Phosphatidylcholine synthesis in castor bean endosperm: the localization and control of CTP: choline-phosphate cytidylyltransferase activity

The reaction catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been postulated to be a control reaction in the synthesis of phosphatidylcholine (PtdCho) in many animal tissues and some plants. In 3-day-old castor bean (Ricinus communis L. var. Hale) endosperm the majority of cytidylyltransferase activity resided in a 12,000gav 10-min pellet. Following density-gradient fractionation, 60 to 70% of the enzyme activity was associated with the endoplasmic reticulum (ER) fraction, with the remainder in the particulate fraction being in an unidentified membrane band (band A), less than occurred in the soluble fractions. The properties and kinetics of the forward and reverse reactions are described. About 40% of the total ER activity could be solubilized by treatment of the fraction with 0.32 M KCl, which resulted in a threefold increase in the specific activity of the enzyme. The Michaelis constants of the solubilized enzyme were similar to those of the ER activity. The activity of the solubilized enzyme was stimulated 35% by addition of phosphatidylglycerol or phosphatidylinositol to the assay. Addition of a number of other phospholipids to the incubation medium caused only a small change in activity (+/- 10%) but the enzyme could be stimulated up to 60% by the addition of 0.01-1 mM sodium oleate. A combination of 0.25 mM PtdCho with oleate in the assay resulted in additional stimulation at all concentrations of oleate. Oleate had no effect on the ER activity. These results are discussed in relation to the regulation of cytidylyltransferase activity in plants.     

The oldest evidence of a sauropod dinosaur in the western united states and other important vertebrate trackways from grand staircase‐escalante national monument,Utah

Recent discoveries of abundant fossil footprints from the new Grand Staircase‐Escalante National Monument of southern Utah, have important implications for the spatial and temporal distribution of Mesozoic vertebrates in Triassic and Jurassic time. Since the monument's creation in 1996, fossil footprints have been reported from at least seven formations in the Mesozoic (Triassic‐Cretaceous) within the monument. By far the most significant of these discoveries are sauropod and theropod tracks from the upper part of the Middle Jurassic Entrada Sandstone and a large Apatopus trackway from the Late Triassic Chinle Formation. Tracks in the Entrada Sandstone are found at the same stratigraphic level as those in the Moab megatracksite, and so considerably extend this large ichnological complex. A wide‐gauge sauropod trackway (cf. Brontopodus) from this unit represents the first reported from the Entrada Sandstone, and so is the oldest known from the western United States. This trackway also reveals a tail trace, which is the first reliable record of a sauropod tail trace.     

The biogeochemical controls of N2O production and emission in landfill cover soils: the role of methanotrophs in the nitrogen cycle

Emissions of N2O from cover soils of both abandoned (> 30 years) and active landfills greatly exceed the maximum fluxes previously reported for tropical soils, suggesting high microbial activities for N2O production. Low soil matrix potentials (<-0.7 MPa) indicate that nitrification was the most likely mechanism of N2O formation during most of the time of sampling. Soil moisture had a strong influence on N2O emissions. The production of N2O was stimulated by as much as 20 times during laboratory incubations, when moisture was increased from -2.0 MPa to -0.6 MPa. Additional evidence from incubation experiments and delta13C analyses of fatty acids (18:1) diagnostic of methanotrophs suggests that N2O is formed in these soils by nitrification via methanotrophic bacteria. In a NH3(g)-amended landfill soil, the rate of N2O production was significantly increased when incubated with 100 ppmv methane compared with 1.8 ppmv (atmospheric) methane. Preincubation of a landfill soil with 1% CH4 for 2 weeks resulted in higher rates of N2O production when subsequently amended with NH3(g) relative to a control soil preincubated without CH4. At one location, at the soil depth (9-16 cm) of maximum methane consumption and N2O production, we observe elevated concentrations of organic carbon and nitrogen and distinct minima in delta15N (+1.0%) and delta13C (-33.8%) values for organic nitrogen and organic carbon respectively. A delta13C value of -39.3% was measured for 18:1 carbon fatty acids in this soil, diagnostic of type II methanotrophs. The low delta15N value for organic nitrogen is consistent with N2 fixation by type II methanotrophs. These observations all point to a methanotrophic origin for the organic matter at this depth. The results of this study corroborate previous reports of methanotrophic nitrification and N2O formation in aqueous and soil environments and suggest a predominance of type II rather than type I or type X methanotrophs in this landfill soil.     

Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants

Degradation of milk components through various enzymatic activities associated with the contamination of dairy products by Pseudomonas spp. can reduce the shelf life of processed milk. Reliable methods for differentiating among Pseudomonas spp. strains are necessary to identify and eliminate specific sources of bacterial contamination from dairy processing systems. To that end, we assessed the genetic diversity and dairy product spoilage potentials among a total of 338 Pseudomonas spp. isolates from raw and pasteurized milk and from environmental samples collected from four dairy processing plants. The majority of isolates were identified as P. fluorescens and P. putida by API 20 NE. A total of 42 different ribotype patterns were identified among a subset of 81 isolates. The presence of many different ribotypes within this collection indicates high genetic diversity among the isolates and suggests multiple origins of contamination within the processing plant and in dairy products. The extracellular enzyme activity patterns among Pseudomonas isolates appeared to be associated with ribotypes. Isolates with the same ribotype frequently had the same extracellular protease, lecithinase, and lipase activities. For example, isolates grouped in ribotype 55-S-6 had the highest extracellular protease activity, while those in ribotypes 50-S-8 and 72-S-3 had the highest extracellular lipase activities. We conclude that ribotyping provides a reliable method for differentiating Pseudomonas strains with dairy food spoilage potential.     

Molecular Ecology of Listeria monocytogenes: Evidence for a Reservoir in Milking Equipment on a Dairy Farm

A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P < 0.05) and samples from other sources (P < 0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P < 0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.Listeria monocytogenes can cause listeriosis in humans. This illness, despite being underreported, is an important public health concern in the United States (23) and worldwide. According to provisional incidence data provided by the Centers for Disease Control and Prevention (CDC), 762 cases of listeriosis were reported in the United States in 2007. In previous years (2003 to 2006), the number of reported annual listeriosis cases in the United States ranged between 696 and 896 cases per year (5).Exposure to food-borne L. monocytogenes may cause fever, muscle aches, and gastroenteritis (30), but does not usually cause septicemic illness in healthy nonpregnant individuals (7, 30). Elderly and immunocompromised people, however, are susceptible to listeriosis (22, 10), and they may develop more-severe symptoms (10). Listeriosis in pregnant women may cause abortion (22, 30) or neonatal death (22).Dairy products have been identified as the source of several human listeriosis outbreaks (4, 7, 10, 22). Listeria is ubiquitous on dairy farms (26), and it has been isolated from cows'' feces, feed (3, 26), and milk (21, 35). In ruminants, L. monocytogenes infections may be asymptomatic or clinical. Clinical cases typically present with encephalitis and uterine infections, often resulting in abortion (26, 39). Both clinically infected and healthy animals have been reported to excrete L. monocytogenes in their feces (20), which could eventually cause contamination of the bulk tank milk or milk-processing premises (39).On-farm epidemiologic research provides science-based information to improve farming and management practices. The Regional Dairy Quality Management Alliance (RDQMA) launched a combined United States Department of Agriculture (USDA)-RDQMA pilot project in January 2004 to scientifically validate intervention strategies in support of recommended best management practices among northeast dairy farms. The primary goal of the project was to track dynamics of infectious microorganisms on well-characterized dairy farms. Target species included Salmonella spp. (6, 36, 37), Mycobacterium avium subsp. paratuberculosis (13, 24), and L. monocytogenes.The objectives of this study were to describe the presence of L. monocytogenes on a dairy farm over time and to perform molecular subtyping by pulsed-field gel electrophoresis (PFGE) on L. monocytogenes isolates obtained from bulk tank milk, milk filters, milking equipment, feces, and the environmental samples to identify diversity among L. monocytogenes strains, persistence, and potential sources of bulk tank milk contamination.     

Alteration of the omega-3 fatty acid desaturase gene is associated with reduced linolenic acid in the A5 soybean genotype

Reducing the linolenic acid (18?:?3^{ω? 3,6,9}) concentration of soybean [Glycine max (L.) Merr.] oil may lessen the need for chemical hydrogenation and enhance flavor stability. Soybean genotypes A5 and A23 have reduced linolenic acid concentration compared with current cultivars. Seed linolenic acid is synthesized primarily by the ω-3 fatty acid desaturase located in the microsomes. The objective of this research was to study whether this enzyme has a role in reducing the fatty acid levels in the soybean genotypes A5 and A23. DNA from A5 and A23 was analyzed by gel-blot hybridization with a cDNA encoding the ω-3 fatty acid desaturase. A5 and lines selected from it have a DNA fragment missing compared to A23 and lines with normal linolenic acid concentration. Seventy F_4:5 lines from a population segregating for linolenic acid concentration were scored for presence or absence of the fragment. The absence of the fragment was significantly (P?0.0001) associated with a reduced linolenic acid level and accounted for 67% of the variation for linolenic acid in the population. These results suggest that the reduced linolenic acid concentration in A5 was at least partially the result of a full or partial deletion of a microsomal ω-3 desaturase gene. No DNA polymorphisms were found for the desaturase gene in A23, so no mutations could be studied in this line.