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O Grau

Publications and source records attributed to O Grau.

At least 19 recordsLinked to original sources
PubMed2004

The complete nucleotide sequence of a Spanish isolate of Citrus psorosis virus: comparative analysis with other ophioviruses.

The complete genomic sequence (11278 nt) of Citrus psorosis virus (CPsV), isolate P-121 from Spain, was determined and compared with those from isolate CPV-4 and from other ophioviruses. The three RNAs of P-121 had similar size and identical organization as those of CPV-4. The 24K and the RdRp proteins were potentially encoded in the viral complementary (vc) strand of RNA 1, the 54K protein potentially encoded in vcRNA 2 and the coat protein encoded in vcRNA 3. These four proteins from P-121 and CPV-4 had 87, 92, 93 and 94% amino acid identity, respectively, but only 22, 38, 25 and 33% identity with their homologous proteins from Mirafiori lettuce big vein virus (MLBVV), the only other ophiovirus completely sequenced. Biological and genetic differences between CPsV and MLBVV (and the other ophioviruses), would support their future allocation in different genera within a tentative family Ophioviridae.

Base Sequence↗
PubMed2001

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

Brucella abortus↗
PubMed2000

A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA.

Reverse transcription and polymerase chain reaction (RT-PCR) are being used increasingly for detection and typing RNA viruses. For this purpose, metal block thermal cyclers (MBTC) are considered to provide higher DNA yield, whereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 s denaturation, 0 s annealing, and 4-8 s elongation) was developed to amplify genomic segments from two RNA viruses, which allowed increasing the number of cycles without a parallel increase of non-specific DNA fragments. Under these conditions, 80-90 cycles with the ATC provided a DNA yield close to that of a standard 40-cycles MBTC protocol in about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily.

Citrus↗
PubMed2000

A highly sensitive heminested RT-PCR assay for the detection of citrus psorosis virus targeted to a conserved region of the genome.

Psorosis is a widespread and damaging disease of citrus in many parts of the world. The causal agent is a multipartite virus with RNA genome present in very low concentration in infected citrus tissue. Diagnosis is made by biological indexing on indicator citrus seedlings, but it is a slow and costly procedure and therefore it is not used generally. No sensitive wide-spectrum assay for Citrus Psorosis virus (CPsV) has been reported based on RT-PCR. A highly sensitive heminested RT-PCR assay is described for the detection of CPsV. Fragments of 313 bp amplified from RNA 1 of different isolates were cloned and sequenced. Very high homology was found among six isolates from the citrus producing region of Argentina: 96.6-100% in nucleotide sequence. The consensus sequence obtained was used for the design of the primers for heminested PCR assay. It has been tested on different Argentine isolates, employing various methods for RNA extraction from infected tissue. This test is able to detect CPsV in dilutions of 10(10) of the original sample.

Base Sequence↗
PubMed2000

Cloning and nucleotide sequence of the DNA gyrase (gyrA) gene from Mycoplasma hominis and characterization of quinolone-resistant mutants selected in vitro with trovafloxacin.

We report the cloning and characterization of the gyrA gene of the Mycoplasma hominis DNA gyrase, which was previously shown to be associated with quinolone resistance in this organism. The 2,733-bp gyrA gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kDa. As expected, M. hominis GyrA exhibits higher homology with the GyrA subunits of the gram-positive bacteria Clostridium acetobutylicum, Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus than with its Escherichia coli counterpart. Knowing the entire sequence of the gyrA gene of M. hominis could be very useful for confirming the role of the GyrA subunit in fluoroquinolone resistance. Twenty-nine mutants of M. hominis were selected stepwise for resistance to trovafloxacin, a new potent fluoroquinolone, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. Three rounds of selection yielded 3 first-step, 12 second-step, and 14 third-step mutants. The first-step mutants harbored a single substitution, Glu460-->Lys (E. coli coordinates), in ParE. GyrA changes, Ser83-->Leu, Glu87-->Lys, and Ala119-->Glu or Val, were found only in the second round of selection. At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resistance to fluoroquinolones, appeared. Thus, high-level resistance to trovafloxacin required three steps and was associated with alterations in both fluoroquinolone targets. According to these genetic data, in M. hominis, as in Staphylococcus aureus and Streptococcus pneumoniae, topoisomerase IV seems to be the primary target of trovafloxacin.

Amino Acid Sequence↗
PubMed1999

Human chemokine receptors CCR5, CCR3 and CCR2B share common polarity motif in the first extracellular loop with other human G-protein coupled receptors implications for HIV-1 coreceptor function.

Chemokine receptors (CRs) are 7-helix membrane proteins from the family of G-protein coupled receptors (GPCRs). A few human CRs act as cofactors for macrophage-tropic (M-tropic) human immunodeficiency virus type-1 (HIV-1) entry into cells, while others do not. In this study, we describe an application of molecular modeling techniques to delineate common molecular determinants that might be related to coreceptor activity, and the use of the data to identify other GPCRs as putative cofactors for M-tropic HIV-1 entry. Subsequently, the results were confirmed by an experimental approach. The sequences of extracellular domains (ECDs) of CRs were employed in a compatibility search against a database of environmental profiles derived for proteins with known spatial structure. The best-scoring sequence-profile alignments obtained for each ECD were compared in pairs to check for common patterns in residue environments, and consensus sequence-profile fits for ECDs were also derived. Similar hydrophobicity motifs were found in the first extracellular loops of the CRs CCR5, CCR3, and CCR2B, and are all used by M-tropic HIV-1 for cell entry. In contrast, other CRs did not reveal common motifs. However, the same environmental pattern was also delineated in the first extracellular loop of some human GPCRs showing either high (group 1) or low (group 2) degree of similarity of their polarity patterns with those in HIV-1 coreceptors. To address the question of whether the delineated molecular determinant plays a critical role in the receptor-virus binding, three of the identified GPCRs, bradykinin receptor (BRB2) and G-protein receptor (GPR)-CY6 from group 1, and GPR8 from group 2, were cloned and transfected into HeLa-CD4 cells, which are nonpermissive to M-tropic HIV-1 infection. We demonstrate that, similar to CCR5, the two selected GPCRs from group 1 were capable of mediating M-tropic HIV-1 entry, whereas GPR8 from group 2 did not serve as HIV-1 coreceptor. The potential biological significance of the identified structural motif shared by the human CCR5, CCR3, CCR2B and other GPCRs is discussed.

Amino Acid Sequence↗
PubMed1998

A longitudinal study of seroreactivity against Mycoplasma penetrans in HIV-infected homosexual men: association with disease progression.

We investigated the relationships between a putative cofactor of HIV infection, Mycoplasma penetrans, and the evolution of HIV disease. The evolution of titers of anti-M. penetrans antibodies in 58 randomly selected HIV-seropositive adult homosexual men was investigated. The median length of follow-up was 38 months. Thirty-six individuals was investigated. The median length of follow-up was 38 months. Thirty-six individuals (62.1%) remained M. penetrans seronegative (group 0). Fourteen patients (24.1%) had consistently low antibody titers or low antibody titer(s) in at least one sample and negative test(s) in the other(s). This pattern was possibly associated with latent or earlier infection (group 1). Eight patients (13.8%) had moderate to high antibody titers for long periods, indicating an active and persistent M. penetrans infection (group 2); four patients in this group presented a serological reactivation and thus probably developed an acute infection during the study; two had a stable and moderate level of antibody throughout the study; in two patients the antibody titers decreased substantially. Interestingly, CD4 cell counts declined more rapidly in group 2 than in group 0 (medians of -4.5 versus -2.1 cells/mm3/month, p < 0.05 and -0.16 versus 0 cell percentage/month, p < 0.05), whereas there was no significant difference between groups 1 and 0 (medians of -2.0 versus -2.1 cells/mm3/month and -0.15 versus 0 cell percentage/month). In patients with serological reactivation, the viral load was higher in sera with higher M. penetrans antibody titers. These findings suggest an association between active M. penetrans infection and progression of HIV disease.

AIDS-Related Opportunistic Infections↗
PubMed1998

Genotyping isolates of the entomopathogenic fungus beauveria bassiana by RAPD with fluorescent labels

Random amplified polymorphic DNA (RAPD) with incorporation of fluorescent deoxynucleotides was used to examine the genetic diversity among Beauveria bassiana isolates from Argentina and Brazil. High-resolution DNA fingerprints were generated on line, during polyacrylamide gel electrophoresis of amplification products, by automated laser fluorescence analysis. Each isolate displayed a distinct genotype. Cluster analysis showed a high level of variability among these genotypes. No correlation with geographical origin or host was detected. Nevertheless, a phenetic group of 80% similarity represented mainly the isolates exhibiting high virulence against the sugar cane borer, Diatraea saccharalis. Fluorescence-based RAPD fingerprints provide a useful tool for identifying entomopathogenic fungi, and this technique is specially applicable to screening many isolates in population studies. Copyright 1998 Academic Press.

Journal Article↗
PubMed1997

Cladistic analysis of Tospovirus using molecular characters.

The genus Tospovirus was thought to be composed only of the tomato spotted wilt virus (TSWV), but now at least four Tospovirus species have been proposed based on serological and molecular data. A classification of tospoviruses has been proposed taking into account global similarities of the N gene and N protein sequences of 7 isolates of Tospovirus. Because phylogenetic analyses based on global similarities can lead to classifications which do not mirror the genealogy of the group, we have employed a cladistic analysis using parsimony of this genus with RNA sequences of 450 nucleotides of the N gene from 14 new Argentinean isolates and 4 previously described isolates. Representatives of the Bunyaviridae family, Rift Valley Fever Virus (Phlebovirus) and Bunyamwera (Bunyavirus), were used as the outgroup in separate analyses.

Argentina↗
PubMed1997

High prevalence of antibodies to Mycoplasma penetrans in human immunodeficiency virus-seronegative and -seropositive populations in Brazzaville, Congo.

In industrialized countries, the prevalence of antibodies to Mycoplasma penetrans is higher among human immunodeficiency virus (HIV)-seropositive homosexuals than other HIV-seropositive and HIV-seronegative groups. In an African heterosexual population, we found a higher prevalence of M. penetrans antibodies in HIV-seronegative blood donors (15.5%) than in France (0.9%) or the United States (0.3%) and a prevalence of 13.4% in HIV-seropositive individuals. HIV-seropositive individuals with less than 5% CD4 cells had a higher prevalence of M. penetrans antibodies than individuals with 5% or more CD4 cells (25.0 versus 8.5%).

Adult↗
PubMed1996

Clustering of Argentinean tospoviruses with existing species in the genus by sequence analysis of a 450-nucleotide RNA region of the N gene.

The genomic diversity of Argentine Tospoviruses from different geographical areas, and from several distinct crops was analysed here. For each isolate, RT-PCR, cloning and sequencing of a 450 nt fragment of the N gene were performed. Comparisons of RNA and predicted amino acid sequence identity and similarity were made. A partial sequence of the N gene was able to classify our local isolates within three Tospovirus species previously described (Tomato spotted wilt virus, TSWV; tomato chlorotic spot virus, TCSV and groundnut ringspot virus, GRSV). With the sequence data currently available, we performed a cladistic phylogenetic analysis which gave a possible genealogy among members of the Tospovirus genus.

Amino Acid Sequence↗
PubMed1996

Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products.

Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.

Base Sequence↗
PubMed1996

PCR screening for carriers of bovine leukocyte adhesion deficiency (BLAD) and uridine monophosphate synthase (DUMPS) in Argentine Holstein cattle.

BLAD (Bovine Leukocyte Adhesion Deficiency) and DUMPS (Deficiency of Uridine Monophosphate Synthase) are monogenic autosomal, recessive inherited diseases of Holstein cattle. Single nucleotide changes (point mutations) responsible for the genetic disorders were detected by polymerase chain reaction coupled with restriction fragment length polymorphism assays (PCR-RFLP). Using oligonucleotide primers, DNA fragments of predicted sizes were amplified, and the products' specificity was assessed by nucleotide sequencing. Mutations were detected in DNA samples from bovine blood and semen by the presence or absence of restriction sites within the PCR amplification products (Taq I, Hae III for BLAD, Ava I for DUMPS). The test included 104 bulls and 950 cows of Argentinean Holstein breed. Defective alleles frequencies were as follows: 2.88% BLAD in bulls used in artificial insemination, 1.79% in cows; 0.96% DUMPS in bulls and 0.11% in cows.

Animals↗
PubMed1996

Search for the presence of six Mycoplasma species in peripheral blood mononuclear cells of subjects seropositive and seronegative for human immunodeficiency virus.

The prevalence of Mycoplasma fermentans, Mycoplasma pirum, Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma penetrans was investigated by using specific PCR assays with peripheral blood mononuclear cells from subjects infected or not infected with the human immunodeficiency virus (HIV). Only M. fermentans was detected in 5.8% of 154 HIV-seropositive and 11.1% of 90 HIV-seronegative subjects.

AIDS-Related Opportunistic Infections↗
PubMed1995

Association of Mycoplasma penetrans with human immunodeficiency virus infection.

A cross-sectional study was done to determine the seroprevalence of Mycoplasma penetrans in human immunodeficiency virus (HIV) type 1-seropositive and -seronegative persons recruited in France. The data were analyzed with respect to the sociodemographic, clinical, and biologic status of the patients. M. penetrans seropositivity was associated with HIV infection (18.2% of HIV-seropositive vs. 1.3% of HIV-seronegative persons were M. penetrans-seropositive; P < .001). M. penetrans infection was predominantly but not exclusively associated with homosexual practices in HIV-seropositive subjects and thus presumably sexually transmitted. M. penetrans seroprevalence increased with progression of HIV-associated disease. No association was found between M. penetrans and Kaposi's sarcoma. Thus, there is an unambiguous association between M. penetrans and HIV, particularly among homosexual persons, but its clinical significance remains to be investigated.

AIDS-Related Opportunistic Infections↗
PubMed1995

Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based assay.

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%.

Argentina↗
PubMed1995

Bacteriophage T4 gene 28.

The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.

Amino Acid Sequence↗