Pyrolytic Methylation-Gas Chromatography of Whole Bacterial Cells for Rapid Profiling of Cellular Fatty Acids

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.     

Comparitive study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice

Milk fat globule membranes and mammary tumour virus particles (d = 1.17 g/cm³) have been obtained from the milk of a Swiss albino mice strain. Comparitive biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.     

Superior immunoreactivity of 125I (Des-Tyr-betaAla)-secretin with rabbit anti-secretin sera compared to 125I-secretin and 125 I-6-Tyrosyl secretin.

A secretin analogue in which the normal amino acid sequence had been elongated by a (Des-Tyr-betaAla)-residue was studied as tracer for secretin radioimmunoassay. 125I-(DATA)-secretin exhibited superior immunoreactivity with several rabbit anti-secretin sera compared to 125I-6-Tyr-secretin and also to secretin iodinated at its N-terminal histidyl residue. This may be due, at least in part, to higher conformational integrity of the secretin moiety in the 125I-(DATA)-secretin molecule. Thus, at present, 125I-(DATA)-secretin appears to be most suitable as tracer for sensitive secretin radioimmunoassay.     

Influence of the hormone analogue 13-NLE-molitin and of 1-methyl-3-isobutylxanthine on tone and cyclic 3',5'-AMP content of antral and duodenal muscles in the rabbit.

1. By the action of 1-methyl-3-isobutylxanthine (isobutyltheophylline, 2 - 3 × 10⁻⁴ M), the content of cyclic 3', 5'-AMP in the antral and duodenal muscles of the rabbit is increased by 72 % and 126 %, respectively; by 1.8 × 10⁻⁷ M 13-norleucine-motilin and 1.8 × 10⁻⁶ M acetylcholine it is not changed. 13-norleucine-motilin is an analogue of the recently discovered duodenal tissue hormone motilin and has identical effects. 1-methyl-3-isobutylxanthine has a more powerful inhibiting effect on phosphodiesterase than has theophylline.2. 3 × 10⁻⁴ M isobutyltheophylline reduces the tone of the duodenal muscle while simultaneously increasing the content of cyclic AMP and negates the tone-enhancing effect of nle-motilin on the duodenal muscle, while nle-motilin increases the muscle tone lowered by isobutyltheophylline.3. The basic tone of the antral muscle is not reduced by isobutyltheophylline. However, the contraction-promoting effect of nle-motilin after an increase in cyclic AMP due to isobutyltheophylline is significantly lower.4. It is assumed that the changes in the tone or in the response of the antral and duodenal muscles to nle-motilin observed after the administration of isobutyltheophylline, are due to the increase of cyclic AMP in the tissue.5. The antagonistic effects of cyclic AMP and motilin on the gastro-intestinal muscles might be of physiological importance for the regulation of the gastro-intestinal motor activity.     

Gene delivery to pancreatic exocrine cells in vivo and in vitro

Background

Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein).

Results

The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1?C2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17?-oestradiol (E₂), the synthetic oestrogen 17??- ethinyloestradiol (EE₂), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE₂; 10?ng/L, E₂; 100?ng/L, after 72?h exposures). For the NP exposures, GFP expression was observed at 10???g NP/L after 72?h (100???g NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.

Conclusion

Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.     

Evaluating the effectiveness of road mitigation measures

The last 20 years have seen a dramatic increase in efforts to mitigate the negative effects of roads and traffic on wildlife, including fencing to prevent wildlife-vehicle collisions and wildlife crossing structures to facilitate landscape connectivity. While not necessarily explicitly articulated, the fundamental drivers behind road mitigation are human safety, animal welfare, and/or wildlife conservation. Concomitant with the increased effort to mitigate has been a focus on evaluating road mitigation. So far, research has mainly focussed on assessing the use of wildlife crossing structures, demonstrating that a broad range of species use them. However, this research has done little to address the question of the effectiveness of crossing structures, because use of a wildlife crossing structure does not necessarily equate to its effectiveness. The paucity of studies directly examining the effectiveness of crossing structures is exacerbated by the fact that such studies are often poorly designed, which limits the level of inference that can be made. Without well performed evaluations of the effectiveness of road mitigation measures, we may endanger the viability of wildlife populations and inefficiently use financial resources by installing structures that are not as effective as we think they are. In this paper we outline the essential elements of a good experimental design for such assessments and prioritize the parameters to be measured. The framework we propose will facilitate collaboration between road agencies and scientists to undertake research programs that fully evaluate effectiveness of road mitigation measures. We discuss the added value of road mitigation evaluations for policy makers and transportation agencies and provide recommendations on how to incorporate such evaluations in road planning practices.     

Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus

In hippocampal slices arachidonic acid released after NMDA post-synaptic receptor activation is thought to act as a retrograde trans-synaptic messenger which facilitates the pre-synaptic release of L-glutamate to be involved in the expression of long-term synaptic potentiation (LTP). We measured the mass amount of arachidonic acid released from hippocampal slices incubated under conditions which maintain the electrophysiological responsiveness of the slice. Melittin released arachidonic, oleic and docosahexaenoic acids by phospholipase A₂ activation but not palmitic or stearic acids. Of greater interestl-glutamate, N-methyl-d-aspartate and incubation conditions known to induce LTP selectively and rapidly increased the release of archidonic acid in amounts over basal levels of 200–300 ng/mg protein. This is the first direct determination of the mass amount of arachidonic acid released following NMDA receptor activation in the hippocampus.Special issue dedicated to Dr. Louis Sokoloff.     

Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity.