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Biomedical subjects

R A Norton

Publications and source records attributed to R A Norton.

At least 19 recordsLinked to original sources

High genetic divergences indicate ancient separation of parthenogenetic lineages of the oribatid mite Platynothrus peltifer (Acari, Oribatida).

Theories on the evolution and maintenance of sex are challenged by the existence of ancient parthenogenetic lineages such as bdelloid rotifers and darwinulid ostracods. It has been proposed that several parthenogenetic and speciose taxa of oribatid mites (Acari) also have an ancient origin. We used nucleotide sequences of the mitochondrial gene cytochrome oxidase I to estimate the age of the parthenogenetic oribatid mite species Platynothrus peltifer. Sixty-five specimens from 16 sites in North America, Europe and Asia were analysed. Seven major clades were identified. Within-clade genetic distances were below 2 % similar to the total intraspecific genetic diversity of most organisms. However, distances between clades averaged 56 % with a maximum of 125 %. We conclude that P. peltifer, as it is currently conceived, has existed for perhaps 100 million years, has an extant distribution that results from continental drift rather than dispersal and was subject to several cryptic speciations.

Animals↗

No evidence for the 'Meselson effect' in parthenogenetic oribatid mites (Oribatida, Acari).

It has been hypothesized that in ancient apomictic, nonrecombining lineages the two alleles of a single copy gene will become highly divergent as a result of the independent accumulation of mutations (Meselson effect). We used a partial sequence of the elongation factor-1alpha (ef-1alpha) and the heat shock protein 82 (hsp82) genes to test this hypothesis for putative ancient parthenogenetic oribatid mite lineages. In addition, we tested if the hsp82 gene is fully transcribed by sequencing the cDNA and we also tested if there is evidence for recombination and gene conversion in sexual and parthenogenetic oribatid mite species. The average maximum intra-specific divergence in the ef-1alpha was 2.7% in three parthenogenetic species and 8.6% in three sexual species; the average maximum intra-individual genetic divergence was 0.9% in the parthenogenetic and 6.0% in the sexual species. In the hsp82 gene the average maximum intra-individual genetic divergence in the sexual species Steganacarus magnus and in the parthenogenetic species Platynothrus peltifer was 1.1% and 1.2%, respectively. None of the differences were statistically significant. The cDNA data indicated that the hsp82 sequence is transcribed and intron-free. Likelihood permutation tests indicate that ef-1alpha has undergone recombination in all three studied sexual species and gene conversion in two of the sexual species, but neither process has occurred in any of the parthenogenetic species. No evidence for recombination or gene conversion was found for sexual or parthenogenetic oribatid mite species in the hsp 82 gene. There appears to be no Meselson effect in parthenogenetic oribatid mite species. Presumably, their low genetic divergence is due to automixis, other homogenizing mechanisms or strong selection to keep both the ef-1alpha and the hsp82 gene functioning.

Animals↗

A longitudinal study of Salmonella and Campylobacter jejuni isolates from day of hatch through processing by automated ribotyping.

Comparisons of bacterial populations over long periods of time allow researchers to identify clonal populations, perhaps those responsible for contamination of farms or humans. Salmonella and Campylobacter can cause human illness, and our objective was to use a library typing system to track strains that persist in the poultry house and through the processing plant. Two farms, over four consecutive flocks, were studied. Multiple samples were taken of the poultry house environment, feed mill, transport crates, and carcasses in the processing plant. Sample collection on the farm took place on chick placement day, midgrowout, and the day of harvest. This study found that 80.3% of isolates belonged to a single strain of Salmonella Kentucky that persisted in several environmental samples for all flocks at both farms, from chick placement day to the final product at the plant. Surgical shoe covers produced most isolates (n = 26), and processing day yielded the highest recovery (n = 68). Additional serotypes were recovered, but the Salmonella Kentucky-positive eggshells and chick mortality appeared to be the source of the organism for both farms. All Campylobacter isolates recovered were identified as C. jejuni. Most Campylobacter isolates (90.1%) belonged to one of three core strains. C. jejuni was not recovered on chick placement day. Cecal droppings yielded all nine strains. Most isolates (98.2%) were from one farm. Cluster analysis grouped C. jejuni and Salmonella isolates into four and six distinct clusters, respectively, on the basis of a similarity level of 80%.

Animal Husbandry↗

Food security issues--a potential comprehensive plan.

The need for a comprehensive plan to protect the food production system has emerged as a critical issue over the last several years. To address this need, a comprehensive food security plan has been developed at Auburn University. The proposed program, entitled the Consolidated American Network for Agriculture Resource Intelligence (CANARI) system is one of several systems being proposed to deal with potential agricultural bioterrorism or agroterrorism events. Unlike other systems, which hastily emerged in many agencies after the tragedy of September 11, 2001, the system has been planned over the last 5 yr with the input of the agricultural industries, is comprehensive in its conception, and is designed to coordinate all components (existing and planned) necessary to prevent, detect, and respond to potential agroterrorism events. The plan uses the principle that the first line of defense must be within the states and agricultural companies for the detection of agroterrorism incidents to be rapid and the response effective, organized, and timely. CANARI is designed to integrate the previously disparate elements by fostering a cooperative network of local, state, and federal agencies as well as commodity entities and interested non-governmental organizations. Using a market-driven approach, the system encourages commodity membership and cooperation through positive incentives rather than regulatory duress. A centralized command structure is envisioned, which would be provided through the creation of a National Agroterrorism Defense Center. The responsibility of this Center would be to coordinate all of the activities presently available in components at the local, state, and federal levels and develop and manage new and emerging activities provided by the stakeholders. CANARI offers a new paradigm by which all of its constituent members act collectively and cooperatively to lessen the risk of an attack and better ensure the continued availability of a safe, abundant, and economical food supply.

Agriculture↗

Major histocompatibility complex effect on cellulitis among different chicken lines.

The chicken major histocompatibility complex (MHC) has been implicated in conferring resistance/susceptibility to several bacterial, parasitic, and viral diseases. Investigators have shown that the chicken MHC plays a major role in determining the outcome of a Marek's disease infection, in that standard B(13) is susceptible to the virus while B(21) confers resistance to the virus. Previous work with a broiler line has shown that B(21) is susceptible to an Escherichia coli-induced cellulitis infection and that B(13) conferred resistance to the infection. For this experiment, a broiler and a Leghorn chicken line shown to contain standard B(13) and B(21) were examined in a challenge model for cellulitis. The birds were challenged with a cellulitis-causing E. coli isolate. Homozygous B(21) had the highest incidence of cellulitis development compared with either homozygous B(13) or the heterozygous B(13)/B(21) for both the broiler and Leghorn lines. Additionally, cellulitis lesion severity was measured in both lines and shown to be independent of MHC type.

Animals↗

Isoform patterns of chitinase and beta-1,3-glucanase in maturing corn kernels (Zea mays L.) associated with Aspergillus flavus milk stage infection.

Isoform patterns of chitinase and beta-1,3-glucanase of maturing kernels of yellow dent corn (Pioneer 3394) infected with Aspergillus flavus at the milk stage were investigated through polyacrylamide gel electrophoresis (PAGE). Proteins on the sodium dodecyl sulfate (SDS) gel with an apparent molecular mass range of 23-46 kDa were differentially present in the kernels infected with both aflatoxin-producing and non-aflatoxin-producing strains of A. flavus. From in-gel (native PAGE) enzyme activity assays, three bands corresponding to chitinase isoforms and two bands corresponding to beta-1,3-glucanase isoforms were detected in the infected kernels. One chitinase isoform of 29 kDa was present only in the infected kernels, and another one of 28 kDa was present in both infected and noninfected kernels. They were judged to be acidic on the basis of their migration on an acrylamide isoelectric focusing (IEF) gel. For the beta-1,3-glucanase, one isoform of 35 kDa was present in both infected and noninfected kernels, but another one, a 33 kDa isoform, was present only in the infected kernels. Both acidic and basic beta-1,3-glucanase isoforms were detected in the IEF gel. The results of this study are the first to demonstrate patterns of enhanced or inducible proteins in maturing corn kernels in response to A. flavus infection at the milk stage. The results also indicate that only particular isoforms of the two hydrolytic enzymes are involved in the maturing corn kernels infected at the milk stage with A. flavus.

Aspergillus↗

Inhibition of aflatoxin B(1) biosynthesis in Aspergillus flavus by anthocyanidins and related flavonoids.

Anthocyanidins and precursors or related flavonoids were tested at concentrations from 0.3 to 9.7 mM ( approximately 0.1-3.0 mg/mL) for activity against growth and aflatoxin B(1) biosynthesis by Aspergillus flavus Link:Fr. NRRL 3357. Aflatoxin B(1) production was inhibited by all anthocyanidins tested, and 3-hydroxy compounds were more active than 3-deoxy forms. Monoglycosides of cyanidin were 40% less inhibitory than the aglycon, whereas a monoglucoside and a diglucoside of pelargonidin were 80 and 5%, respectively, as active as the aglycon. Of eight flavonoids tested, only kaempferol was moderately active, whereas luteolin and catechin were weakly inhibitory. Binary combinations of delphinidin and three other aflatoxin inhibitors acted independently of each other. Results with an aflatoxin pathway mutant indicated that anthocyanidin inhibition occurred before norsolorinic acid synthesis.

Aflatoxin B1↗

Biotransformation of 24 alpha-methylcholesterol and 24 beta-methylcholesterol by yeast mutant GL7.

Incubation of 24 alpha- and 24 beta-methylcholesterols with yeast mutant GL7 afforded their corresponding C-22-desaturated products under the catalysis of sterol delta 22(23)-desaturase. The metabolites were identified to be 22-dehydro-24 alpha-methylcholesterol (2% yield from 24 alpha-methylcholesterol) and 22-dehydro-24 beta-methylcholesterol (51% yield from 24 beta-methylcholesterol) respectively on the basis of their chromatographic and spectral properties. It was concluded that the sterol delta 22(23)-desaturase prefers the 24 beta-methyl sterols and is highly stereospecific.

Biotransformation↗

Detection of human herpesvirus 6 by reverse transcription-PCR.

The role of human herpesvirus 6 (HHV-6) in disease beyond primary infection remains unclear. We have developed and validated a new reverse transcription-PCR (RT-PCR) assay for HHV-6 that can determine the presence of HHV-6 in clinical specimens and differentiate between latent and replicating virus. Peripheral blood mononuclear cells from 109 children were evaluated for HHV-6 by RT-PCR, DNA PCR, and viral culture. Of these samples, 106 were suitable for analysis. A total of 20 samples were positive for HHV-6 by culture and DNA PCR, of which 19 were positive by RT-PCR (sensitivity, 95%). All 28 samples from children that were negative by viral culture, but positive by DNA PCR, were negative for viral transcripts by our RT-PCR assay. One positive RT-PCR result was observed in 56 samples that were negative by tissue culture and DNA PCR. This indicates a low rate of false-positive results (1.2%) and a specificity of 98.8%. This RT-PCR assay can reliably differentiate between latent and actively replicating HHV-6 and should allow insight into the pathogenesis of this ubiquitous virus.

Base Sequence↗

Sterol utilization and metabolism by Heliothis zea.

Heliothis zea (corn earworm), an insect that fails to synthesize sterols de novo, was reared on an artificial diet treated with 18 different sterol supplements. Larvae did not develop on a sterol-less medium. delta 5-Sterols with a hydrogen atom, a methylene group, an E- or Z-ethylidene group, or an alpha- or beta-ethyl group (cholesterol, ostreasterol, isofucosterol, fucosterol, sitosterol, and clionasterol, respectively) at position C-24, and delta 5-sterols doubly substituted in the side chain at C-24 with an alpha-ethyl group and at C-22 with a double bond (stigmasterol) supported normal larval growth to late-sixth instar (prepupal: maturity). The major sterol isolated from each of these sterol treatments was cholesterol, suggesting that H. zea operates a typical 24-dealkylation pathway. The sterol requirement of H. zea could not be met satisfactorily by derivatives of 3 beta-cholestanol with a 9 beta, 19-cyclopropyl group, gem dimethyl group at C-4, a delta 5,7-bond or delta 8-bond, or by side chain modified sterols that possessed a delta 25(27)-24 beta-ethyl group, delta 23(24)-24-methyl group or 24-ethyl group, or delta 24(25)-24-methyl or 24-ethyl group. The major sterol recovered from the larvae (albeit developmentally arrested larvae) treated with a nonutilizable sterol was the test compound. Sterol absorption was related to the degree of sterol utilization. The most effective sterols absorbed by the insect ranged from 27 to 66 micrograms per insect, whereas the least effective sterols absorbed by the insect ranged from 0.6 to 6 micrograms per insect. Competition experiments using different proportions of cholesterol and 24-dihydrolanosterol (from 9:1 to 1:9 mixtures) indicated that abnormal development of H. zea may be induced on less than a 1 to 1 mixture of utilizable (cholesterol) to nonutilizable (24-dihydrolanosterol) sterols. The results demonstrate new structural requirements for sterol utilization and metabolism by insects, particularly with respect to the position of double bonds in the side chain and functionalization in the nucleus. The novel sterol specificities observed in this study appear to be associated with the dual role of sterols as membrane inserts (nonmetabolic) and as precursors to the ecdysteroids (metabolic).

Animals↗

The challenge of irritable bowel syndrome.

Irritable bowel syndrome is a common disorder with symptom complexes that can include diarrhea, constipation, pain and bloating. After other disorders have been excluded, treatment should be directed at the predominant symptoms and gauged to their severity. A careful balance between a thoughtful investigation and the expense and dangers of overtesting must always be considered. Most patients continue to have persistent symptoms several years after the original diagnosis.

Adult↗

A novel glass fiber disc culture system for testing of small amounts of compounds on growth and aflatoxin production by Aspergillus flavus.

A new method for growing Aspergillus flavus for experimental studies is presented. The system consists of a humidified vial with a thick septum pierced by a pin on which a glass fiber disc is affixed. The disc contains the test solution and inoculum plus medium. The method has been used to assess the effect of variations in culture conditions on production of aflatoxin B1 (AFB1). The AFB1 level was affected by the amount of medium placed on the disc and type of disc material. The results for different types of glass fiber and quartz discs were compared with AFB1 produced by fungus grown in liquid medium or on paper discs. When compared to a liquid medium culture there was a 15 to 20-fold increase in AFB1 for one type of disc. Incubations with less than 14 microliters of medium gave satisfactory results. A crude phosphatidylcholine preparation at a concentration of 0.7% of the medium resulted in a 4-fold increase in AFB1.

Aflatoxin B1↗

Quantitation of steryl ferulate and p-coumarate esters from corn and rice.

The principal steryl ferulate and p-coumarate esters of different fractions from processed corn brans and corn oils, unrefined and refined, and from rice bran and rice bran oil were quantified by high-performance liquid chromatography. The results show that hexane-extracted corn oils yield more than five times the amount of esters compared to expeller processed oils. The yields of esters from bran and related products ranged from 0.07 to 0.54 mg/g of bran. Unrefined corn oils had levels from 0.18 to 8.6 mg/g for oil from hexane-extracted bran. By comparison, rice bran had ester levels of 3.4 mg/g of bran, and rice bran oil had levels of 15.7 mg/g of oil. The predominant esters from corn were sitostanyl and campestanyl ferulate, and sitostanyl and campestanyl p-coumarate. The principal esters from rice bran were cycloartenyl, 24-methylenecycloartanyl, and campesteryl ferulate. Rice bran oils had low levels of 24-methylenecycloartanyl but high levels of cyclobranol esters. The data presented provide a direct comparison of steryl ferulate and p-coumarate levels in the two cereals, and will aid in selecting the most suitable sources for the isolation of these compounds from corn products.

Chromatography, High Pressure Liquid↗

Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor.

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.

Cell Division↗

Efficacy of different anticoccidials against experimental coccidiosis in large white turkeys.

Two trials were conducted to compare the efficacy of currently approved anticoccidials for turkeys against challenge using a field isolate of mixed Eimeria species; E. adenoides, E. gallopavonis, and E. meleagrimitis. Poults in wire-floored cages were fed unmedicated diets from day-old to 3 wk of age. Diets were supplemented with either amprolium (AMP, 125 mg/kg), butynorate (BUT, 375 mg/kg), monensin (MON-60, 60 mg/kg; MON-100, 100 mg/kg), halofuginone (HAL; 3 mg/kg), zoalene (ZOA; 125 mg/kg), or sulfadimethoxine plus ormetoprim (SUL + ORM, 62.5 mg/kg and 37.5 mg/kg, respectively). After 2 days on the test diets, poults were individually weighed and inoculated with sporulated coccidial oocysts from the field isolate. Total fecal collections were obtained for Days 0 to 5 and 6 to 10 to estimate oocyst output. At 10 days postinoculation, the birds were individually weighed and killed to determine severity of intestinal lesions. The HAL and MON were most effective and AMP, ZOA, and SUL + ORM were least effective in maintaining weight and in reducing the severity of intestinal lesions. All the coccidiostats tested reduced oocyst passage, but poults fed HAL produced fewer oocysts. The results demonstrated differences in efficacy among anticoccidials with the more recently approved drugs providing the best protection against coccidiosis.

Amprolium↗

Research note: use of fenbendazole for the treatment of turkeys with experimentally induced nematode infections.

Turkeys were raised under parasite-free conditions until 25 days of age at which time the birds were administered infective ova of Capillaria obsignata, Heterakis gallinarum, and Ascaridia dissimilis. At 28 days postinfection, four groups of birds were placed on rations medicated with fenbendazole at 15,30,45, or 60 ppm. These rations were given ad libitum for 6 consecutive days. At 31 days postinfection, five additional groups of birds were placed on rations medicated with fenbendazole at 15,30,45,60 or 120 ppm. These latter rations were given ad libitum for 3 consecutive days. One group of turkeys served as an unmedicated, infected control. Treatment group size ranged from 17 to 19 birds. All birds were necropsied 5 days after the medicated rations were withdrawn and nematode recovery was performed. Control birds harbored an average of 3.29 A. dissimilis, 12.06 H. gallinarum, and 65.94 C. obsignata. All but one of the fenbendazole-medicated groups showed 100% removal of A. dissimilis. The exception was that group that received fenbendazole at 15 ppm for 6 days, and that showed a 98.5% efficacy. The removal rate for H. gallinarum ranged from 78.6% (15 ppm for 3 days) to 100% (120 ppm for 3 days and 45 ppm for 6 days). The C. obsignata infections proved dose-limiting, with a removal rate ranging from 30.8% (15 ppm for 3 days) to 97.8% (45 ppm for 6 days).

Administration, Oral↗

Zymosterol is located in the plasma membrane of cultured human fibroblasts.

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.

Acetates↗