of the recombinant yeast after mechanical disruption. It was purified by a simple method and was obtained N-glycosylated with purity of approximately 70% as deduced from densitometry scan analysis. The recombinant product was antigenically similar to the native capsid protein as determined with polyclonal antibodies obtained from rabbits vaccinated with VP60 protein purified from native virus. The immunogenicity of VP60 protein purified from P. pastoris was demonstrated by ELISA in a vaccination experiment conducted with two groups of rabbits subcutaneously immunized. Animals vaccinated with VP60 in Freund's incomplete adjuvant developed a significant (p<0.01) virus-specific antibody response while the group injected with placebo remained seronegative. Preliminary results showed that the antigen administered within the cell debris fraction of the transformed yeast protected rabbits immunized by the oral route against an intramuscular challenge with 100 LD50 (16,000 hemagglutination units) of homologous virus.

Animals↗

PubMed2005

2-Alkoxydihydrocinnamates as PPAR agonists. Activity modulation by the incorporation of phenoxy substituents.

Herein we describe a series of potent and selective PPARgamma agonists with moderate PPARalpha affinity and little to no affinity for other nuclear receptors. In vivo studies in a NIDDM animal model (ZDF rat) showed that these compounds are efficacious at low doses in glucose normalization and plasma triglyceride reduction. Compound 1b (LY519818) was selected from our SAR studies to be advanced to clinical evaluation for the treatment of type II diabetes.

Animals↗

PubMed2004

Nisin-controlled expression of Norwalk virus VP60 protein in Lactobacillus casei.

A food-grade strain with nisRK stably integrated into the genome, was constructed in order to implement the nisin-controlled expression system (NICE) in Lactobacillus casei ATCC393. Expression of beta-glucuronidase (gus) reporter gene was employed to optimize the system, which has been successfully used to produce the main antigenic protein from Norwalk virus, opening new perspectives for producing edible vaccines.

Anti-Bacterial Agents↗

PubMed2004

Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft.

Norwalk virus is a major cause of acute gastroenteritis for which effective treatments are sorely lacking. To provide a basis for the rational design of novel antiviral agents, the main replication enzyme in Norwalk virus, the virally encoded RNA-dependent RNA polymerase (RdRP), has been expressed in an enzymatically active form, and its structure has been crystallographically determined both in the presence and absence of divalent metal cations. Although the overall fold of the enzyme is similar to that seen previously in the RdRP from rabbit hemorrhagic disease virus, the carboxyl terminus, surprisingly, is located in the active site cleft in five independent copies of the protein in three distinct crystal forms. The location of this carboxyl-terminal segment appears to interfere with the binding of double-stranded RNA in the active site cleft and may play a role in the initiation of RNA synthesis or mediate interactions with accessory replication proteins.

Animals↗

PubMed2004

Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on (-)sense genomic RNA: mapping of a subgenomic promoter.

Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3' one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3'-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (-)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (-)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae.

Animals↗

PubMed2004

Etiology of sporadic cases of pediatric acute gastroenteritis in asturias, Spain, and genotyping and characterization of norovirus strains involved.

From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%), Campylobacter spp. (28.8%), and Salmonella spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and Yersinia spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.

Acute Disease↗

PubMed2004

A recombinant antigen recognized by Fasciola hepatica-infected hosts.

This work reports the detection of specific immunoglobulins (Ig) against rFh8, a recombinant Fasciola hepatica adult worm excretion-secretion antigen, in sera from experimentally (rabbit, Wistar rat, cattle, and sheep), or naturally (human) infected hosts. In the case of laboratory experimental models the study revealed significant differences between rabbits, which recognized the recombinant antigen all along the infection, and Wistar rats, which showed high anti-rFh8 Ig levels only for a short period of the infection. Available sera from experimentally infected cattle and sheep, as well as sera from naturally F. hepatica-infected humans, also contained significant levels of Ig against rFh8, suggesting that Fh8 was produced by F. hepatica at a very early stage of infection in all hosts so far analyzed and that the rFh8 antigen could be used as a tool for the diagnosis of F. hepatica infections.

Animals↗

PubMed2003

Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica.

A Fasciola hepatica cDNA clone of 1752 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 489 codons which encoded a 55 kDa polypeptide, showing a high degree of homology to protein disulfide isomerases. This putative antioxidant protein cDNA was expressed in Escherichia coli as a GST fusion protein. The cleaved recombinant protein was shown to be biologically active in vitro by mediating the oxidative refolding of reduced RNase. Immunoblotting studies using a specific antiserum raised against the recombinant protein showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The extracellular location of this protein was also supported by the specific immune responses found against this protein in F. hepatica experimentally infected rabbits.

Amino Acid Sequence↗

PubMed2003

Oral immunization using tuber extracts from transgenic potato plants expressing rabbit hemorrhagic disease virus capsid protein.

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.

Animals↗

PubMed2002

Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC.

Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp. mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster. In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals. Using this strategy, a genome fragment has been isolated and characterised. The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E. coli. The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp. capricolum (Mcc). Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster. The full recombinant EF-Tu was produced in E. coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.

Amino Acid Sequence↗

PubMed2002

Psychotropic drugs and the ECG: focus on the QTc interval.

The QT interval measuring depolarisation and repolarisation has, when lengthened, been implicated as a risk factor for the development of torsades de pointes and sudden death, particularly in patients predisposed to these complications due to cardiovascular impairment. Since some of the medications used in psychiatry have been implicated, an extensive review of available literature was made of the major classes, including antipsychotics, antidepressants, lithium, anticonvulsants and benzodiazepines. Further, where no publications were found on a particular medication, the pharmaceutical firms responsible for these items were contacted concerning possibly unpublished data. Results of the survey indicate that there may be difficulty in one of three situations: immediate (in the first minutes to hours after oral or parenteral administration), short-term use of 4 - 12 weeks or long-term use of 6 months. Based on this approach, the greatest concern is directed at the immediate application of haloperidol, droperidol, pimozide and trazodone, the short-term use of thioridazine, pimozide, sertindole, nortriptyline, clomipramine, doxepin and the long-term use of clozapine, olanzapine and carbamazepine. It is of interest that a reduction in QTc is reported with aripiprazole. Among the antidepressants, the tertiary tricyclic antidepressants (imipramine, amitriptyline and doxepin) appear to have a more general impact, while the secondary tricyclic antidepressants (nortriptyline, desipramine) may impact more on children and the elderly. Among other antidepressants, the only reports of torsades de pointes appeared to occur with mirtazapine. It was also of interest to find data showing no effect or reductions in QTc produced by sertraline, citalopram, paroxetine and bupropion in multiple studies. Effects of medications on other heart parameters are also briefly reviewed. In particular, the safety of sertraline in post-MI patients and of bupropion in heart disease patients is highlighted. Little information was available on other classes of medications used in psychiatric disorders. What is available concerning lithium, the anticonvulsants and the benzodiazepines indicates little effect on the QTc, although there may be effects on other cardiovascular parameters.

Adolescent↗

PubMed2001

Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase.

The structure of the RNA-dependent RNA polymerase (RdRP) from the rabbit hemorrhagic disease virus has been determined by x-ray crystallography to a 2.5-A resolution. The overall structure resembles a "right hand," as seen before in other polymerases, including the RdRPs of polio virus and hepatitis C virus. Two copies of the polymerase are present in the asymmetric unit of the crystal, revealing active and inactive conformations within the same crystal form. The fingers and palm domains form a relatively rigid unit, but the thumb domain can adopt either "closed" or "open" conformations differing by a rigid body rotation of approximately 8 degrees. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important to enzymatic function in RdRPs. Comparisons between the structures of the alternate conformational states of rabbit hemorrhagic disease virus RdRP and the structures of RdRPs from hepatitis C virus and polio virus suggest novel structure-function relationships in this medically important class of enzymes.

Animals↗