Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.     

Natural killer T cells: rapid responders controlling immunity and disease

Natural killer T (NKT) cells are a subset of T cells that share properties of natural killer cells and conventional T cells. They are involved in immediate immune responses, tumor rejection, immune surveillance and control of autoimmune diseases. Most NKT cells express both an invariant T cell antigen receptor and the NK cell receptor NK1.1, and are referred to as invariant NKT cells. This invariant T cell receptor is restricted to interactions with glycolipids presented by the non-classical MHC, CD1d. These NKT cells rapidly produce high levels of interleukin (IL)-2, IFN-gamma, TNF-alpha, and IL-4 upon stimulation through their TCR. Most also have cytotoxic activity similar to NK cells. NKT cells are involved in a number of pathological conditions, and have been shown to regulate viral infections in vivo, and control tumor growth. They may also play both protective and harmful roles in the progression of certain autoimmune diseases, such as diabetes, lupus, atherosclerosis, and allergen-induced asthma.     

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.     

Persistence of Escherichia coli O157 on farm surfaces under different environmental conditions

AIMS: To compare the persistence of Escherichia coli O157 on a variety of common faecally contaminated farmyard material surfaces (wood and steel) under different moisture and temperature regimes. METHODS AND RESULTS: Samples of field-conditioned farmyard materials (galvanized steel and wood) were cut into pieces and contaminated with fresh cattle faeces inoculated with nontoxigenic E. coli O157 (strain 3704). Thereafter, they were stored at four different environmental conditions; with temperature (5 and 20 degrees C) and moisture (moist or dry) as variables. Transfer of the pathogen to hands from the surfaces was also evaluated. Escherichia coli O157 numbers declined over time on all surfaces albeit at different rates according to the sample material and environmental conditions. Persistence was greatest on moist wood samples under cooler temperatures with large population numbers remaining after 28 days. Desiccation of surfaces resulted in a more rapid decline in E. coli O157 populations under both temperature regimes. Substantial numbers of colonies may also potentially be transferred to human hands from the surfaces during brief contact. CONCLUSIONS: When environmental conditions are favourable, E. coli O157 may persist for considerable times on a range of surfaces. However, when exposed to higher temperatures and dehydration, survival is notably decreased. Overall, bacterial persistence was significantly greater on wood samples relative to steel. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157 is a prevalent pathogen, common in ruminant faeces. Contact with contaminated faeces may lead to human infection, resulting in possible severe illness. Although our study used only one strain of bacteria, our findings indicates that E. coli O157 has the potential to persist for long periods of time on gates, stiles and other farmyard surfaces under a range of environmental conditions. These farmyard surfaces therefore pose a potential infection pathway particularly where there is a high risk of direct human contact (e.g. child petting zoos, open farms).     

The overexpression of presenilin2 and Alzheimer's-disease-linked presenilin2 variants influences TRPC6-enhanced Ca2+ entry into HEK293 cells

Mutations in the presenilin (PS) genes are linked to the development of early-onset Alzheimer's disease by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP). Recent work indicates that Alzheimer's-disease-linked mutations in presenilin1 and presenilin2 attenuate calcium entry and augment calcium release from the endoplasmic reticulum (ER) in different cell types. However, the regulatory mechanisms underlying the altered profile of Ca(2+) signaling are unknown. The present study investigated the influence of two familial Alzheimer's-disease-linked presenilin2 variants (N141I and M239V) and a loss-of-function presenilin2 mutant (D263A) on the activity of the transient receptor potential canonical (TRPC)6 Ca(2+) entry channel. We show that transient coexpression of Alzheimer's-disease-linked presenilin2 mutants and TRPC6 in human embryonic kidney (HEK) 293T cells abolished agonist-induced TRPC6 activation without affecting agonist-induced endogenous Ca(2+) entry. The inhibitory effect of presenilin2 and the Alzheimer's-disease-linked presenilin2 variants was not due to an increase in amyloid beta-peptides in the medium. Despite the strong negative effect of the presenilin2 and Alzheimer's-disease-linked presenilin2 variants on agonist-induced TRPC6 activation, conformational coupling between inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) and TRPC6 was unaffected. In cells coexpressing presenilin2 or the FAD-linked presenilin2 variants, Ca(2+) entry through TRPC6 could still be induced by direct activation of TRPC6 with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, transient coexpression of a loss-of-function PS2 mutant and TRPC6 in HEK293T cells enhanced angiotensin II (AngII)- and OAG-induced Ca(2+) entry. These results clearly indicate that presenilin2 influences TRPC6-mediated Ca(2+) entry into HEK293 cells.     

Identification of a new cross-link and unique histidine adduct from bovine serum albumin incubated with malondialdehyde

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.     

The role of chromatic and achromatic signals for fruit detection by birds

Fruit color changes during ripening are typically viewed asan adaptation to increase signal efficacy to seed dispersers.Plants can increase signal efficacy by enhancing chromatic (wavelengthrelated) and/or achromatic (intensity related) contrasts betweenfruit and background. To assess how these contrasts determinethe detectability of fruit signals, we conducted 2 experimentswith free-flying crows (Corvus ossifragus) under seminaturalconditions in a 2025 m² aviary. Crows searched first for artificialred and black fruits and detected red fruits from a larger distance.Because artificial red fruits had higher chromatic and lowerachromatic contrasts against foliage than artificial black fruits,crows apparently prioritized chromatic contrasts. Thus, thecommon change in fruit color from red to black during ripeningdoes not increase signal efficacy to crows. In a second trial,crows searched for UV-reflecting and black blueberries (Vaccinummyrtillus) against backgrounds of foliage and sand. Againstfoliage, UV-reflecting berries had higher chromatic and achromaticcontrasts than black berries, and crows detected them from alarger distance. Against sand, UV-reflecting berries had lowachromatic contrasts and black berries low chromatic contrasts.Crows detected both fruit types equally, suggesting that theyused chromatic contrasts to detect UV-reflecting berries andachromatic contrasts to detect black berries. Birds prioritizedchromatic contrasts when searching for artificial red fruitsin foliage but not when searching for blueberries on sand. Wesuggest that the relative importance of chromatic and achromaticcontrasts is contingent on the chromatic and achromatic varianceof the background. Models of signal perception can be improvedby incorporating background-specific effects.     

Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk

Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth.Since Friend disease was first reported in 1957 (19), the acute erythroleukemia induced by the various strains of Friend virus have provided an excellent model to study multistage carcinogenesis (5). In the first stage, the virus infects erythroid progenitor cells and a viral glycoprotein, gp55, interacts with both the erythropoietin receptor (EpoR) and a naturally occurring truncated form of the stem cell-derived tyrosine kinase (Stk), Sf-Stk, resulting in the Epo-independent (Epo^ind) expansion of erythroid progenitor cells. The late stage of erythroleukemia in Friend disease is marked by inactivation of the p53 locus (6, 28, 38, 39, 51) and proviral integration into the Spi-1 locus (36, 43, 44), resulting in enhanced expression of Pu.1, which causes a block in erythroid differentiation and promoting the onset of acute erythroleukemia.Friend virus is a complex of two viruses, the spleen focus-forming virus (SFFV), which is a replication-defective C-type retrovirus, and the ecotropic Friend murine leukemia virus (F-MuLV). SFFV is responsible for the rapid splenomegaly and acute erythroleukemia induced by Friend virus infection (7, 64, 65, 67), while F-MuLV provides helper function and can be substituted for by other murine leukemia viruses (35). Specifically, the glycoprotein gp55, encoded by the SFFV env gene, acts as the transforming viral oncoprotein (2, 65).Several loci in the mouse genome that control Friend virus susceptibility have been identified. Fv1, Fv3, and Fv4 affect the ability of Friend virus to infect early erythroid progenitor cells. The Fv1 gene product inhibits Friend virus infection by interacting with the viral capsid protein (60). The Fv3 gene encodes cytidine deaminase Apobec3, which broadly inhibits retrovirus infection (42, 53, 57). The Fv4 gene product affects viral binding by competing for receptors on the cell membrane (59). Another set of genes, W, Sl, f, and Fv2, are required for the development or expansion of infected progenitor cells. Our previous work demonstrated that W, Sl, and f, which encode the kit receptor, its ligand SCF, and Smad5, respectively, also play key roles in the BMP4-dependent stress erythropoiesis pathway(46, 47, 55). Analysis of those mutants showed that Friend virus activates this pathway, leading to acute amplification of stress progenitors, which are targets of Friend virus in the spleen, and resulting in rapid onset of disease.The Friend virus susceptibility gene Fv2 encodes the stem cell-derived tyrosine kinase (Stk) receptor (48). A naturally occurring N-terminally truncated form of Stk, short-form Stk (Sf-Stk), is required for Friend virus susceptibility. Fv2^r/r mice, including C57BL/6, lack expression of Sf-Stk and are resistant to Friend virus infection, while full-length Stk expression is unaffected in these mice. An internal promoter within the Stk locus drives Sf-Stk expression, and Fv2^r/r mice harbor mutations in the internal promoter. Sf-Stk lacks the N-terminal ligand binding domain of full-length Stk but retains the transmembrane and tyrosine kinase domains. In vitro and in vivo expression of Sf-Stk in C57BL/6 bone marrow cells has been shown to confer Friend virus susceptibility to Fv2^r/r mice (18).Sf-Stk covalently interacts with gp55, resulting in constitutive activation of Sf-Stk (41). However, the mechanism by which this occurs is currently unknown. Here, we identify cysteines in the extracellular domains of Sf-Stk and gp55 that mediate this interaction. Furthermore, we demonstrate that while the association with gp55 is not required for the dimerization of Sf-Stk, the interaction of gp55 with Sf-Stk promotes tyrosine phosphorylation of Sf-Stk. In addition, while the extracellular cysteines in Sf-Stk promote retention of Sf-Stk in the cytoplasm in the absence of gp55, the interaction of Sf-Stk with gp55 through these cysteines results in enhanced cell surface localization of Sf-Stk. These changes in receptor activation and subcellular localization mediate the ability of Sf-Stk to induce gene expression and promote the Epo^ind growth of primary erythroblasts.