<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T₁ generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid - bar: Bialaphos resistance gene - GUS: -Glucuronidase - PPT: Phosphinothricin - ubi: Ubiquitin Communicated by J.M. Widholm     

Mechanistic studies on AMD6221: a ruthenium-based nitric oxide scavenger

Nitric oxide is a mediator of many disease states. Previous studies have demonstrated that ruthenium(III) polyaminocarboxylates can react with NO to form stable complexes reducing the levels of nitrite in the culture medium of stimulated RAW264 macrophages and reverse the NO-mediated hypotension in animal models of septic shock. It was necessary to confirm that these observations were due to NO scavenging and not inhibition of the NO metabolic pathway. Using RAW264 cells it was confirmed that [Ru(H(3)dtpa)(Cl)] (AMD6221) was neither acting at the level of iNOS induction, nor as an inhibitor of iNOS by measuring iNOS mRNA by RT-PCR and protein by Western blot and enzyme activity. Using HPLC, the nitrosyl adduct of reaction of AMD6221, [Ru(H(2)dtpa)NO], was identified in the medium of stimulated RAW264 cells co-incubated with AMD6221, concomitant with a stoichiometric reduction in nitrite/nitrate levels, thus confirming that the ruthenium(III) polyaminocarboxylates exert their pharmacological effect by scavenging NO.     

Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in Escherichia coli O157:H7

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.     

Novel Carboxypeptidase A6 (CPA6) Mutations Identified in Patients with Juvenile Myoclonic and Generalized Epilepsy

Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy.     

Fungal network responses to grazing

Mycelial networks operate on scales from microscopic to many m² and naturally persist for extended periods. As fungi exhibit highly adaptive development, it is important to test behavioural responses on natural substrata with realistic nutrient levels across a range of spatial scales and extended time periods. Here we quantified network responses over 7.5 months in large (57 × 57 cm) microcosms to test whether grazing shifts the network to a more resilient architecture. Resource limitation constrained any ability to respond at all, with both grazed and ungrazed networks gradually thinning out over time. Added resources sustained further exploratory growth, but only transiently increased cross-connectivity and network resilience, when tested by simulated damage in silico. Grazed networks were initially weaker and emergence of new exploratory growth was curtailed. However, increased interstitial proliferation led to new cross-links, consolidating the existing mycelial network and increasing the resilience of the network to further attack.     

Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.