Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM flow cytometry - FITC fluorescein-iso-thiocyanate - LB Luria broth - MM minimal salt medium - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.     

Influence of phenolics on the sensitivity of free and immobilized bioluminescent Acinetobacter bacterium

In this work, the constructed bioluminescent Acinetobacter strain DF4/PUTK2 was employed to assess the toxicity of phenolic compounds and the 5 min EC50 values were calculated. The results of the DF4/PUTK2 assay were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay. To develop a bioassay system appropriate to be used in continuous toxicity testing, strain DF4/PUTK2 was subjected for immobilization in microtiter plates into the matrices Ca-alginate, polyacrylamide, agar and agarose. After a choice of materials was tried, Ca-alginate was selected as the most suitable candidate material. Because, it could be stored at least 8 weeks at 4 °C, during which the ability of the bioreporter DF4/PUTK2 to detect the toxicity of phenolics was maintained. However, the stability of the bioluminescence for DF4/PUTK2 cells immobilized into agarose and agar was significantly less than that of cells stored in alginate suspensions. This study recommended that luxCDABE-marked Acinetobacter strain DF4/PUTK2 could be employed to assay the ecotoxicity of environmental samples contaminated with phenols. The host strain of the bioreporter DF4/PUTK2 is Acinetobacter strain DF4. It is known that members of the genus Acinetobacter are widespread in nature and also involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes.     

Pilot-scale production of mosquitocidal toxins by Bacillus thuringiensis and Lysinibacillus sphaericus under solid-state fermentation

Mosquitocidal toxins of Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaericus 14N1 (Ls14N1) were produced under solid-state fermentation using agro-industrial wastes. Sugar beet pulp–sesame meal (1:1) and wheat germ meal–linen meal (1:1) at 9% were the efficient substrate mixtures for the growth and toxin production of Bti and Ls14N1, respectively. Bti was more active after the addition of beef extract (0.2%) or yeast extract (0.5%) to the medium. On the other hand, the addition of yeast extract (0.2%) or NYSM salts (2%) significantly enhanced the toxicity produced by Ls14N1. The optimum conditions for the maximum toxicity of Bti were at pH 7–8, 20–30% moisture, 4–10% inoculum and 7 days incubation. For Ls14N1, the best conditions were pH 6.5–7.5, 20–30% moisture, 4–10% inoculum and 5 days incubation. It was found that the best thickness of carrier-substrates in the plate (15?cm in diameter) for the maximum mosquitocidal activity was about 0.5?cm for Bti and 0.5–1?cm for Ls14N1. Pilot-scale production in aluminium trays applying the above conditions showed a decrement of toxicity of fermented cultures and some plates were contaminated. These problems were dissolved by reducing the moisture content to 15%, increasing inoculum to 10% and manual agitation of trays every-day.     

Hypocone reduction and Carabelli's traits in contemporary Jordanians and the association between Carabelli's trait and the dimensions of the maxillary first permanent molar

The objective of this study is to determine the prevalence of expression and bilateralism of two dental morphological traits in contemporary Jordanians: The hypocone reduction trait on the maxillary second permanent molar and Carabelli's trait on maxillary permanent first and second molars. Furthermore, inter-trait correlation and the relationship of Carabelli's traits with upper first molar dimensions were investigated. Three hundred subjects of school children at their 10th grade and of an average age of 15.5 +/- 0.4 years were involved. Alginate impressions for the maxillary arch were taken, dental casts were reproduced. The selected accurate casts were of 132 male- and 155 female-students. The frequencies of hypocone reduction trait on the maxillary second molar and Carabelli's trait on the maxillary molars were examined. Buccolingual and mesiodistal diameters of the maxillary first molar were measured and recorded. Paired Sample t test and Nonparametric Correlation analysis were used for data analysis. Hypocone reduction trait on the maxillary second molar was found in 29.8% of the examined students. Positive forms of Carabelli's trait on first and second molars were observed in 65.0% and 3.8%, respectively. Nonparametric correlation analysis revealed positive association between Carabelli's trait on first molar and hypocone reduction trait on the maxillary second molar. The presence of Carabelli's trait on first molar was strongly associated with the increase of buccolingual, but not the mesiodistal, diameter. Bilateralism was found highly significant in the tested traits and both genders (p < 0.001). This finding might be a sign of relatively low environmental stresses in the living Jordanian population and/or great ability of its individuals to buffer the adverse effects of such stresses.     

A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm™ inserts

Micropatterning techniques provide direct control over the spatial organization of cells at the sub-mm scale. Regulation of these spatial parameters is important for controlling cell fate and cell function. While micropatterning has proved a powerful technique for understanding the impact of cell organization on cell behaviour, current methods for micropatterning cells require complex, specialized equipment that is not readily accessible in most biological and bioengineering laboratories. In addition, currently available methods require significant protocol optimization to ensure reliable and reproducible patterning. The inaccessibility of current methods has severely limited the widespread use of micropatterning as a tool in both biology and tissue engineering laboratories. Here we present a simple, cheap, and fast method to micropattern mammalian cells into stripes and circular patterns using Parafilm?, a common material found in most biology and bioengineering laboratories. Our method does not require any specialized equipment and does not require significant method optimization to ensure reproducible patterning. Although our method is limited to simple patterns, these geometries are sufficient for addressing a wide range of biological problems. Specifically, we demonstrate i) that using our Parafilm? insert method we can pattern and co-pattern ARPE-19 and MDCK epithelial cells into circular and stripe micropatterns in tissue culture polystyrene (TCPS) wells and on glass slides, ii) that we can contain cells in the desired patterns for more than one month and iii) that upon removal of the Parafilm? insert we can release the cells from the containment pattern and allow cell migration outward from the original pattern. We also demonstrate that we can exploit this confinement release feature to conduct an epithelial cell wound healing assay. This novel micropatterning method provides a reliable and accessible tool with the flexibility to address a wide range of biological and engineering problems that require control over the spatial and temporal organization of cells.