Opportunistic testing of medically underserved women for cervical cancer in South Africa

OBJECTIVE: To determine the yield of opportunistic Pap smears taken in an unscreened and medically underserved population in the Transkei Region of South Africa. STUDY DESIGN: Cross-sectional study of 22,160 cervical cytology specimens from an unscreened population attending gynecologic outpatient clinics between January 1990 and December 1996. RESULTS: The overall prevalence of atypical squamous cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL) was, respectively, 34.7%, 8.3% and 2.4%. The ASCUS: SIL ratio was 3:1. The prevalence of invasive squamous cell carcinoma was 1.6%. The yield of opportunistic Pap smears was 10.7% including only LSIL and HSIL. CONCLUSION: The pathologic process of precursor lesions of cervical cancer appears to start at an early age since > 20% of cases are diagnosed before the age of 30 years. In the absence of a national screening program, opportunistic testing of medically underserved women needs to be maintained and encouraged.     

Liquid serial dilution is inferior to solid media for isolation of cultures representative of the phylum-level diversity of soil bacteria

Representatives of only four well-characterized bacterial phyla were isolated from a pasture soil by using liquid serial dilution culture. In contrast, members of Acidobacteria, Verrucomicrobia, and Gemmatimonadetes and of other poorly represented bacterial lineages were isolated in earlier experiments with solidified versions of the same media. We conclude that, contrary to expectation, liquid serial dilution culture is inferior to culturing on solid media for isolating representatives of many bacterial phyla from soil.     

A basal membrane-like structure surrounding tumour nodules may prevent intraepithelial leucocyte infiltration in colorectal cancer

Epithelial tumours consist of an epithelial compartment and a stromal compartment, which are sometimes separated by a basal membrane-like structure. We sought to determine whether these factors have prognostic value in 84 curatively resected stage II and III colorectal cancer by immunohistochemically staining tumours for leucocytes (CD45) and extracellular matrix, and to assess the presence of a basal membrane-like structure. Leucocyte infiltration was also assessed in hematoxylin-eosin (HE) stained sections. Most leucocytes were located in the tumour stroma. A relatively high intraepithelial leucocyte infiltration was significantly correlated with a lower level of tumour recurrence (P=0.03) and a longer disease-free survival (P=0.05), whereas leucocytes located in the tumour stroma (P=0.92) or at the advancing margin (p=0.06) were not. Intraepithelial leucocyte infiltration was also significantly correlated with leucocyte infiltration in the tumour stroma (P=0.02) and at the advancing tumour margin (P=0.005), and as assessed in HE-stained tumour sections (P=0.05), but each of these parameters on its own did not have a prognostic value in predicting disease-free survival. Moreover, the presence of a basal membrane-like structure surrounding the tumour epithelium was inversely correlated with the number of intraepithelial leucocytes (P=0.05), suggesting that this membrane-like structure functions as a barrier to intraepithelial leucocyte infiltration. We conclude that leucocytes must be in the direct vicinity of tumour cells to affect tumour growth. The presence of an extracellular matrix barrier seems to prevent this interaction.     

Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.     

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.     

Cleave to leave: structural insights into the dynamic organization of the nuclear pore complex

A detailed understanding of the fine structure of the nuclear pore complex has remained elusive. Now, studies on a small protein domain have shed light on the dynamic organization of this massive assembly.     

Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages

In order to study the prevalence and diversity of tetracycline resistant lactic acid bacteria (Tc(r) LAB) along the process line of two different fermented dry sausage (FDS) types, samples from the raw meat, the meat batter and the fermented end product were analysed quantitatively and qualitatively by using a culture-dependent approach. Both the diversity of the tet genes and their bacterial hosts in the different stages of FDS production were determined. Quantitative analysis showed that all raw meat components of both FDS types (FDS-01 and FDS-08) contained a subpopulation of Tc(r) LAB, and that for FDS-01 no Tc(r) LAB could be recovered from the samples after fermentation. Qualitative analysis of the Tc(r) LAB subpopulation in FDS-08 included identification and typing of Tc(r) LAB isolates by (GTG)5-PCR fingerprinting, plasmid profiling, protein profiling and a characterization of the resistance by PCR detection of tet genes. Two remarks can be made when the results of this analysis for the different samples are compared. (i) The taxonomic diversity of Tc(r) LAB varies along the process line, with a higher diversity in the raw meat (lactococci, lactobacilli, streptococci, and enterococci), and a decrease after fermentation (only lactobacilli). (ii) Also the genetic diversity of the tet genes varies along the process line. Both tet(M) and tet(S) were found in the raw meat, whereas only tet(M) was found after fermentation. A possible relationship was found between the disappearing of species other than lactobacilli and tet(S), because tet(S) was only found in lacotocci, enterococci, and streptococci. These data suggest that fermented dry sausages are among those food products that can serve as vehicles for Tc(r) LAB and that the raw meat already contains a subpopulation of these bacteria. Whether these results reflect the transfer of resistant bacteria or of bacterial resistance genes from animals to man via the food chain is difficult to ascertain and may require a combination of cultivation-dependent and cultivation-independent approaches.     

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.