Purification and Characterization of 4-Hydroxyphenylpyruvate Dioxygenase from Maize

The proposed target enzyme for benzoylcyclohexanedione herbicides, 4-hydroxyphenylpyruvate dioxygenase (HPPD) was purified from etiolated maize seedlings with a purification factor of 105. Enzyme activity was measured by detection of carbon dioxide formed from radiolabelled substrate. The enzyme has a pH optimum of 7·3 and an apparent molecular mass of 43 kDa, similar to that of the mammalian liver enzyme. Activity needs the presence of a reducing system glutathione/dichlorophenol indophenol or ascorbate and catalase. Surprisingly, a commercial catalase preparation of low specific activity—generally used for the enzyme assay—showed HPPD activity which was separable from the catalase activity on a gel filtration column. According to kinetic studies with purified maize HPPD, experimental herbicides from the family mentioned were strong competitive inhibitors of the plant enzyme in nanomolar range withK_i values of 5 and 15 nM for 2-(2-nitro-4-chlorobenzoyl)-5-(2-methoxyethyl) cyclohexane- 1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)- cyclohexane-1,3-dione (SC-0051; sulcotrione), respectively.     

Chemical catalysis of the isomerisation of peroxidising herbicidal thiadiazolidines

Model reactions with various -SH, -OH and -NH nucleophiles in an aprotic solvent were used to characterise the nature of the enzymatic conversion of thiadiazolidines to triazolidines. This conversion results in greater inhibition of protoporphrinogen oxidase (protox). It is inferred that GST-isoforms probably promote formation of one of the intermediates.     

Models to capture the potential for disease transmission in domestic sheep flocks

Successful control of livestock diseases requires an understanding of how they spread amongst animals and between premises. Mathematical models can offer important insight into the dynamics of disease, especially when built upon experimental and/or field data. Here the dynamics of a range of epidemiological models are explored in order to determine which models perform best in capturing real-world heterogeneities at sufficient resolution. Individual based network models are considered together with one- and two-class compartmental models, for which the final epidemic size is calculated as a function of the probability of disease transmission occurring during a given physical contact between two individuals. For numerical results the special cases of a viral disease with a fast recovery rate (foot-and-mouth disease) and a bacterial disease with a slow recovery rate (brucellosis) amongst sheep are considered. Quantitative results from observational studies of physical contact amongst domestic sheep are applied and results from the differently structured flocks (ewes with newborn lambs, ewes with nearly weaned lambs and ewes only) compared. These indicate that the breeding cycle leads to significant changes in the expected basic reproduction ratio of diseases. The observed heterogeneity of contacts amongst animals is best captured by full network simulations, although simple compartmental models describe the key features of an outbreak but, as expected, often overestimate the speed of an outbreak. Here the weights of contacts are heterogeneous, with many low weight links. However, due to the well-connected nature of the networks, this has little effect and differences between models remain small. These results indicate that simple compartmental models can be a useful tool for modelling real-world flocks; their applicability will be greater still for more homogeneously mixed livestock, which could be promoted by higher intensity farming practices.     

Is the Function of the Porcine Sperm Reservoir Restricted to the Ovulatory Period?

The uterotubal junction (UTJ) and caudal isthmus are recognized as a functional pre-ovulatory sperm reservoir (SR). Spermatozoa are released from the SR in a complex and concerted action. However, whether this functionality is restricted only to the ovulatory period is still open to debate. Our study was aimed to analyze the presence of spermatozoa within the UTJ (SR), isthmus (ISTH) and ampulla (AMP) after laparoscopic intrauterine insemination (LIUI) either in the peri- (PERI) or post-ovulatory (POST) period or at mid cycle (MID). Each uterine horn of estrus synchronized gilts (n=12) was inseminated with 20 ml sperm (29.5×10⁶ cells/ml). Oviducts were recovered 7 h after LIUI and separated into the UTJ, ISTH and AMP, and sections were flushed with 10 ml PBS+EDTA solution. After centrifugation, the sperm pellet was evaluated by Čeřovský staining. The median sperm numbers in the PERI, POST and MID groups were 578, 171 and 789 in the UTJ; 545, 233 and 713 in the ISTH; and 496, 280 and 926 in the AMP, respectively, and there were differences between the POST and MID groups (P<0.05) but not between the oviductal sections of each group (P>0.05). Compared with the MID group, the percent of intact sperm cells was higher (P<0.01) in the PERI and POST groups (32.8 vs. 66.4 and 76.8%). Also, the percentages of aberrations in the acrosome and tail were higher (P<0.05) in the MID group. Based on this, it can be assumed that the sperm reservoir is active during different phases of the estrus cycle. However, the mid-cycle oviduct environment considerably impairs sperm cell quality.     

Alkylating reactivity and herbicidal activity of chloroacetamides

The relationship between S- and N-alkylating reactivity and herbicidal activity within a series of chloroacetamides, including several commercial herbicides and newly synthesised analogues was studied. The S-alkylating reactivity of selected chloroacetamides, as well as those of atrazine and chlorfenprop-methyl, was determined by in vitro GSH conjugation at a ratio of GSH to alkylating agent of 25:1. A spectrophotometric reaction using 4-(4-nitrobenzyl)pyridine was used to characterise the N-alkylating reactivity of the chemicals. Our results indicate that a reduced level of N-alkylating reactivity correlates with an improved herbicidal efficacy at a practical rate. However, the phytoxicity of the molecules is not simply dependent on chemical reactivities, but strictly related to the molecular structure, indicating that lipophilicity, uptake, mobility and induction of detoxifying enzymes may also be decisive factors in the mode of action.     

Specific flavonoids as interconnecting signals in the tripartite symbiosis formed by arbuscular mycorrhizal fungi, Bradyrhizobium japonicum (Kirchner) Jordan and soybean (Glycine max (L.) Merr.)

Many legume plants benefit from the tripartite symbiosis of arbuscular mycorrhizal fungi (AMF) and rhizobia. Beneficial effects for the plant have been assumed to rely on increased P supply through the mycorrhizas. Recently, we demonstrated that P does not regulate the establishment of the tripartite symbiosis. Flavonoids appear to play a role as early signals for both rhizobia and AMF. Four soybean lines known to express different concentrations of the isoflavones genistein, daidzein and glycitein in the seed were used to test three hypotheses: (i) The establishment of the tripartite symbiosis is not dependent of a nutrient mediated effect; (ii) There is a positive correlation between seed isoflavone concentrations of different soybean lines and the progress of the tripartite symbiosis; (iii) Specific flavonoids control the establishment of the tripartite symbiosis in that a change in flavonoid root accumulation resulting from the development of one microbial partner can stimulate colonization of soybean roots by the other. Disturbed versus undisturbed soil treatments were produced to vary the potential for indigenous AMF colonization of soybean. In contrast, the potential for Bradyrhizobium was kept identical in both soil disturbance treatments. The uptake of P and Zn and the concentration of flavonoids in mycorrhizal soybean roots at 10 d after emergence were analysed either separately of Bradyrhizobium or in context of the tripartite symbiosis. Zinc nutrition did not differ between AMF treatments which supports the first hypothesis. The concentration of daidzein was at least four times greater in the root than in the seed reaching 3958±249 μg g⁻¹ dry across soybean lines. Coumestrol, which was absent in the seed, was synthesized to reach 2154±64 μg g⁻¹ dry. Conversely, the concentration of genistein was approximately three times smaller in the root that in the seed (301±15 μg g⁻¹ dry), while glycitein and formononetin were never detected. The establishment of the tripartite symbiosis was identical across soybean lines which does not support the second hypothesis. Concentrations of flavonoids were significantly greater in roots under disturbed soil, for which both symbioses were not as developed as in plants from undisturbed soil. This clearly supports the third hypothesis. This research provides the first data linking the function of different flavonoids to the establishment of the tripartite symbiosis, and suggests that these compounds are produced and released into the rhizosphere as a function of the colonization process.     

Coenzyme Q9 provides cardioprotection after converting into coenzyme Q10

Coenzyme Q10 (CoQ10) has been extensively studied as adjunctive therapy for ischemic heart disease, and its cardioprotective ability is well-established. The mitochondrial respiratory chain contains several coenzymes, including CoQ1, CoQ2, CoQ4, CoQ6, CoQ7, CoQ8, CoQ9, and CoQ10. It is not known whether other CoQs, especially CoQ9, is equally cardioprotective as CoQ10. The present study was designed to determine if CoQ 9 could protect guinea pig hearts from ischemia reperfusion injury. Guinea pigs were randomly divided into three groups: groups I and II were fed CoQ 9 and CoQ10, respectively, for 30 days while group III served as control. After 30 days, the guinea pigs were sacrificed and isolated hearts were perfused via working mode were subjected to 30 min ischemia followed by 2 h of reperfusion. Cardioprotection was assessed by evaluating left ventricular function, ventricular arrhythmias, myocardial infarct size, and cardiomyocyte apoptosis. Samples of hearts were examined for the presence of CoQ9 and CoQ10. The results demonstrated that both CoQ9 and CoQ10 were equally cardioprotective, as evidenced by their abilities to improve left ventricular performance and to reduce myocardial infarct size and cardiomyocyte apoptosis. High performance liquid chromatographic (HPLC) analysis revealed that a substantial portion of CoQ9 had been converted into CoQ10. The results indicate that CoQ9 by itself, or after being converted into CoQ10, reduced myocardial ischemia/reperfusion-induced injury.     

Highly ciprofloxacin resistant Salmonella enterica serovar Kentucky isolates in turkey meat and a human patient

In recent years in France, England, Wales, Denmark and the USA about 500 human infections occurred, which were caused by multidrug-resistant Salmonella enterica Serovar (S.) Kentucky isolates displaying high-level resistance to fluoroquinolones (ciprofloxacin, MIC > or = 4 mg/l). The responsible clone was referred to as ST198-X1.To determine whether this clone is also present in German S. Kentucky isolates, the National Reference Laboratory for Salmonella (NRL-Salm) at the BfR analyzed the trend of S. Kentucky isolates received over the past years. Since 2010 the first entries of highly ciprofloxacin resistant S. Kentucky isolates, especially from turkey meat products, were recorded. 15 isolates originating from animal or food as well as one human isolate displayed MIC values of > or = 8 mg/l to ciprofloxacin. Molecular biological typing methods showed the in Germany isolated S. Kentucky isolates to be identical to the clone described by Le Hello et al. (2011) and to carry a multidrug resistance conferring region (SGI1). Since fluoroquinolones are considered by the WHO in human and veterinary medicine as drugs of critical importance, this trend demands attention. The implementation of mitigation strategies for this highly resistant clone seems to be required.     

Comparing validation of four ELISA-systems for detection of Salmonella derby- and Salmonella infantis-infected pigs

The objective of this study was the comparative evaluation of four indirect Salmonella ELISA tests at study time approved in Germany to detect Salmonella infection in pigs.Three tests are based on a LPS-antigen mix and directed against specific IgG antibodies. The fourth test is based on a purified S. Typhimurium whole-cell lysate antigen and discriminates between Salmonella-specific IgM-, IgA-, and IgG- antibodies. In a longitudinal study, two groups of six weeks old hybrid piglets were orally infected with a porcine S. Infantis or S. Derby strain. Clinical and bacteriological parameters were monitored weekly during an observation period of 130 days after infection and serum samples were investigated in parallel with the respective ELISAs. Apparently, the LPS-based ELISA systems used in this study failed to recognize S. Infantis-infected pigs although those animals shed the pathogen in high amounts throughout the study until day 81 post infection (p. i.). In contrast, the isotype-specific Salmonella Typhimurium whole-cell-lysate based ELISA was capable of detecting Salmonella-infected pigs from day ten p. i. at all tested serotypes and revealed the highest sensitivity in detection of S. Infantis-infected pigs. Furthermore, it became apparent that the often used surveillance cut-off value of 40 OD% is not appropriate for intra-vitam detection of S. Infantis- and S. Derby-infected pigs. In contrast, the cut-off values of the ELISAs given by the suppliers result in considerable higher detection rates.