Stimulation of the Cyclic GMP Pathway by NO Induces Expression of the Immediate Early Genes c-fos and junB in PC12 Cells

Abstract: Stimulation of several second messenger pathways induces the expression of immediate early genes such as c- fos , c- jun , junB , and junD , but little is known about their induction via the stimulation of the cyclic GMP pathway. Here we looked at the expression of early genes in pheochromocytoma PC12 cells after activation of cytosolic guanylate cyclase by sodium nitroprusside. This compound spontaneously releases NO, a molecule known to be involved in cell communication. We found that expression of c- fos and junB but not of c- jun or junD is increased upon activation of cyclic GMP pathway. c- fos mRNA expression was the most activated (fourfold at 30 min), whereas junB response was more modest (2.2-fold activation at 60 min). Nuclear extracts of stimulated cells show increased binding capacity to the AP1 binding site consistent with the dose-response curve. The activating effect of nitroprusside could be reproduced by dipyridamole, a selective cyclic GMP phosphodiesterase inhibitor and by 8- p -chlorophenylthio-cyclic GMP, a permeant selective cyclic GMP-dependent protein kinase activator, and abolished by KT5823, an inhibitor of that kinase. The results show that NO promotes early gene activation and AP1 binding enhancement through the stimulation of the cyclic GMP pathway.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Responses of the Fructose-1,6-bisphosphatase and Glutamate Dehydrogenase Activities of Alfalfa to Boron,Gypsum, and Limestone Amendments of Soil

Plants cultivated on acid soils that contain toxic levels of Al³⁺ usually produce low yields. A multi-factorial treatment of gypsum (G), boron (B), and limestone (Lm) was applied to such soil in order to determine the biochemical basis of the best management plan for ameliorating the soil acidity for sustainable growth of alfalfa. The alfalfa shoots were subjected to analysis for hexose, protein, nucleotide, and chlorophyll (Chl) contents, fructose 1,6-bisphosphatase (FBPase) activity, and the RNA synthetic activity of glutamate dehydrogenase (GDH). Hexose and protein contents of control alfalfa without B and G, but with Lm (672 g m^–2) amendment were 0.87 and 38.30 g, respectively, per kg shoot. Increasing the G doses at fixed moderate doses of 0.15 and 0.30 g m^–2 B decreased the FBPase activity by 53 and 31 %, respectively. However, increasing the B doses at higher fixed G (1 kg m^–2= G_1.0) increased the FBPase activity by 91 % thus indicating that G₁ optimized the saccharide metabolism by neutralizing the soil acidity. In the absence of B, increasing the G doses also maximized the hexose and Chl contents, but minimized the nucleotide amount. In the absence of G, increasing the B doses maximized the RNA synthetic activity of GDH, but lowered the hexose and Chl contents as well as the FBPase activity without affecting the protein contents, thereby permitting the selection of B (0.45 g m^–2) with Lm as the best amendment for the sustainable growth of alfalfa. Treatment with 0.45 g B and 0.5 kg G (= G_0.5) induced the strongest B-Ca antagonism by maximizing the hexose and Chl contents but severely suppressing the FBPase activity and the RNA synthetic activity of GDH. Therefore, the coordinate optimization of saccharide metabolism through the G-dependent neutralization of soil acidity, and of RNA metabolism through the B-dependent detoxification of Al³⁺ are the biochemical options for the mitigation of the adverse effects of soil acidity for the optimization of sustainable alfalfa production.     

Synergy between LRH-1 and beta-catenin induces G1 cyclin-mediated cell proliferation

LRH-1 is an orphan nuclear receptor predominantly expressed in tissues of endodermal origin, where it controls development and cholesterol homeostasis. We show here that LRH-1 induces cell proliferation through the concomitant induction of cyclin D1 and E1, an effect that is potentiated by its interaction with beta-catenin. Whereas beta-catenin coactivates LRH-1 on the cyclin E1 promoter, LRH-1 acts as a potent tissue-restricted coactivator of beta-catenin on the cyclin D1 promoter. The implication of LRH-1 in cell proliferation highlights an unanticipated crosstalk between LRH-1 and the beta-catenin/Tcf4 signaling pathway, which is relevant for the renewal of intestinal crypt cells.     

Isolation and characterization of new microsatellite markers in shea tree (Vitellaria paradoxa C. F. Gaertn)

Vitellaria paradoxa is one of the major components of African parkland agroforestry systems. In order to assess the genetic diversity and population structure of this species, we isolated and characterized 14 polymorphic nuclear microsatellite loci. Primers developed to amplify these loci were used to analyse 200 individuals of a shea tree population in Mali. Loci have shown a high number of alleles ranging from four to 26, and display an observed level of heterozygosity between 0.37 and 0.85. These new very polymorphic microsatellite markers will be useful for genetic and ecological studies of V. paradoxa.     

Passive recovery of small vertebrates following livestock removal in the Australian rangelands

In arid environments, grazing by exotic herbivores, including domestic livestock, can greatly influence native, small vertebrate assemblages. Whether the removal of livestock facilitates passive recovery of these assemblages depends on habitat condition and the species present. We explore changes in small mammal and reptile species richness, abundance, and composition in a degraded chenopod shrubland dominated by Acacia victoriae ssp. and open Acacia aneura (mulga) woodland destocked in 1976 and 1984, respectively. Data were obtained between 1997 and 2007, from two grazed and two ungrazed sites in each community. Species richness increased at a faster rate in ungrazed open A. aneura woodland, but did not differ significantly between ungrazed and grazed degraded chenopod shrubland. Subsequent analyses at a finer‐scale detected disparate responses in richness and abundance for microhabitat. At this scale, a greater number of species‐specific responses were also detected, including increased abundance of generalist species and decreased abundance of species requiring low cover. These results reiterate the potential for species‐specific responses to livestock that are more apparent in particular microhabitats. Furthermore, this investigation provides evidence for the gradual passive recovery of small mammal and reptile assemblages in both communities, which is facilitated by the removal of livestock in open A. aneura woodland in fair condition, but not degraded chenopod shrubland in poor condition.     

Assessment of Shiga Toxin-Producing Escherichia coli Isolates from Wildlife Meat as Potential Pathogens for Humans

A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx₂, stx_2d, and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.Shiga toxin-producing Escherichia coli (STEC) strains represent an important emerging group of food-borne zoonotic pathogens causing diarrhea, hemorrhagic colitis (HC), and the life-threatening hemolytic uremic syndrome (HUS) in humans (30). Production of potent cytotoxins, which are called Shiga toxins (Stx) or Vero toxins (VT) and are encoded on the genomes of temperate lambdoid bacteriophages, is the major virulence determinant of STEC strains. Additional virulence factors, such as genes encoding the attaching and effacing function and virulence plasmid-encoding genes, contribute to the pathogenicity of STEC strains. These virulence genes are closely associated with a subgroup of STEC strains that are frequently isolated from patients with hemorrhagic diseases (HC and HUS) and were therefore designated enterohemorrhagic E. coli (EHEC) strains. Strains belonging to serogroups O157, O26, O103, O111, and O145 are the EHEC types most frequently isolated from humans with HC and HUS (33).STEC strains are part of the gut flora of different animal species, and ruminants, particularly cattle, have been identified as a major reservoir of STEC strains that are highly virulent to humans (27). Today, it is evident that STEC strains can be transmitted from their animal reservoirs to humans via ingestion of contaminated food and water or by contact with STEC-excreting animals or the environment (9).Recent reports indicate that wildlife animals play an important role as carriers and transmitters of STEC strains in nature. EHEC O157 strains (13, 32, 36, 40, 46) and other STEC strains were isolated from feces of different ruminant deer species at different geographic locations (2, 20, 28, 34, 36, 42). Deer have been suggested to play a role as transmitters of EHEC O157 strains to cattle by fecal contamination of farmland (43). Wild migrating birds have been identified as STEC excretors and participate in the spread of EHEC O157 and other STEC strains over long distances (17, 37, 47). To date, only a few reports have been published on the contamination of raw game meat and other game products with STEC strains. A study conducted in Belgium indicated that about half of meat samples from wildlife ruminants contained STEC strains (38). Deer meat and jerky were identified as sources of EHEC O157 infections in humans in the United States (31, 39). In Germany, different types of STEC strains were isolated from venison samples (34), and surveys performed in the Federal Institute for Risk Assessment revealed a contamination rate of wild meat samples with STEC strains of 9.0% to 14.8% between 2005 and 2006. In this time period, the proportion of STEC-contaminated samples from game was considerably higher than that found with beef samples (1.3% to 4.5% STEC positives) (23, 24).Current data suggest that wild-living animals and their meat products are underestimated as natural reservoirs for STEC strains and as possible sources for human infections. Game meat is popular in Germany, since it is considered to be a high-quality product, and per capita consumption is rising steadily (report from the Federal Institute for Risk Assessment [http://www.bfr.bund.de/cd/7134]). To meet the demand for game meat, a total of 36,126 tons of wild animals were hunted from 2005 to 2006. These were divided into 19,000 tons of wild boar (n = 461,881 animals), 11,300 tons of roe deer (n = 905,387), and about 4,000 tons of red deer (n = 60,664) (Deutscher Jagdschutz-Verband [http://www.jagd-online.de]). Taking these data as a basis for estimation, the average amount of annual wild meat consumption is about 0.45 kg/person and accounts for 0.8% of the total meat consumption in Germany (22).About 62% of retailed game meat originates from animals hunted in the wild in Germany. Only 3% of the meat is from animals that are grown in captivity, with fallow deer the most frequently grown captive game animal. Imported game accounts for 35% of retailed meat (26). In compliance with the legal regulations, hunters are educated in meat inspection, and hygiene rules request evisceration of hunted game immediately after killing (C. Commichau [http://www.tiho-hannover.de/einricht/lmmikro/wild1.doc]). Inspected and acceptable carcasses are allowed to proceed to immediate sale to individuals, restaurants, and food handlers. For safety reasons, processing of game meat must occur separately from processing of other meat; when processing of game meat is conducted on a larger scale, it is performed in special meat-processing plants. Only a small portion of hunted game meat is inspected by official meat inspection authorities (26).At present, little is known about the characteristics of STEC strains other than O157 strains from wildlife meat. In order to provide data for estimating the impact of game as a potential source of human pathogenic STEC types, we characterized 140 STEC strains found in meat isolates from deer, wild boar, and hare. The strains were examined for their serotypes, for properties related to virulence of E. coli for humans, and for their genetic relationship to STEC isolates from farm animals, from their food products, and from human patients. The aim was to determine the similarities between STEC strains from wildlife meat and those from other sources, including humans. Our data indicate that game is a natural reservoir for and a potential source of human pathogenic EHEC and STEC types.     

Isolation and preliminary characterization of amino acid substitution mutations that increase the activity of the osmoregulated ProP protein of Salmonella enterica serovar Typhimurium

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.     

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources

AIMS: To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. METHODS AND RESULTS: The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. CONCLUSIONS: The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.     

The isomerization of glutamate dehydrogenase in response to lead toxicity in maize

Maize (Zea mays) was cultivated on lead-adultrated soil up to 600 mg(Pb) kg-1. At maturity, the maize seeds were harvested. The glutamate dehydrogenase (GDH) was fractionated to its isoenzyme population by Rotofor isoelectric focusing (IEF). The increasing Pb concentration progressively enhanced the more acidic isoenzymes (pI 6.3 - 6.5), and at the same time suppressed the less acidic isoenzymes (pI 7.3 - 7.8) and at the 600 mg(Pb) kg-1(soil) only the most acidic couple of isoenzymes (pI 6.3, and 6.5) were detectable. The NH4+ Km values of the GDH increased progressively from 6.2 in the control to 100 mM and the total glutathione content of maize seeds from 60 to 240 nmol g-1 in the 600 mg(Pb) kg-1(soil) treated maize. The orderly, and sequential isomerization of GDH in response to Pb suggests that the enzyme functions as a sensor in the monitoring of environmentally induced stress. This revised version was published online in August 2006 with corrections to the Cover Date.