Dane R. Hansen

Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids.

SummaryAn efficient DNA-mediated transformation system for the pathogen of chestnut, Cryphonectria parasitica, is reported. Ten vectors, each containing a promoter from Cochliobolus heterostrophus, Aspergillus nidulans, Ustilago maydis, Cephalosporium acremonium, Neurospora crassa or cauliflower mosaic virus, were creened for their ability to confer resistance to hygromycin B, benomyl or G418 sulfate. Transformants were obtained with all vectors screened and, in each case, transformation occurred by integration of the foreign DNA into the host genome. The initial transformation efficiency ranged from approximately 1–60 transformants/μg circular DNA. Under optimized transformation conditions, the transformation rate of the vector pDH25, which contains the trpC promoter and terminator of A. nidulans, exceeded 105 transformants/μg DNA. The ease with which C. parasitica is transformed should greatly facilitate the genetic manipulation of this fungal plant pathogen.

Fatty acid responses in taste cells from obesity-prone and -resistant rats

One of the transduction mechanisms for the chemoreception of fat has been proposed to involve the inhibition of delayed rectifying potassium (DRK) channels by polyunsaturated free fatty acids (PUFAs). In the present study we have compared the responsiveness of fungiform taste receptor cells (TRCs) to fatty acids in obesity-prone (Osborne-Mendel; O-M) and obesity-resistant (S5B/Pl) rat strains using patch clamp recording. TRCs from S5B/Pl rats were markedly more responsive to PUFAs than those from O-M, yet with identical inhibition constants. Moreover, addition of PUFAs to subthreshold concentrations of saccharin enhanced preference for the mixture in two-bottle preference tests compared to the saccharin alone in S5B/Pl but not O-M rats. The correlation between electrophysiological and behavioral effects of PUFAs suggested that differences in fatty acid-sensitive DRK expression may underlie the phenotypic differences between S5B/Pl and O-M rats. Consistent with this hypothesis, O-M rats exhibit a greater DRK current density and express quantitatively more DRK channels as assayed using quantitative real-time PCR. No differences were found when comparing expression of fatty acid activated two pore domain potassium channels. We propose that the ratio of fatty acid-sensitive DRK channels to fatty acid-insensitive DRK channels may be important to contributing to overall peripheral fatty acid sensitivity and in that way influence the strength of the resulting chemosensory response to fat.

Naked dsRNA Associated with Hypovirulence of Endothia parasitica Is Packaged in Fungal Vesicles

Summary Hypovirulence in Endothia parasitica is known to be associated with dsRNA. The characteristics of this cytoplasmically transmissible element suggest it might be viral in nature. Attempts to isolate viral particles indicate the presence of two particulate fractions in hypovirulent (HV) strains and a similar one in virulent (V) strains. Composition analyses of the fractions show these to be similar except that those from HV strains include dsRNA. Also present in these particles are carbohydrate, protein and chloroform-methanol-extractable substances. The protein composition is too low to indicate the presence of a capsid. The neutral sugars that are present (arabinose, mannose, galactose, glucose) in these particles are also present in the fungal cell wall. Silver- and ethidium bromide-stained polyacrylamide gels resolve several minor as well as the known major dsRNA components. These components are arbitrarily divided into four mol. wt. groups: 4.5 × 106 to 5.0 × 106, 1.2 × 106 to 1.5 × 106, 5.8 × 105 to 6.2 × 105 and < 4.6 × 105. Not all segments are present at all times, although the phenotype of the culture is not affected. Proteins are not detectable in vesicles of HV strains, but six proteins are detectable from V strains by gel electrophoresis. Assays for [32P]UMP incorporation indicate the presence of a RNA polymerase closely associated with the dsRNA. Our results suggest that the transmissible element responsible for hypovirulence is a naked dsRNA genome packaged within vesicles formed by the host.

TRPM5 is critical for linoleic acid-induced CCK secretion from the enteroendocrine cell line, STC-1.

Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked. LA leads to activation of monovalent cation-specific transient receptor potential channel type M5 (TRPM5) in STC-1 cells. LA-induced TRPM5 currents are significantly reduced when expression of TRPM5 or GPR120 is reduced using RNA interference. Furthermore, the LA-induced rise in intracellular calcium and CCK secretion is greatly diminished when expression of TRPM5 channels is reduced using RNA interference, consistent with a role of TRPM5 in LA-induced CCK secretion in STC-1 cells.

Orosensory Detection of Fatty Acids by Obesity-Prone and Obesity-Resistant Rats: Strain and Sex Differences

A series of brief-access (15s) behavioral assays following the formation of a conditioned taste aversion (CTA) to linoleic acid were performed in order to follow up on observations showing differences in the chemosensory responses to dietary fat in obesity-prone (Osborne-Mendel [O-M]) and obesity-resistant (S5B/Pl) rat strains. Strong aversions to linoleic acid (conditioned stimulus 100 microM) were generated in both O-M and S5B/Pl rats to concentrations as low as 2.5 microM. Observed strain differences were in contrast to expectations based upon electrophysiological studies previously showing greater fatty acid-induced inhibition of delayed rectifying K+ channels in S5B/Pl rats. In the CTA assays, the O-M rats showed aversions at lower fatty acid concentrations with more resistance to extinction in brief-access orosensory tests, suggesting that the obesity-prone strain may be more sensitive in the detection and subsequent avoidance of linoleic acid than the obesity-resistant strain. The independent variable of sex produced even greater differences in the avoidance of linoleic acid following conditioning than the effects of strain. Female rats of both strains were significantly more sensitive to fatty acids, showed greater cross-generalization from linoleic to oleic acid, and showed greater avoidance of linoleic acid than male counterparts. These findings suggest genetic influences on yet to be identified mechanisms potentially within the gustatory system that affect the sensitivity to detect the fatty acid chemicals found in dietary fat during brief-access orosensory testing.

Activation of oral trigeminal neurons by fatty acids is dependent upon intracellular calcium

The chemoreception of dietary fat in the oral cavity has largely been attributed to activation of the somatosensory system that conveys the textural properties of fat. However, the ability of fatty acids, which are believed to represent the proximate stimulus for fat taste, to stimulate rat trigeminal neurons has remained unexplored. Here, we found that several free fatty acids are capable of activating trigeminal neurons with different kinetics. Further, a polyunsaturated fatty acid, linoleic acid (LA), activates trigeminal neurons by increasing intracellular calcium concentration and generating depolarizing receptor potentials. Ion substitution and pharmacological approaches reveal that intracellular calcium store depletion is crucial for LA-induced signaling in a subset of trigeminal neurons. Using pseudorabies virus (PrV) as a live cell tracer, we identified a subset of lingual nerve-innervated trigeminal neurons that respond to different subsets of fatty acids. Quantitative real-time PCR of several transient receptor potential channel markers in individual neurons validated that PrV labeled a subset but not the entire population of lingual-innervated trigeminal neurons. We further confirmed that the LA-induced intracellular calcium rise is exclusively coming from the release of calcium stores from the endoplasmic reticulum in this subset of lingual nerve-innervated trigeminal neurons.

Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

High-Fat Diet Alters the Orosensory Sensitivity to Fatty Acids in Obesity- Resistant but not Obesity-Prone Rats

Gene-environment interactions play a role in the development of obesity but specific effects of diet on the orosensory detection of fatty acids have yet to be clarified. The objective of this study is to characterize the effect of prolonged (5-week) exposure to a high-fat (60%) diet on the behavioral sensitivity to the fatty acid linoleate following a conditioned taste aversion in obesity-prone and obesity-resistant rats. Exposure to the high-fat diet significantly enhanced the sensitivity of obesity-resistant (S5B/Pl) rats to linoleate while producing no effect on the fatty acid sensitivity for obesity-prone rats. Specifically, high-fat diet fed S5B/Pl rats showed stronger initial avoidance of linoleate and slower extinction rates than their normal diet cohorts. Our study suggests that prolonged dietary fat consumption may alter the behavioral sensitivity to fatty acids particularly in obesity-resistant animals.

Fatty acid responses in an enteroendocrine cell line: chemosensory cues for dietary fat.

Nutrient-induced stimulation of enteroendocrine cells (EECs) in the small intestine leads to release of the hormones glucagon-like peptide-1 and cholecystokinin that contribute to satiety. Our research has been focusing on the mechanisms that underlie the ability of fat to stimulate enteroendocrine cells during food intake. Recently, using RT-PCR we identified a rich array of potential fatty acid (FA)-activated proteins in the enteroendocrine cell line STC-1 including FA-sensitive potassium channels and several FA-activated G protein coupled receptors (GPCRs) including the long-chain FA-activated receptors GPR40 and GPR120 and the short chain FA-activated receptors, GPR41 and GPR43. To explore functional responses to FAs in STC-1 cells, we have undertaken a series of experiments using Ca2+ imaging with the ratiometric Ca2+ indicator, fura-2. Long chain unsaturated FAs elicit concentration-dependent increases in intracellular Ca2+ in STC-1 cells. Using pharmacological approaches to explore the route for FA-induced Ca2+ changes, our data are consistent with the interpretation that Ca2+ influx through voltage-gated Ca2+ channels and not release from intracellular stores is the major source of Ca2+ change. In addition, removal of extracellular Na+ also reduces the magnitude of the FA response suggesting that Ca2+-activated cation channels may contribute to the depolarization that occurs in the FA transduction pathway. We will present a model based upon available data linking GPCRs, cation channels, potassium channels and voltage-gated Ca2+ channels in the chemosensory responses of EECs to FAs. Supported by NIH-DK-59611 and International Flavors & Fragrances (TAG).