Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway

The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 ?) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 ?. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.     

Count Your Eggs Before They Invade: Identifying and Quantifying Egg Clutches of Two Invasive Apple Snail Species (Pomacea)

Winning the war against invasive species requires early detection of invasions. Compared to terrestrial invaders, aquatic species often thrive undetected under water and do not garner notice until too late for early action. However, fortunately for managers, apple snails (Family Ampullariidae, Genus Pomacea) provide their own conspicuous sign of invasion in the form of vibrantly colored egg clutches. Managers can potentially use egg clutches laid in the riparian zone as a means of early detection and species identification. To facilitate such efforts, we quantified differences in characteristics (length, width, depth, mass, egg number) of field-laid clutches for the two most common invasive species of apple snail, P. canaliculata and P. maculata, in native and non-native populations. Pomacea canaliculata native and non-native populations differed noticeably only in width. Native P. maculata clutches possessed significantly greater width, mass and eggs numbers compared with native P. canaliculata. Non-native P. maculata clutches significantly exceeded all other populations in all measured characteristics. Consequently, these traits may successfully distinguish between species. Fecundity data also allowed us to develop models that accurately estimated the number of eggs per clutch for each species based on clutch dimensions. We tested one, two and three dimensional models of clutches, including rendering a clutch as either a complete ellipsoid or an ellipsoid intersected by a cylinder to represent the oviposition site. Model comparisons found the product of length and depth, with a different function for each population, best predicted egg number for both species. Comparisons of egg number to clutch volume and mass implied non-native P. canaliculata may be food limited, while non-native P. maculata appeared to produce such enormous clutches by having access to greater nutrients than the native population. With these new tools, researchers and managers can quickly identify, quantify and begin eradication of new non-native apple snail populations.     

Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and β generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC β inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC β as another PKC β inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC β, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.     

Effect of bait formulation and number of traps on detection of navel orangeworm (Lepidoptera: Pyralidae) oviposition using egg traps

Egg traps are the primary tool for monitoring egg deposition of the navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), and for timing treatments for this pest in almonds, Prunus amygdalus Batsch, and pistachios, Pistacia vera L. We compared, in almond and pistachio orchards, the number of eggs per trap in traps baited with almond meal, pistachio meal, or the current standard commercial bait. When considering cumulative eggs captured over an extended period, traps baited with pistachio meal prepared from previous-crop nuts generally captured a similar number of eggs compared with the commercial bait, and more eggs than those baited with almond meal prepared from previous-crop nuts. However, differences in eggs per trap between bait formulations were not as evident when examining individual weeks, particularly in weeks with few eggs per trap, as is typical when treatment decisions are made. The variance in eggs per trap was generally greater than the mean and increased with the mean and, when mean eggs per trap was low, most traps did not have eggs. We discuss implications of these findings for the relative importance of bait type and trap numbers for monitoring, and for experiments comparing egg trap performance.     

Kentsin: tetrapeptide from hamster embryos produces naloxone-sensitive effects without binding to opioid receptors

The opioid nature of kentsin (Thr-Pro-Arg-Lys) and its ability to alter pain perception and intestinal transit were examined. Kentsin (30,000 nM) did not inhibit electrically stimulated contractions of the guinea pig ileum (GPI) or mouse vas deferens (MVD), nor did it cause a rightward displacement of the inhibitory concentration-response curves of the mu-selective opioid agonist PL017 in the GPI or the delta-selective agonist DPDPE in the MVD. Kentsin (10,000 nM) did not displace [3H] naloxone from rat brain homogenates. These results indicate that kentsin lacks opioid agonist and mu and delta opioid antagonist properties and does not bind to opioid receptors. In vivo, kentsin produced dose-dependent analgesia in both the hotplate and abdominal stretch tests when administered intracerebroventricularly (ICV) and intrathecally but not intravenously. The central analgesic effect of kentsin was partially antagonized by the opioid antagonist naloxone. Kentsin inhibited intestinal transit in a dose-dependent manner after ICV administration only. The intestinal antitransit effect of kentsin was not blocked by pretreatment with naloxone. These results suggest that kentsin acts centrally to produce both opioid and non-opioid effects. Further, the opioid-mediated analgesic effects of kentsin involve mechanisms other than direct interaction with opioid receptors.     

RepSeq – A database of amino acid repeats present in lower eukaryotic pathogens

Background  

Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses.     

Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Rationale and design of the Sonolysis study

Background

The CONSORT Statement provides recommendations for reporting randomized controlled trials. We assessed the extent to which leading medical journals that publish reports of randomized trials incorporate the CONSORT recommendations into their journal and editorial processes.

Methods

This article reports on two observational studies. Study 1: We examined the online version of 'Instructions to Authors' for 165 high impact factor medical journals and extracted all text mentioning the CONSORT Statement or CONSORT extension papers. Any mention of the International Committee of Medical Journal Editors (ICMJE) or clinical trial registration were also sought and extracted. Study 2: We surveyed the editor-in-chief, or editorial office, for each of the 165 journals about their journal's endorsement of CONSORT recommendations and its incorporation into their editorial and peer-review processes.

Results

Study 1: Thirty-eight percent (62/165) of journals mentioned the CONSORT Statement in their online 'Instructions to Authors'; of these 37% (23/62) stated this was a requirement, 63% (39/62) were less clear in their recommendations. Very few journals mentioned the CONSORT extension papers. Journals that referred to CONSORT were more likely to refer to ICMJE guidelines (RR 2.16; 95% CI 1.51 to 3.08) and clinical trial registration (RR 3.67; 95% CI 2.36 to 5.71) than those journals which did not. Study 2: Thirty-nine percent (64/165) of journals responded to the on-line survey, the majority were journal editors. Eighty-eight percent (50/57) of journals recommended authors comply with the CONSORT Statement; 62% (35/56) said they would require this. Forty-one percent (22/53) reported incorporating CONSORT into their peer-review process and 47% (25/53) into their editorial process. Eighty-one percent (47/58) reported including CONSORT in their 'Instructions to Authors' although there was some inconsistency when cross checking information on the journal's website. Sixty-nine percent (31/45) of journals recommended authors comply with the CONSORT extension for cluster trials, 60% (27/45) for harms and 42% (19/45) for non-inferiority and equivalence trials. Few journals mentioned these extensions in their 'Instructions to Authors'.

Conclusion

Journals should be more explicit in their recommendations and expectations of authors regarding the CONSORT Statement and related CONSORT extensions papers.     

The Dynamics, Prevalence and Impact of Nematode Infections in Organically Raised Sheep in Sweden

A three-year survey (1997–99) was carried out on organically reared sheep flocks throughout Sweden. The aim was to determine the prevalence and intensity of nematode infections and to establish relationships between sheep management practices and parasite infections. Faecal samples from ewes and lambs were collected from 152 organic flocks around lambing-time and during the grazing-period for analysis. Results were compared with the different management practices that farmers use to prevent parasitism in their flocks. A high proportion of the flocks was infected with nematodes. The most prevalent species were Haemonchus contortus, Teladorsagia circumeincta, Trichostrongylus axei, T. colubriformis and Chabertia ovina and infections progressively increased during summer in lambs grazing on permanent pastures. Severity of parasitic infection in lambs was highly dependent on egg output from the ewes. H. contortus was found in 37% of the flocks, even at latitudes approximating the Polar Circle. Nematodirus battus was recorded for the first time in Sweden during the course of this study. Lambs turned out onto permanent pasture showed higher nematode faecal egg counts (epg) than lambs that had grazed on pastures, which had not carried sheep the previous year. This beneficial effect of lambs grazing non-infected pastures persisted if the ewes were treated with an anthelmintic before turn-out and if the lambs were kept on pastures of low infectivity after weaning. In lambs, the prevalence and the magnitude of their egg counts were higher during autumn in flocks where lambs were slaughtered after 8 months of age, compared with flocks where all lambs were slaughtered before this age. These results will be used in providing advice to farmers of ways to modify their flock management in order to minimise the use of anthelmintics, but at the same time efficiently produce prime lambs.     

The potential for using red claw crayfish and hybrid African catfish as biological control agents for Schistosoma host snails

The potential of red claw crayfish and hybrid African catfish (Clarias gariepinus and Clarias ngamensis) as predators for Schistosoma host snails was evaluated in 2014 by monitoring the consumption of snails by crayfish and catfish in experimental tanks over time under laboratory conditions. After 15 days, both crayfish and catfish had significantly reduced the populations of Bulinus globosus. Crayfish consumed 6.9 snails d^?1, whereas catfish consumed 5.9 snails d^?1. However, when supplied with an alternative prey, Melanoides tuberculata (100 individuals per tank), crayfish clearly preferred M. tuberculata (100% consumed over seven days) to B. globosus (100 individuals per tank) (54% consumed over the same period). Catfish, conversely, did not have a clear preference for either prey species, consuming 77% and 88% of M. tuberculata and B. globosus, respectively. It was also observed that young catfish were more efficient predators than older ones, because of ontogenetic shifts in their diet with age. Hybrid catfish retain the molluscivorous characteristics of their parent stock and red claw crayfish also preys on Schistosoma host snails. However, the effectiveness of both predators is affected by the presence of an alternative prey. Therefore, under suitable conditions, these species can be considered for biological control of schistosomiasis transmission.