Rapid pre-screening of cervical smears as a method of internal quality control in a cervical screening programme

Objective:  To evaluate the performance of rapid pre-screening (RPS) as a method of internal quality control in the cytopathological examination of cervical smears for cervical cancer screening.
Methods:  The sample consisted of 6135 cervical smears submitted to RPS and routine screening (RS) methods. The smears classified as negative in RPS and RS were considered final diagnoses, and were not, therefore, submitted to any additional review. The smears identified as suspect or unsatisfactory according to RPS were analysed separately by two different cytologists irrespective of the diagnosis reached in RS. Smears considered abnormal or unsatisfactory at RS were also reviewed. When both cytologists issued concordant diagnoses, this was considered the final diagnosis. Discordant results were analysed by a third cytologist and a consensus meeting was held to define the final diagnosis.
Results:  Taking abnormalities detected by RS as the denominator, RPS had a sensitivity of 63.0% for the detection of all abnormal smears and 96.7% for high grade squamous intraepithelial lesion (HSIL). When compared with the final diagnosis, sensitivity of RPS for all abnormal smears was 74.9% and for HSIL 95.0%. Of the 529 abnormal smears confirmed in the final diagnosis, 2.15% were detected only by the RPS.
Conclusion:  RPS is an effective alternative method of internal quality control with high sensitivity for the detection of more severe lesions. It also permits monitoring of the laboratory rate of false-negative results, and allows constant evaluation of the performance both of the pre-screening and RS cytologists.     

Bothrops moojeni venom kills Leishmania spp. with hydrogen peroxide generated by its L-amino acid oxidase

Leishmaniasis is an endemic tropical disease in South America, with few therapeutic approaches. Snake venoms are complex protein mixtures with biological actions that could be used as tools for drug development. Here we show that Bothrops moojeni crude venom presented a killing effect in vitro against Leishmania spp. promastigotes, but not with amastigotes, as determined by a viability assay using the mitochondrial oxidative function. Purification of active fractions from crude venom was performed by molecular exclusion and ion exchange chromatography. Anti-Leishmania and l-amino acid oxidase (L-AAO, EC.1.4.3.2.) activities co-eluted in the same fractions. The molecular weight of the active enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa by SDS--PAGE, with a 4.8 isoelectric point. Using substrate subtraction and catalase for scavenging, the action of L-AAO was demonstrated to be hydrogen-peroxide-dependent.     

Effect of heme iron valence state on the conformation of cytochrome c and its association with membrane interfaces. A CD and EPR investigation

Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochrome c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300-600 and 200-320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe(3+). The studies also include the effect of P(i), NaCl, and CaCl(2). Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome c with charged interfaces promotes conformational changes in the alpha-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochrome c. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl(2), NaCl, and P(i), in response to the increase of the ionic strength.     

Evaluation of the effect of 90Sr beta-radiation on human blood cells by chromosome aberration and single cell gel electrophoresis (comet assay) analysis

Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.     

Zinc nutritional status and its relationships with hyperinsulinemia in obese children and adolescents

A perturbation of zinc metabolism has been noted in subjects with obesity. The present work intends to investigate whether the zinc nutritional status is associated with hyperinsulinemia in obesity. A study was carried out in a group of obese children and adolescents (n=23) and compared to a control group (n=21), both between 7 and 14 yr of age. Software analyzed diet information from 3-d food records. Body composition was evaluated by body mass index (BMI), bioelectrical impedance, and skinfold measurements. Zinc nutritional status was evaluated by Zn determination in plasma, erythrocyte, and 24-h urine, by atomic absorption spectrophotometry (λ=213.9 nm). Insulin was measured by radioimmunoassay (Linco Res). Diets consumed by both groups had marginal concentrations of zinc. Zinc concentrations in plasma and erythrocytes were significantly lower in the obese group. Urinary zinc excretion and serum insulin were significantly higher in the same group, although the insulinemia and the parameters of zinc nutritional status were not significantly correlated. As a result, considering that zinc is part of the synthesis and secretion of this hormone, an assessment is necessary of the possible participation of the oligoelement in the mechanisms of insulin resistance, commonly present in obese patients.     

Effect of abietane diterpenes from Plectranthus grandidentatus on T- and B-lymphocyte proliferation

Five known abietane diterpenes of the royleanone and coleon type, namely, fatty acid esters of 7alpha-acyloxy-6beta-hydroxyroyleanone (1), grandidone A (2), 7alpha-acetoxy-6beta-hydroxyroyleanone (3), 6beta,7alpha-dihydroxyroyleanone (4) and coleon U (5), isolated from Plectranthus grandidentatus, were evaluated for their effect on the proliferation of human lymphocytes induced by the mitogen PHA. All except 4, showed a dose-dependent suppressor effect, with 3 yielding the most potent antiproliferative activity, followed by 5. These two compounds, that represent diterpenes of the royleanone and coleon type respectively, were also shown to be potent inhibitors of mouse splenocyte proliferation induced by ConA or LPS mitogens. However, the sensitivity of ConA-stimulated splenocytes to their suppressive effect was higher, suggesting a preferential inhibition of T-lymphocyte proliferation. The antiproliferative activity of 3 seemed to be exerted without affecting the expression of the lymphocyte activation marker CD69. On the contrary, 5 was shown to reduce the expression of CD69 of TCD8(+) and B-cells, suggesting a relationship between its antiproliferative effect and the expression of this early marker of activation on these cell populations. The capacity of 5 to induce apoptosis on ConA-stimulated splenocytes could also be related with its antiproliferative activity.     

Rapid population decline in red knots: fitness consequences of decreased refuelling rates and late arrival in Delaware Bay

Most populations of migrant shorebirds around the world are in serious decline, suggesting that vital condition-dependent rates such as fecundity and annual survival are being affected globally. A striking example is the red knot (Calidris canutus rufa) population wintering in Tierra del Fuego, which undertakes marathon 30,000 km hemispheric migrations annually. In spring, migrant birds forage voraciously on horseshoe crab eggs in Delaware Bay in the eastern USA before departing to breed in Arctic polar deserts. From 1997 to 2002 an increasing proportion of knots failed to reach threshold departure masses of 180-200 g, possibly because of later arrival in the Bay and food shortage from concurrent over-harvesting of crabs. Reduced nutrient storage, especially in late-arriving birds, possibly combined with reduced sizes of intestine and liver during refuelling, had severe fitness consequences for adult survival and recruitment of young in 2000-2002. From 1997 to 2002 known survivors in Delaware Bay were heavier at initial capture than birds never seen again, annual survival of adults decreased by 37% between May 2000 and May 2001, and the number of second-year birds in wintering flocks declined by 47%. Population size in Tierra del Fuego declined alarmingly from 51,000 to 27,000 in 2000-2002, seriously threatening the viability of this subspecies. Demographic modelling predicts imminent endangerment and an increased risk of extinction of the subspecies without urgent risk-averse management.     

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.     

Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.