Regulation of yeast mRNA 3' end processing by phosphorylation

Recent studies have found that the phosphatase Glc7 associates with the yeast cleavage/polyadenylation factor (CPF), but the role of Glc7 in 3' end processing has not been investigated. Here, we report that depletion of Glc7 causes shortened poly(A) tails in vivo and accumulation of phosphorylated Pta1, a CPF subunit. Removal of Glc7 also gives extract defective for poly(A) addition but normal for cleavage at the poly(A) site. Polyadenylation is rescued by addition of Glc7 or Pta1, but not by phosphorylated Pta1. Moreover, Ypi1, a Glc7-specific inhibitor, or the Cka1 kinase blocks poly(A) addition in wild-type (wt) extract. Pta1 interacts physically and genetically with Glc7, suggesting that Pta1 may also regulate Glc7 or recruit it to CPF. A weakened association of Fip1 with phosphorylated CPF may explain the specific effect on polyadenylation. These results support a model in which poly(A) synthesis is controlled by cycles of phosphorylation and dephosphorylation that require the action of Glc7.     

Declines of zoonotic agents in liquid livestock wastes stored in batches on-farm

AIM: To measure the decline rates of zoonotic agents introduced into liquid livestock wastes in on-farm storage tanks. METHODS AND RESULTS: Salmonella spp., Escherichia coli O157, Campylobacter jejuni, Listeria monocytogenes and Cryptosporidium parvum, propagated in laboratory-controlled conditions, were inoculated into 35,000-l volumes of fresh livestock wastes (pig slurries, cattle slurries and dirty waters). D-values for bacteria were six to 44 days, and for C. parvum were 133 to 345 days. Campylobacter jejuni declined significantly more rapidly than the other bacterial pathogens, while E. coli O157 declined significantly more slowly. On average, bacterial declines were not affected by the season of waste deposition and storage or by the dry matter content of the wastes, but were more rapid in dirty waters than in pig slurries. The physiciochemical composition of wastes in each category varied significantly. CONCLUSIONS: Zoonotic agents can survive for several months during storage of liquid livestock wastes. Livestock wastes should be batch-stored and not subjected to continuous additions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that batches of liquid livestock waste, if contaminated with bacterial pathogens, should be stored for 6 months to reduce contamination levels. Alternative strategies for reducing C. parvum levels in liquid livestock wastes should be explored.     

Decline of zoonotic agents in livestock waste and bedding heaps

AIMS: To measure the rates of decline of zoonotic agents introduced into heaps of spent bedding and faecal wastes generated by commercially farmed livestock and managed in a similar way to that of a working farm. METHODS AND RESULTS: Livestock isolates of Salmonella, pathogenic Listeria, Campylobacter and Escherichia coli O157 were laboratory cultured and used to inoculate 5 m3 heaps of cattle, sheep or pig wastes mixed with bedding materials. Decline of each of the infectious agents was monitored with time as was the temperature inside each heap. Temperatures of >50 degrees C were typically achieved at the core of the heaps. Pathogen decline was rapid, typically <3 days for a 1-log reduction in levels. The longest time that zoonotic agents were isolated from the heaps was 93 days. CONCLUSIONS: Movement of heaps of livestock bedding waste from animal pens to a secondary store, and storing them under conditions conducive for increased temperature is a simple and cost-effective treatment for rapidly lowering levels of zoonotic agents in solid farm wastes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a simple and cheap treatment that can be used to help prevent the spread of zoonotic agents through agricultural environments.     

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.     

Flaxseed oil and inflammation-associated bone abnormalities in interleukin-10 knockout mice

Interleukin-10-/- (IL-10) knockout (KO) mice develop an intestinal inflammation that closely mimics human inflammatory bowel disease (IBD) which is accompanied by inflammation-associated bone abnormalities and elevated serum proinflammatory cytokines. The objective of this study was to use the IL-10 KO mouse model to determine whether flaxseed oil (FO) diet, rich in alpha-linolenic acid (ALA), attenuates intestinal inflammation and inflammation-associated bone abnormalities, compared to a corn oil (CO) control diet. Male wild-type (WT) or IL-10 KO mice were fed a 10% CO or 10% FO diet from weaning (postnatal day 28) for 9 weeks. At necropsy, serum, intestine, femurs and lumbar vertebrae were collected and analyzed. IL-10 KO mice fed CO had lower femur bone mineral content (BMC; P<.001), bone mineral density (BMD; P<.001), peak load (P=.033) and lumbar vertebrae BMD (P=.02) compared to WT mice fed either diet. Flaxseed oil had a modest, favorable effect on IL-10 KO mice as femur BMC, BMD and peak load were similar to WT mice fed CO or FO. In addition, lumbar vertebra BMD was similar among IL-10 KO mice fed FO and WT mice fed CO or FO. The fact that FO attenuated serum tumor necrosis factor-alpha (TNF-alpha) among IL-10 KO mice suggests that the positive effects of FO on femur BMC, BMD, peak load and vertebral BMD in IL-10 KO mice may have been partly mediated by changes in serum TNF-alpha. In conclusion, these findings suggest that a dietary level of ALA attainable from a 10% flaxseed oil diet results in modest improvements in some bone outcomes but does not attenuate intestinal inflammation that is characteristic of IL-10 KO mice.     

Nitrogen availability of anaerobic swine lagoon sludge: sludge source effects

Increased numbers of swine producers will be removing sludge from their anaerobic waste treatment lagoons in the next few years, due to sludge exceeding designed storage capacity. Information on availability of nitrogen (N) in the sludge is needed to improve application recommendations for crops. The objective of this study was to investigate possible effects of different companies and types of swine operations on the availability of N in sludge from their associated lagoons. A laboratory incubation study was conducted to quantify the availability of N (i.e. initial inorganic N plus the potentially mineralizable organic N) in the sludge. Nine sludge sources from lagoons of sow, nursery and finishing operations of three different swine companies were mixed with a loamy sand soil (200 mg total Kjeldahl N kg(-1) soil) and incubated at a water content of 0.19 g. water g(-1) dry soil and 25+/-2 degrees C for 12 weeks. Samples were taken at eight times over the 12-week period and analyzed for inorganic N (i.e. NH(4)-N and NO(3)-N) to determine mineralization of organic N in the sludge. Company and type of swine operation had no significant effects (P < 0.05) on the pattern of inorganic N accumulation over time. Thus, inorganic N accumulation from all sludge sources was fit to a first order equation [Nt = Ni + No (1-e(-kt)]. This relationship indicated that of the 200 mg of total sludge N added per kg soil, 23.5% was in the form of potentially mineralizable organic N (No) and 17.5% was in the form of inorganic N (Ni). The sum of these two pools (41%) represents an estimate of the proportion of total N in the applied sludge in plant available form after the 12 week incubation. While plant N availability coefficients were not measured in this study, the lack of significant company or type of swine operation effects on sludge N mineralization suggests that use of the same plant N availability coefficient for sludge from different types of lagoons is justifiable. The validity of this interpretation depends on the assumption that variation in other components of different sludge sources such as Cu and Zn does not differentially alter N uptake by the receiver crops.     

Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse

The melanocortin system is involved in hypothalamic regulation of energy homeostasis. The melanocortin-4 receptor (MC4R) has been linked to both obesity and reproductive dysfunction. Deletion of the MC4R from the mouse genome has resulted in phenotypes including adult onset obesity, hyperphagia, and difficulty in reproducing when homozygote parents are bred. Additionally, polymorphisms of the human MC4R have been identified in morbidly obese children and adults. Herein, we have identified that voluntary exercise, provided via the presence of a running wheel, impedes the monogenetic obesity (at 20 weeks of age running wheel housed body weight=31+/-1.8 g versus conventionally housed body weight=41+/-2.3 g, a 25% decrease in body weight p<0.01), hyperphagia (average cumulative food intake is not statistically different than wild type mice housed in running wheel cages), and reproductive dysfunction phenotypes associated with the MC4R knockout mice housed by conventional means. These data demonstrate the novel finding that voluntary exercise at a young age may hinder genetically induced obesity.     

FTIR spectroscopic studies of lipid dynamics in phytosphingosine ceramide models of the stratum corneum lipid matrix

IR spectroscopic studies are reported for N-stearyl-d-erythro-phytosphingosine (Cer NP) and N-stearyl-2-hydroxy-d-erythro-phytosphingosine (Cer AP) in a hydrated model of the skin lipid barrier comprised of equimolar mixtures of each ceramide with cholesterol and d₃₅-stearic acid. Examination of the methylene stretching, rocking and bending modes reveal some rotational freedom and hexagonal packing in both the ceramide and stearic acid chains. Analysis of the acid carbonyl stretch and the ceramide Amide I modes show both shift to higher frequencies, indicating weaker hydrogen bonding, in the mixed systems compared to the pure materials. For both systems, the fatty acid chain disordering temperatures are significantly increased from those of the pure acids. The observed behaviors of these phytosphingosine ceramide systems are fundamentally different from the previously reported analogous sphingosine ceramide systems. The implications of these observations for lipid organization in the stratum corneum are briefly discussed.