Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.     

Relationship between dental morphology, sex, body length and age in Pontoporia blainvillei and Sotalia fluviatilis (Cetacea) in Northern Rio de Janeiro, Brazil

The relationship between dental morphology, sex, body length and age of small cetaceans can be used to determine ontogeny, sexual dimorphism and geographical variation. The objective of this study was to determine the relationship between dental morphology, sex, body size and age. A total of 91 specimens of P. blainvillei and 80 specimens of S. fluviatilis accidentally captured in fisheries or stranded in northern Rio de Janeiro (21 masculine37'-22 masculine25'S), from September 1988 to November 1996 were analysed. The teeth root diameter in P. blainvillei was significantly different between the sex; the values for females were larger than males. In neither species aid we observed significant in variations dimension and number of teeth, thickness of dentine and cemental layers and in the maximum width of cement as a function of body size. Age was related to increases in tooth length, root and cingulum diameters, and maximum width of cement in individuals of P. blainvillei, and tooth and crown lengths and maximum width of cement in individuals of S. fluviatilis. The observation of a linear growth between maximum width of cement and age in both species indicates that the equations obtained can be used to estimate relative age in P. blainvillei and S. fluviatilis in northern of Rio de Janeiro.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Introduced brown algae in the North East Atlantic, with particular respect toUndaria pinnatifida (Harvey) suringar

The recent introduction of the macroalgaUndaria pinnatifida (Harvey) Suringar into the North Atlantic is the latest of a large number of introductions, which have occurred over many years. Some have been deliberate introductions for mariculture or research, while most have been accidental, via vectors such as shipping and shellfish imports. Not all newly recorded species are introductions; some are thought to be merely extensions of distribution, e.g.Laminaria ochroleuca, while others may have been overlooked previously, e.g.Scytosiphon dotyi. Subsequent to its accidental introduction into the waters around the Mediterranean French coast at Sete, most likely with imported oysters,Undaria was deliberately introduced into the North Atlantic, to Brittany, in 1983 by IFREMER for commercial exploitation.Undaria has since spread from the original sites in Brittany, and is now established at several sites on the south coast of England. This paper discusses the introduced brown algae in the North Atlantic and outlines the establishment ofUndaria in the UK.     

Ethanol production at 45 °C by Kluyveromyces marxianus IMB3 during growth on molasses pre-treated with Amberlite® and non-living biomass

The use of high concentrations of molasses as a fermentation feed-stock for ethanol production is normally precluded by the presence of inhibitory compounds. Use of the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 in fermentations containing high concentrations of molasses resulted in sub-optimal production of ethanol. The results suggested that this was caused by the presence of inhibitory materials rather than an intolerance to increased concentrations of ethanol. In the current study we describe the pretreatment of molasses preparations with either an Amberlite^® monobed mixed ion-exchange resin or non-living microbial biomass from a local distillery. In the study molasses samples diluted to yield a final sugar concentration of 160?g/l were used as the substrate. Control fermentations using the untreated molasses dilutions yielded a maximum ethanol concentration of 40?g/l, representing 49% of the maximum theoretical yield. Fermentations using molasses samples pre-treated with Amberlite^® or non-living biomass yielded maximum ethanol concentrations of 58 and 54?g/l, representing 71 and 66% of the maximum theoretical yield, respectively. The results suggest that pre-treatment brings about removal of toxic or inhibitory materials from the fermentation feed-stock and we believe that such pre-treatments, particularly using the less expensive non-living biomass preparations may find a role in processes concerned with the commercial production of ethanol from molasses using this microorganism.     

Environmental Effects on Disulfide Bonding Patterns of Bovine κ-Casein

Bovine -casein, the stabilizing protein of the colloidal milk protein complex, has a unique disulfide bonding pattern. The protein exhibits varying molecular sizes on SDS-PAGE ranging from monomer to octamer and above in the absence of reducing agents. Heating the samples with SDS prior to electrophoresis caused an apparent decrease in polymeric distribution: up to 60% monomer after 30min at 90°C as estimated by densitometry of SDS-PAGE. In contrast, heating the samples without detergent at 90 or 37°C caused a significant increase in high-molecular-weight polymers as judged by electrophoresis and analytical ultracentrifugation. In 6 M urea, the protein could be completely reduced, but upon dialysis, varying degrees of polymer reformation occurred depending on the dialysis conditions. Spontaneous reoxidation to polymeric forms is favored at low pH (<5.15) and low ionic strength. The results are discussed with respect to the influence of the method of preparation on the polymer size of -caseins and on their resultant physical chemical properties.     

Coronary vascular volume remodelling in rainbow trout Oncorhynchus mykiss

The 34% increase in relative ventricular mass (M_rv) resulting from chronic anaemia (induced by an intraperitoneal injection of phenylhydrazine hydrochloride) was accompanied by a 117% increase in coronary vascular volume of diploid rainbow trout Oncorhynchus mykiss. Coronary vascular volume of normocythemic triploid fish was similar to that of normocythemic diploid fish despite a larger M_rv. These observations, in combination with previous studies, suggest that the vascularity of compact myocardium in O. mykiss can vary independently of M_rv.     

Critical evaluation of municipal solid waste composting and potential compost markets

Mechanical biological treatment (MBT) of mixed waste streams is becoming increasingly popular as a method for treating municipal solid waste (MSW). Whilst this process can separate many recyclates from mixed waste, the resultant organic residue can contain high levels of heavy metals and physical and biological contaminants. This review assesses the potential end uses and sustainable markets for this organic residue. Critical evaluation reveals that the best option for using this organic resource is in land remediation and restoration schemes. For example, application of MSW-derived composts at acidic heavy metal contaminated sites has ameliorated soil pollution with minimal risk. We conclude that although MSW-derived composts are of low value, they still represent a valuable resource particularly for use in post-industrial environments. A holistic view should be taken when regulating the use of such composts, taking into account the specific situation of application and the environmental pitfalls of alternative disposal routes.     

RUNX3-mediated repression of RUNX1 in B cells

Are there three different RUNX genes in the human genome just to allow differential expression in different cell types or are the RUNX proteins functionally different? Much previous evidence has suggested that the RUNX proteins were functionally redundant but, in human B cells, RUNX1 and RUNX3 have clearly been shown to have different functions at a key developmental stage. RUNX1 is expressed in mature resting B cells but proliferating B cell lines express RUNX3. Repression of RUNX1 by RUNX3 is required to allow the cell proliferation and this provides an experimental system to investigate the mechanism of the different activities of RUNX1 and RUNX3 in these cells. J. Cell. Physiol. 221: 283–287, 2009. © 2009 Wiley-Liss, Inc.