Effect of E-beam treatment on expression of virulence and stress-response genes of Listeria monocytogenes in dry-cured ham.

Various adverse conditions can trigger defensive mechanisms in Listeria monocytogenes that can increase the virulence of surviving cells. The objective of this study was to evaluate the expression of one stress-response (sigB) and three virulence (plcA, hly, and iap) genes in L. monocytogenes exposed to a sub lethal dose of E-beam irradiation in dry-cured ham. To accomplish this, dry-cured ham slices (10 g) were immersed in a 109 CFU/mL suspension of L. monocytogenes strain S4-2 and subsequently irradiated with 1, 2, or 3 kGy. After irradiation, samples were stored at 7 °C or 15 °C for 30 days. Absolute gene expression levels were determined by RT-qPCR, and numbers of surviving Listeria cells were assessed by microbial counts after different storage times (0, 7, 15, and 30 days). At 7 °C, after E-beam treatment at doses of 2 or 3 kGy, Listeria gene expression significantly increased (p ≤ 0.05) up to day 15. Listeria counts decreased with increasing dosage. The relationship between absolute gene expression and the number of surviving Listeria cells could indicate that sublethal doses of E-beam irradiation can increase expression of the genes studied. We observed no significant influence of storage time or temperature on gene expression (p > 0.05). Listeria that survives E-beam treatment may display increased virulence, constituting a significant potential public health risk.

Evaluation of metal concentrations in mentha herbal teas (Mentha piperita, Mentha pulegium and Mentha species) by inductively coupled plasma spectrometry.

Phytopharmaceuticals are gaining popularity worldwide; however, cases of adverse effects and drug interactions have also increased. One reason is in the high metal content both as ingredients but also as contaminants. Metal monitoring in food, like herbal teas, provides basic information on safety aspects in regulatory processes as well as nutritional values. In the present work, Cd, Pb, K, Na, Ca, Mg, Al, B, Ba, Co, Cr, Cu, Fe, Mn, Zn, Li, Ni, and Mo were determined by inductively coupled plasma spectrometry (ICPS) in 36 samples of Mentha sp. Mint tea bags and loose leaves were randomly obtained from supermarkets, traditional markets, herbal stores, and pharmacies in Tenerife (Canary Islands, Spain). Metal contents varied significantly, dependent on the stores the products were purchased in and on tea packaging (loose leaves versus tea bags). Pb analyses revealed levels (0.65±0.71mg/kg) below legal limits. The maximum permissible limit for Cd, 0.3mg/kg, set by the WHO for medicinal plants, was exceeded by 19.44% of the samples (0.22±0.13mg/kg), but all values were below the limit given in the European Pharmacopoeia for this metal (4mg/kg). We observed high Al (151.24±162.73mg/kg) and Li (5.46±3.94mg/kg) levels. B, Ba, Co, Cr, Cu, Fe, Mn, Ni, Zn, and Mo mean levels were 20.51, 14.15, 0.26, 1.65, 10.65, 406.00, 55.05, 1.72, 33.67, and 0.73mg/kg, respectively. Mean Ca, Mg, K, and Na were detected in concentrations of 10.32, 3.83, 7.23 and 1.17g/kg, respectively. In conclusion, metal exposure through herbal mint teas does not seem to be of health concern, as to most of the studied metals, but regulatory limits for Al contents should be imposed.

Vocally correlated seasonal auditory variation in the house sparrow (Passer domesticus).

Songbirds exhibit seasonal plasticity in a broad variety of behavioral and morphological traits associated with reproduction. Changes in song production are well described while changes in song reception are not. In the present study, we test for seasonal variation in auditory processing of the house sparrow (Passer domesticus L.) using auditory brainstem responses (ABRs) to tone bursts. We measured amplitude and latency of the first ABR peak in spring, summer and autumn at stimulus frequencies from 0.8 to 6.4 kHz and intensity levels from 24 to 80 dB SPL. ABR thresholds were determined at each frequency using cross-correlation. Amplitude was greater in spring than in autumn at frequencies from 3.2 to 6.4 kHz whereas latency and thresholds exhibited no seasonal variation. The results indicate an increase in the number or temporal synchrony of responses from peripheral auditory neurons during the early breeding season. Changes in peripheral auditory processing may enhance temporal coding of the fine structure and envelope of song; thereby, improving assessment of encoded information in both sexes (e.g. individual identity and dominance status) and auditory feedback during song production in males. Peripheral auditory changes may be mediated by reproductive hormones, and could involve changes in hair cell density on the basilar papilla. Our results suggest that peripheral auditory processing of songbirds changes seasonally in parallel with other behavioral and morphological traits, such as song production.

U.S. laboratory and field trials of metofluthrin (SumiOne) emanators for reducing mosquito biting outdoors.

Metofluthrin (SumiOne is a novel, vapor-active pyrethroid that is highly effective against mosquitoes. Laboratory and field trials were conducted in the United States to evaluate the mosquito repellent activity of metofluthrin-treated paper substrates ("emanators"). Initial studies were conducted to evaluate the field performance of 900-cm(2) paper fan emanators impregnated with 160 mg metofluthrin, where Aedes canadensis was the predominant species. Emanators reduced landing rates on human volunteers by between 85% and 100% compared to untreated controls. Subsequent tests with 4,000-cm(2) paper strip emanators impregnated with 200 mg metofluthrin were conducted in a wind tunnel as a precursor to conducting field trials using human bait and laboratory-reared Aedes aegypti. Paper strips, which were pre-aged in a fume hood to determine duration of protection, gave 89-91% reductions in landing rates compared with controls. Similar reductions in biting activity were also noted. Following these tests, field trials to assess effect on landing rates were conducted with emanators positioned 1.22 m on either side of volunteers protected from biting by Tyvek suits, with pre- and posttreatment counts being made. In Florida (predominantly Ochlerotatus spp.) 91-95% reductions were noted 10-30 min after emanators were deployed, while in Washington State (mostly Aedes vexans) 95-97% reductions were observed. These results demonstrate that metofluthrin-treated emanators are highly effective at repelling mosquitoes.

A comparative study of avian auditory brainstem responses: correlations with phylogeny and vocal complexity, and seasonal effects.

We conducted a comparative study of the peripheral auditory system in six avian species (downy woodpeckers, Carolina chickadees, tufted titmice, white-breasted nuthatches, house sparrows, and European starlings). These species differ in the complexity and frequency characteristics of their vocal repertoires. Physiological measures of hearing were collected on anesthetized birds using the auditory brainstem response to broadband click stimuli. If auditory brainstem response patterns are phylogenetically conserved, we predicted woodpeckers, sparrows, and starlings to be outliers relative to the other species, because woodpeckers are in a different Order (Piciformes) and, within the Order Passeriformes, sparrows and starlings are in different Superfamilies than the nuthatches, chickadees, and titmice. However, nuthatches and woodpeckers have the simplest vocal repertoires at the lowest frequencies of these six species. If auditory brainstem responses correlate with vocal complexity, therefore, we would predict nuthatches and woodpeckers to be outliers relative to the other four species. Our results indicate that auditory brainstem responses measures in the spring broadly correlated with both vocal complexity and, in some cases, phylogeny. However, these auditory brainstem response patterns shift from spring to winter due to species-specific seasonal changes. These seasonal changes suggest plasticity at the auditory periphery in adult birds.

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes.

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans.

BACKGROUND: Isocitrate lyase catalyses the first committed step of the carbon-conserving glyoxylate bypass, the Mg(2+)-dependent reversible cleavage of isocitrate into succinate and glyoxylate. This metabolic pathway is an inviting target for the control of a number of diseases, because the enzymes involved in this cycle have been identified in many pathogens including Mycobacterium leprae and Leishmania. RESULTS: As part of a programme of rational drug design the structure of the tetrameric Aspergillus nidulans isocitrate lyase and its complex with glyoxylate and a divalent cation have been solved to 2.8 A resolution using X-ray diffraction. Each subunit comprises two domains, one of which adopts a folding pattern highly reminiscent of the triose phosphate isomerase (TIM) barrel. A 'knot' between subunits observed in the three-dimensional structure, involving residues towards the C terminus, implies that tetramer assembly involves considerable flexibility in this part of the protein. CONCLUSIONS: Difference Fourier analysis together with the pattern of sequence conservation has led to the identification of both the glyoxylate and metal binding sites and implicates the C-terminal end of the TIM barrel as the active site, which is consistent with studies of other enzymes with this fold. Two disordered regions of the polypeptide chain lie close to the active site, one of which includes a critical cysteine residue suggesting that conformational rearrangements are essential for catalysis. Structural similarities between isocitrate lyase and both PEP mutase and enzymes belonging to the enolase superfamily suggest possible relationships in aspects of the mechanism.

The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine/acylcarnitine carrier.

The Aspergillus nidulans acuH gene, required for growth on acetate and long-chain fatty acids, was cloned by complementation of the acuH13 mutation. Northern blotting analysis showed that transcription of the acuH gene occurs in acetate-grown mycelium and at higher levels in oleate-grown mycelium, but not during growth on glucose minimal medium. The acuH gene encodes a protein of 326 amino acids that belongs to the mitochondrial carrier family. The ACUH protein contains three related segments of approximately 100 amino acids in length, each segment comprising two hydrophobic domains that are probably folded into two transmembrane alpha-helices linked by an extensive polar region. Sequence comparisons suggest that the acuH gene of A. nidulans encodes the homologue of the carnitine/acylcarnitine carrier of rat and man. The uncharacterised proteins YOR100C of Saccharomyces cerevisiae, COLT of Drosophila melanogaster, and DIF-1 of Caenorhabditis elegans also seem to be homologues of ACUH. In addition to the motifs present in all members of the mitochondrial carrier family, we propose the highly conserved motif R(A,S)(V,F)PANAA(T,C)F within the sixth hydrophobic domain of these proteins as the characteristic feature of the carnitine carrier subfamily. The proposed function of the ACUH protein is the transport of acetylcarnitine molecules from the cytosol to the mitochondrial matrix, a process required during growth on acetate or on long-chain fatty acids.

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy.

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide.

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method.

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 x 10(7) viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole-) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole- mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim- (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 x g supernatant, but produced wild-type levels of penicillin.

Purification and properties of beta-galactosidase from Aspergillus nidulans.

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

Induction of beta-oxidation enzymes and microbody proliferation in Aspergillus nidulans.

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced beta-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the beta-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The beta-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans.

Variability of normal coronary anatomy: implications for the interpretation of thallium-SPECT myocardial perfusion images in single-vessel disease.

UNLABELLED: Standard criteria for assigning perfusion defects to a specific vascular territory often result in mistaken identification of the affected coronary artery due to the normal variability of coronary anatomy. A retrospective study was performed to determine the frequency of this type of error and to identify the most common perfusion patterns associated with specific coronary lesions. METHODS: Records were reviewed of all patients with single-vessel coronary artery disease (CAD) who had exercise or dipyridamole thallium SPECT myocardial perfusion studies since 1987. Patients with coronary artery bypass grafts and an interval between the two studies greater than 6 wk or interval change in medical status were excluded. Ninety-three studies were available for review. The size, severity and location of all perfusion defects were noted by three observers who had no knowledge of the angiographic data. Significant CAD was defined as luminal diameter stenosis greater than 50%. RESULTS: The diseased vessel was correctly identified in 85% of positive studies. Thallium SPECT, however, mistakenly predicted additional vessel involvement in 29% of those studies. Another 15% correctly predicted single-vessel disease but identified the wrong artery. Using standard criteria, thallium SPECT correctly predicted the arteriogram findings in only 56% of studies. Most of these findings could be correlated with variations in individual coronary anatomy. CONCLUSION: The accurate localization of coronary stenoses by thallium SPECT imaging requires close correlation with arteriography owing to the significant variability in normal coronary anatomy.

Mu1-related transposable elements of maize preferentially insert into low copy number DNA.

The Mutator transposable element system of maize was originally identified through its induction of mutations at an exceptionally high frequency and at a wide variety of loci. The Mu1 subfamily of transposable elements within this system are responsible for the majority of Mutator-induced mutations. Mu 1-related elements were isolated from active Mutator plants and their flanking DNA was characterized. Sequence analyses revealed perfect nine base target duplications directly flanking the insert for 13 of the 14 elements studied. Hybridizational studies indicated that Mu1-like elements insert primarily into regions of the maize genome that are of low copy number. This preferential selection of low copy number DNA as targets for Mu element insertion was not directed by any specific secondary structure(s) that could be detected in this study, but the 9-bp target duplications exhibited a discernibly higher than random match with the consensus sequence 5'-G-T-T-G-G/C-A-G-G/A-G-3'.

Analysis of the regulation of the Aspergillus nidulans acuD gene, encoding isocitrate lyase, by construction of a hybrid promoter.

In order to confirm functionally that a 208 bp fragment of the 5'-flanking sequence of the acuD gene of Aspergillus nidulans is the region responsible for acetate inducibility and catabolite repression, a hybrid promoter was constructed by insertion of this fragment into the promoter of the (highly expressed) oliC gene of A. nidulans. Analysis of expression of the lacZ reporter gene fused to the oliC/acuD promoter showed induction by acetate at much higher levels than wild-type acuD expression. Acetate inducibility of the hybrid promoter was dependent on the facB gene, demonstrating that a facB-dependent upstream activating sequence (UAS) for acetate must be located in the 208 bp acuD fragment. In parallel, partial relief of the transcriptional repression of acetate inducibility by sucrose and glucose was observed in a creA- background, showing that the 208 bp acuD fragment also responds to the creA gene. In addition, the results show that combination of a regulatory element from a low-expression promoter (acuD) with a high-expression constitutive promoter (oliC) leads to amplification of the level of regulated expression.

Regulation of the expression of the isocitrate lyase gene (acuD) of Aspergillus nidulans.

Analysis of the promoter region of the acetate-induced isocitrate lyase gene (acuD) of Aspergillus nidulans is described. Transcription start sites were detected at positions -163, -170 and approximately -281 upstream of the ATG. Transcription analysis showed that the acuD gene is transcribed during growth on acetate but not on hexoses or glycerol. Expression of the acuD gene was studied under inducing and repressing conditions in cre+, creA, creB and creC mutant strains, showing that the creA(d)-1 mutation led to slight derepression of isocitrate lyase. Regulation of expression of the acuD gene was also studied using an in-frame fusion with the lacZ gene of Escherichia coli. Several deletions were made in order to identify the regions responsible for acetate induction and repression. A deletion of the -412 to -200 bp upstream region resulted in loss of all promoter activity and a smaller deletion within this region abolished most of the acetate inducibility.

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans.

The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creAd-30 strain showed that the creA gene is not involved in this process.

Breed group differences in testicular growth patterns in spring-born ram lambs.

Three groups of spring-born ram lambs were used to assess breed group differences in testicular growth patterns through 5 mo of age. Group 1 included 20 3/4-Finnish Landrace (Finn), 1/4-Rambouillet rams and 31 1/8-Finn, 7/8-Rambouillet rams. Group 2 included 23 3/4-Finn, 1/4-Dorset rams and 19 1/8-Finn, 7/8-Dorset rams. Group 3 included 21 black-faced (BF) rams (six Hampshire and 15 Suffolk) and 46 whitefaced (WF) rams (nine Dorset, 20 Barbados Blackbelly X Dorset, 10 Finn X Dorset and six Dorset X Finn). Scrotal circumference (C) was measured at 19, 43, 72, 100, 128 and 153 d in Group 1; at 20, 48, 76, 93 and 153 d in Group 2 and at 40, 60, 81, 103, 124 and 158 d in Group 3. Breed group X age interaction was tested to assess breed group differences in testicular growth patterns, and logistic curves were used to describe increases in scrotal circumference with age. In Group 1, 3/4-Finn and 1/8-Finn rams were similar in estimated final scrotal circumference at 150 d of age (260 and 259 mm, respectively), but rapid testicular growth began earlier in 3/4-Finn rams. The estimated age at which the growth rate in scrotal circumference was maximum was 81 d for 3/4-Finn rams and 93 d for 1/8-Finn rams. No differences were observed in testicular growth in Group 2 rams. However, in Group 3, smaller WF rams matured more rapidly than larger BF rams. Growth in scrotal circumference was most rapid at 99 d in BF rams and 88 d in WF rams. Within WF rams, Barbados Blackbelly X Dorset and Finn X Dorset rams matured earlier than Dorsets. In all groups, the primary breed difference was associated with age differences during the period of rapid testicular growth.