Total internal reflection with fluorescence correlation spectroscopy

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes of living cells. This protocol gives a general overview of the steps necessary to construct and test a TIR-FCS system using either through-prism or through-objective internal reflection geometry adapted for FCS. The expected forms of the autocorrelation function are discussed for the cases in which fluorescent molecules in solution diffuse through the depth of the evanescent field, but do not bind to the surface of interest, and in which reversible binding to the surface also occurs.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Effects of size and sex on the courting success and foraging behaviour of Embiotoca jacksoni

To test life-history theory that body size and sex should influence how animals allocate time to foraging versus reproductive activities, we measured the effects of size and sex on courting success and foraging behaviour of black surfperch Embiotoca jacksoni off Santa Catalina Island, southern California. Observations of focal fish were made while snorkelling, during which the length of each fish (estimated to the nearest cm), total duration of courting encounters and foraging rates were recorded. We made observations during and outside the mating season. Courtship occurred only between pairs and its duration increased with the size of both the male and female. Although males would court females that were smaller or larger than themselves, pairs that were closely matched in size had long courting sessions, whereas those that differed considerably in size courted only briefly. Small fish foraged more than larger fish, both during and outside the mating season. Males and females foraged at similar rates outside of the mating season, but during the mating season males reduced their foraging rates to less than half that seen outside of the mating season, whereas females continued to forage at the same rate. This decrease in foraging rate of males during the mating season was seen in all sizes of males but was proportionally greatest in the largest males. These observations indicate that males trade off time spent on foraging for time spent courting during the mating season, whereas females do not.     

Chemiluminescence Emission during Reactions between Superoxide and Selected Aliphatic and Aromatic Halocarbons in Aprotic Media

The reactions between superoxide free radical anion (.O⁻₂) with the halocarbons CCl₄, CHCl₃, BrCH₂CH₂Br(EDB), decachloro-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O⁻₂ consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.     

Betaine improves growth, but does not induce whole body or hepatic palmitate oxidation in swine (Sus scrofa domestica)

Dietary betaine may reduce carcass fat in growing pigs. We explored the effects of betaine on short-term growth and in vivo and in vitro fatty acid oxidation. Pigs were housed in metabolism crates and fed diets containing either 0% (control), 0.125% or 0.5% betaine at 80% of ad libitum energy intake. Fatty acid oxidation was measured during intravenous infusions of 1-(13)C-palmitate and in hepatocytes incubated in the presence or absence of betaine and carnitine. CO2 and palmitate isotopic enrichments were determined by mass spectrometry. Pigs consuming 0.125% and 0.5% betaine for at least 9 days had growth rates that were 38% and 12% greater than controls, respectively. Feed efficiency was also improved with betaine. Fasting increased palmitate oxidation rates 7-8-fold (P < 0.01), but betaine had no effect in either the fed or fasted state (P > 0.1). For hepatocytes, carnitine but not betaine enhanced palmitate oxidation. This response suggests that previously observed reduction in adipose accretion must be via a mechanism other than oxidation. Betaine had no effect on plasma non-esterified fatty acids or urea nitrogen. Under the confinement conditions in this study, dietary betaine improved animal growth responses, but it had no apparent effect on either whole body or hepatic fatty acid oxidation.     

Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen

Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal-epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33 degrees C, but fail to increase in number at the nonpermissive temperature of 39 degrees C. Serum-deprived OSE-C2 cells are stimulated to grow at 33 degrees C by EGF, whereas they are growth inhibited at 33 degrees C by TGFbeta in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer.