Agronomic performance, chromosomal stability and resistance to velvetbean caterpillar of transgenic soybean expressing cry1Ac gene

The objective of this work was to analyze the agronomic performance and chromosomal stability of transgenic homozygous progenies of soybean [Glycine max (L.) Merrill.], and to confirm the resistance of these plants against Anticarsia gemmatalis. Eleven progenies expressing cry1Ac, hpt and gusA genes were evaluated for agronomic characteristics in relation to the nontransformed parent IAS 5 cultivar. Cytogenetical analysis was carried out on transgenic and nontransgenic plants. Two out of the 11 transgenic progenies were also evaluated, in vitro and in vivo, for resistance to A. gemmatalis. Two negative controls were used in resistance bioassays: a transgenic homozygous line, containing only the gusA reporter gene, and nontransgenic 'IAS 5' plants. The presence of cry1Ac transgene affected neither the development nor the yield of plants. Cytogenetical analysis showed that transgenic plants presented normal karyotype. In detached-leaf bioassay, cry1Ac plants exhibited complete efficacy against A. gemmatalis, whereas negative controls were significantly damaged. Whole-plant feeding assay confirmed a very high protection of cry1Ac against velvetbean caterpillar, while nontransgenic 'IAS 5' plants and homozygous gusA line exhibited 56.5 and 71.5% defoliation, respectively. The presence of cry1Ac transgene doesn't affect the majority of agronomic traits (including yield) of soybean and grants high protection against A. gemmatalis.

Chromosomal evolution and comparative gene mapping in the Drosophila repleta species group

A review of our recent work on the cromosomal evolution of the Drosophila repleta species group is presented. Most studies have focused on the buzzatii species complex, a monophyletic set of 12 species which inhabit the deserts of South America and the West Indies. A statistical analysis of the length and breakpoint distribution of the 86 paracentric inversions observed in this complex has shown that inversion length is a selected trait. Rare inversions are usually small while evolutionary successful inversions, fixed and polymorphic, are predominantly of medium size. There is also a negative correlation between length and number of inversions per species. Finally, the distribution of inversion breakpoints along chromosome 2 is non-random, with chromosomal regions which accumulate up to 8 breakpoints (putative "hot spots"). Comparative gene mapping has also been used to investigate the molecular organization and evolution of chromosomes. Using in situ hybridization, 26 genes have been precisely located on the salivary gland chromosomes of D. repleta and D. buzzatii; another nine have been tentatively identified. The results are fully consistent with the currently accepted chromosomal homologies between D. repleta and D. melanogaster, and no evidence for reciprocal translocations or pericentric inversions has been found. The comparison of the gene map of D. repleta chromosome 2 with that of the homologous chromosome 3R of D. melanogaster shows an extensive reorganization via paracentric inversions and allows to estimate an evolution rate of ~1 inversion fixed per million years for this chromosome

Chromosomal polymorphism in urban populations of Drosophila paulistorum

Drosophila paulistorum populations colonizing the urban area of Porto Alegre, southern Brazil, were studied with the objective of characterizing their chromosomal polymorphism in this new environment. Despite being geographically and ecologically marginal and the fact that the colonization of the urban area seems to be a recent event, the populations showed a large number of inversions on all chromosome arms. Differences regarding inversion frequencies and percentage of heterozygosis were found when we compared the samples with respect to geographical, microenvironmental and temporal aspects. Such differences, however, could be attributed to both selective and stochastic factors

Measuring higher order optical aberrations of the human eye: techniques and applications

In the present paper we discuss the development of "wave-front", an instrument for determining the lower and higher optical aberrations of the human eye. We also discuss the advantages that such instrumentation and techniques might bring to the ophthalmology professional of the 21st century. By shining a small light spot on the retina of subjects and observing the light that is reflected back from within the eye, we are able to quantitatively determine the amount of lower order aberrations (astigmatism, myopia, hyperopia) and higher order aberrations (coma, spherical aberration, etc.). We have measured artificial eyes with calibrated ametropia ranging from +5 to -5 D, with and without 2 D astigmatism with axis at 45º and 90º. We used a device known as the Hartmann-Shack (HS) sensor, originally developed for measuring the optical aberrations of optical instruments and general refracting surfaces in astronomical telescopes. The HS sensor sends information to a computer software for decomposition of wave-front aberrations into a set of Zernike polynomials. These polynomials have special mathematical properties and are more suitable in this case than the traditional Seidel polynomials. We have demonstrated that this technique is more precise than conventional autorefraction, with a root mean square error (RMSE) of less than 0.1 µm for a 4-mm diameter pupil. In terms of dioptric power this represents an RMSE error of less than 0.04 D and 5º for the axis. This precision is sufficient for customized corneal ablations, among other applications.

Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men

The objective of the present study was to determine the frequency of somatic chromosomal anomalies and Y chromosomal microdeletions (azoospermia factor genes, AZF) in infertile males who seek assisted reproduction. These studies are very important because the assisted reproduction techniques (mainly intracytoplasmic sperm injection) bypass the natural selection process and some classical chromosomal abnormalities, microdeletions of AZF genes or some deleterious genic mutations could pass through generations. These genetic abnormalities can cause in the offspring of these patients male infertility, ambiguous external genitalia, mental retardation, and other birth defects. We studied 165 infertile men whose infertility was attributable to testicular problems (60 were azoospermic, 100 were oligospermic and 5 were asthenospermic). We studied 100 metaphases per patient with GTG banding obtained from temporary lymphocyte culture for chromosomal abnormality detection and performed a genomic DNA analysis using 28 Y chromosome-specific sequence-tagged sites for Y AZF microdeletion detection. Karyotyping revealed somatic anomalies in 16 subjects (16/165 = 9.6%). Of these 16, 12 were in the azoospermic group (12/60 = 20%) and 4 were in the oligospermic group (4/100 = 4%). The most common chromosomal anomaly was Klinefelter syndrome (10/165 = 6%). Microdeletions of AZF genes were detected in 12 subjects (12/160 = 7.5%). The frequencies detected are similar to those described previously. These results show the importance of genetic evaluation of infertile males prior to assisted reproduction. Such evaluation can lead to genetic counseling and, consequently, to primary and secondary prevention of mental retardation and birth defects.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

Comparison of I-FISH and G-banding for the detection of chromosomal abnormalities during the evolution of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) patients with a normal karyotype constitute a heterogeneous group from a biological standpoint and their outcome is often unpredictable. Interphase fluorescence in situ hybridization (I-FISH) studies could increase the rate of detection of abnormalities, but previous reports in the literature have been contradictory. We performed I-FISH and conventional karyotyping (G-banding) on 50 MDS patients at diagnosis, after 6 and 12 months or at any time if a transformation to acute myeloid leukemia (AML) was detected. Applying a probe-panel targeting the centromere of chromosomes 7 and 8, 5q31, 5p15.2 and 7q31, we observed one case with 5q deletion not identified by G-banding. I-FISH at 6 and 12 months confirmed the karyotype results. Eight cases transformed to AML during follow-up, but no hidden clone was detected by I-FISH in any of them. The inclusion of I-FISH during follow-up of MDS resulted in a small improvement in abnormality detection when compared with conventional G-banding.

Evaluation of chromosomal abnormalities by cIg-FISH and association with proliferative and apoptotic indexes in multiple myeloma

Eighty-six newly diagnosed multiple myeloma (MM) patients from a public hospital of São Paulo (Brazil) were evaluated by cIg-FISH for the presence of del(13)(q14), t(4;14)(p16.3;q32) and del(17)(p13). These abnormalities were observed in 46.5, 9.3, and 7.0% of the patients, respectively. In order to identify the possible role of del(13)(q14) in the physiopathology of MM, we investigated the association between this abnormality and the proliferative and apoptotic indexes of plasma cells. When cases demonstrating t(4;14)(p16.3;q32) and del(17)(p13) were excluded from the analysis, we observed a trend towards a positive correlation between the proportion of cells carrying del(13)(q14) and plasma cell proliferation, determined by Ki-67 expression (r = 0.23, P = 0.06). On the other hand, no correlation between the proportion of cells carrying del(13)(q14) and apoptosis, determined by annexin-V staining, was detected (r = 0.05, P = 0.69). In general, patients carrying del(13)(q14) did not have lower survival than patients without del(13)(q14) (P = 0.15), but patients with more than 80% of cells carrying del(13)(q14) showed a lower overall survival (P = 0.033). These results suggest that, when del(13)(q14) is observed in a high proportion of malignant cells, it may have a role in determining MM prognosis. Another finding was a statistically significant lower overall survival of patients with t(4;14)(p16.3;q32) (P = 0.026). In the present study, almost half the patients with t(4;14)(p16.3;q32) died just after diagnosis, before starting treatment. This fact suggests that, in São Paulo, there may be even more patients with this chromosomal abnormality, but they probably die before being diagnosed due to unfavorable socioeconomic conditions. This could explain the low prevalence of this chromosomal abnormality observed in the present study.

Genetic aberrations in multiple myeloma characterized by cIg-FISH: a Brazilian context

Genetic abnormalities are critical prognostic factors for patients diagnosed with multiple myeloma (MM). This retrospective, multicenter study aimed to contribute with the genetic and clinical characterization of MM patients in a country with continental dimensions such as Brazil. Genetic abnormalities were assessed by cIg-fluorescent in situ hybridization (cIg-FISH) in a series of 152 MM patients (median age 55 years, 58.5% men). Overall, genetic abnormalities were detected in 52.7% (80/152) of patients. A 14q32 rearrangement was detected in 33.5% (n=51), including t(11;14), t(4;14) and t(14;16) in 18.4, 14.1, and 1% of cases, respectively. del(13q) was identified in 42.7% (n=65) of patients, of whom 49.2% (32/65) presented a concomitant 14q32 rearrangement. del(17p) had a frequency of 5.2% (n=8). del(13q) was associated with high plasma cell burden (≥50%, P=0.02), and del(17p) with advanced ISS stages (P=0.05) and extramedullary disease (P=0.03). t(4;14) was associated with advanced Durie-Salmon stages (P=0.008), renal insufficiency (P=0.01) and was more common in patients over 60 years old. This study reports similar frequencies of genetic abnormalities to most series worldwide, whereas the t(14;16) and del(17p), two high risk factors for newly diagnosed patients, exhibited lower frequencies. Our results expand the knowledge on the molecular features of MM in Brazil, a country where innovative therapies that could overcome a poor prognosis for some genetic abnormalities are not always available.

Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Molecular typing of Candida albicans strains isolated from nosocomial candidemia

Yeasts of the genus Candida have been recognized as important microorganisms responsible for nosocomial fungemia. Six blood-stream and two intravenous central catheter C. albicans strains were isolated from eight patients and studied by electrophoretic karyotyping of chromosomal DNA by pulsed-field gel electrophoresis. Seven chromosomal DNA profiles were identified. Two patients showed isolates with the same profile, suggesting nosocomial transmission. Karyotyping of C. albicans revealed an excellent discriminatory power among the isolates and may therefore be useful in the study of nosocomial candidemia.

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.

Staphylococcus aureus ampicillin-resistant from the odontological clinic environment

The aim of this research was to evaluate the prevalence of Sthaphylococcus spp. and S. aureus in the odontological clinic environment (air), their production of beta-lactamase and antibacterial susceptibility to the major antibiotics utilized in medical particle. During 12 months of samples collect were isolated 9775 CFU by MSA medium suggesting a high amount of Staphylococcus spp. in the clinic environment which can appear through aerosols. A total of 3149 colonies (32.2%) were suggestive of pathogenic staphylococci. Gram coloration, catalase test, colony-mallow growing on chromogenic medium, and coagulase test confirmed the identity of 44 (0.45%) S. aureus isolates. Of these, 35 isolates (79.5%) showed production of beta-lactamase by CefinaseTM discs and resistance to ampicillin, erythromycin (7 isolates) and tetracycline (1 isolate) suggesting the existence of multiresistant isolates. The evaluation of the oxacillin MIC by Etest® assays showed susceptibility patterns suggesting the inexistence of the mecA gene in chromosomal DNA. These results point out to the need of a larger knowledge on the contamination means and propagation of this microorganism into the odontological clinic.