Integrating the molecular and cellular basis of odor coding in the Drosophila antenna

We investigate how the molecular and cellular maps of the Drosophila olfactory system are integrated. A correspondence is established between individual odor receptors, neurons, and odors. We describe the expression of the Or22a and Or22b receptor genes, show localization to dendritic membranes, and find sexual dimorphism. Or22a maps to the ab3A neuron, which responds to ethyl butyrate. Analysis of a deletion mutant lacking Or22a, along with transgenic rescue experiments, confirms the mapping and demonstrates that an Or gene is required for olfactory function in vivo. Ectopic expression of Or47a in a mutant cell identifies the neuron from which it derives and its odor ligands. Ectopic expression in a wild-type cell shows that two receptors can function in a single cell. The ab3A neuron does not depend on normal odor receptor gene expression to navigate to its target in the CNS.     

Kinetics of Congo-red binding by haemin-limited and haemin-excess cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.     

Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.     

Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour

In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.     

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.     

Identification of oxidized proteins in rat plasma using avidin chromatography and tandem mass spectrometry

The objective in much of the proteomics literature today is to establish the difference between healthy and disease states at the protein level using blood plasma. A critical component in this endeavor is to establish what is normal. The focus of the work reported here was to do this with oxidized proteins that might relate to oxidative stress and oxidative stress-related diseases. Oxidative stress is known to increase markedly in cancer, diabetes, heart disease, and neurodegenerative diseases. Since proteins are one of the targets of ROS, generated by oxidative stress, oxidized proteins are excellent biomarker candidates for these diseases. But first it is necessary to identify oxidized proteins that occur in the healthy state. Healthy rat plasma was used in this study as a source for the identification of naturally oxidized proteins. Freshly drawn blood was treated with biotin hydrazide to selectively derivatize carbonyl groups in oxidized proteins. Oxidized proteins thus biotinylated were separated from the other plasma proteins using avidin affinity chromatography. Affinity selected proteins were further fractionated on a C(8) RP column and fractions collected. The collected fractions were then tryptic digested and the peptides identified using a combination of LC/MS/MS and database searches. One hundred forty-six proteins were identified using 700 signature peptides from the tryptic digested chromatographic fractions. The most frequently encountered proteins in the samples were keratins. Brain and liver were among the organs contributing the most oxidized proteins to plasma followed by heart and kidney.     

Isolation of alcohol tolerant,osmotolerant and thermotolerant yeast strains and improvement of their alcohol tolerance by UV mutagenesis

In this study, the yeast strains were isolated from grapes by serial dilution technique to determine their alcohol-, sugar- and thermotolerance. 34 wild type yeast strains were isolated and alcohol-, sugar- and thermotolerance of these strains were determined. The maximum alcohol tolerance was found to be 9% (v/v) in yeast strain which is named Y2. Thermotolerance behavior of 6 strains were investigated. The strains were treated with UV light with intervals of 20, 30, 40 and 50 seconds. Selected resistant colonies were investigated for alcohol tolerance. It was found that alcohol tolerance increased from 9% (v/v) to 12% (v/v) on Y2 strain.     

Glycine betaine as a cryoprotectant for prokaryotes

Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes. These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing. This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant. In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D). GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve. For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA. Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants. GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria. These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens.     

Temperature Elevation Regulates Iron Protoporphyrin IX and Hemoglobin Binding by Porphyromonas gingivalis

Porphyromonas gingivalis, an obligate anerobe with a growth requirement for iron protoporphyrin IX (FePPIX), is exposed to increased temperatures in the inflamed periodontal pocket. In this study, P. gingivalis was grown in a chemostat at 37°C (control), 39°C, and 41°C, and examined for hemagglutinating (HA) activity, hemoglobin binding and degrading activity, and iron protoporphyrin IX binding. HA activity decreased in cells as the growth temperature increased. Binding of μ-oxo bishaem (dimeric haem), and Fe(II)- and Fe(III)-monomeric forms was increased in 39°C-grown cells but decreased in 41°C-grown cells compared with controls. Cellular hemoglobin binding and degradation decreased with increased growth temperature. The decrease in cellular hemagglutination and hemoglobin degradation occurring with increased growth temperature would limit the potential overproduction of toxic monomeric haem molecules. The increased binding of μ-oxo bishaem and monomeric forms of FePPIX at 39°C may reflect a defense strategy against reactive oxidants and a mechanism of dampening down the inflammatory response to maintain an ecological balance. Received: 24 April 2000 / Accepted: 30 May 2000     

Effect of a nutraceutical treatment on diabetic rats with targeted and CE-MS non-targeted approaches

Despite the introduction of hypoglycemic drugs, diabetes and related complications continue being a major medical problem. Diabetes long-term complications are not only related to the genesis of free radicals due to oxidation of glucose and to the non-enzymatic and progressive glycation of proteins but also to the endothelial dysfunction secondary to persistent hyperglycemia that causes cardiovascular complications. In an experimental model of streptozotocin (STZ) diabetic rats, the effect of five doses of an extract containing both an antioxidant (Rosmarinus officinalis) and folic acid were intragastrically administrated. Urine fingerprints of control and diabetic rats, both with and without treatment, were obtained by capillary electrophoresis with mass spectrometry (CE-TOF-MS). In order to have further biochemical knowledge of the effect, after treatment, rats were killed and plasma glucose, triglycerides, cholesterol, total protein, urea were analysed. Vitamin E in plasma and liver was also measured. Among the changes observed, the reduction in diuresis and plasma triglycerides, together with reduction in 2-aminobutyric, leucine/isoleucine, and dimethylglycine have shown that a short term nutraceutical treatment was able to reduce some of the complications in the STZ diabetic rats. In addition, this CE-MS metabolomic approach has permitted to identify metabolites related to metabolism of arginine, histidine, lysine and glycine in urine that can help monitoring the efficiency of treatments against the deleterious effects of type 1 diabetes.