Convenient and Efficient Syntheses of Oligodeoxyribonucleotides Containing O 6-(Carboxymethyl)Guanine and O 6-(4-Oxo-4-(3-Pyridyl)Butyl)Guanine

O ⁶-(carboxymethyl)guanine (O ⁶-CMG) and O ⁶-(4-oxo-4-(3-pyridyl)butyl)guanine (O ⁶-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O ⁶-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O ⁶-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O ⁶-CMG and O ⁶-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O ⁶-pobG and O ⁶-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental file.]     

CO2 elimination as a function of frequency and tidal volume in rabbits during HFO

CO2 elimination (VCO2) was monitored during high-frequency oscillation (HFO) over a frequency (f) range of 2-30 Hz in anesthetized and paralyzed rabbits to determine whether effective gas exchange could be achieved in this species, to determine the f and tidal volume (VT) dependence of gas exchange in this species, and to compare these results with those from dog and human studies. We were able to produce VCO2 levels during HFO that exceeded normal steady-state levels of CO2 production with VT's less than the total dead space volume. VCO2 was related to f in a curvilinear fashion, whereas in some rabbits VCO2 became independent of f at higher frequencies. This curvilinear relationship between f and VCO2 is similar to data from humans but contrasts with the linear relationship found in dogs. Evidence is presented indicating frequency-dependent behavior of gas exchange is correlated with a frequency-dependent decrease in respiratory system resistance. We propose that the frequency-dependent mechanical properties of the rabbit lung may also account for the species differences in HFO gas exchange.     

Monitoring toxicity associated with parenteral sodium stibogluconate in the day-case management of returned travellers with New World cutaneous leishmaniasi

Background

Patients with New World cutaneous leishmaniasis (NWCL) caused by Leishmania Viannia are treated with parenteral sodium stibogluconate (SbV) to reduce the risk of development of mucocutanous leishmaniasis. Our centre manages patients with NWCL on an outpatient-basis. This study was conducted to assess the safety and efficacy of this approach.

Methodology

We reviewed records of 67 consecutive NWCL patients, aged 17–61 years, treated as day-cases with 20 mg/kg/day SbV for up to 28 days at our UK centre. Data had been collected in a standardised format at the time of treatment using a care-record tool. Patients reported adverse-effects daily using a structured questionnaire. Blood tests and electrocardiograms were performed twice weekly to monitor for toxicity.

Principal Findings

Parenteral SbV treatment was associated with an early, significant suppression of mean lymphocyte and platelet counts. By day four of treatment, lymphocytes reduced by 0.53×10⁹/L (CI 0.29×10⁹/L to 0.76×10⁹/L, p<0.001), and platelets by 31,000/µL (CI 16,000/µL to 46,000/µL, p<0.001). SbV was further associated with significant elevation of serum alanine transaminase concentrations, with a mean peak rise of 107 iu/L by day 13 (CI 52 iu/L to 161 iu/L, p<0.001). These disturbances were temporary and did not result in adverse clinical events. Patient-described symptoms were cumulative and at three weeks of treatment, 59.6% of patients experienced myalgia and 29.8% malaise. Treatment adherence and clinical outcomes were comparable to inpatient treatment studies. A total of 1407 individual doses of SbV resulted in only 26 nights'' hospital admission, a saving of 1381 bed-days compared to inpatient treatment.

Conclusions/Significance

In specialist centres, NWCL patients aged below 65 years and without co-morbidities can be safely and effectively treated without hospital admission. This reduces the cost of treatment, and is much preferred by patients. Twice weekly blood and electrocardiographic monitoring may be surplus to requirement in clinically well, low-risk patients.     

A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1.

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.     

Solution structure of mu-conotoxin PIIIA,a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels

Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.     

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors—Part II

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones have been identified as highly potent 5-HT_1A/B/D receptor antagonists with and without additional SerT activity and a high degree of selectivity over hERG potassium channels. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

A biomechanical analysis of prognathous and orthognathous insect head capsules: evidence for a many‐to‐one mapping of form to function

Insect head shapes are remarkably variable, but the influences of these changes on biomechanical performance are unclear. Among ‘basal’ winged insects, such as dragonflies, mayflies, earwigs and stoneflies, some of the most prominent anatomical changes are the general mouthpart orientation, eye size and the connection of the endoskeleton to the head. Here, we assess these variations as well as differing ridge and sclerite configurations using modern engineering methods including multibody dynamics modelling and finite element analysis in order to quantify and compare the influence of anatomical changes on strain in particular head regions and the whole head. We show that a range of peculiar structures such as the genal/subgenal, epistomal and circumocular areas are consistently highly loaded in all species, despite drastically differing morphologies in species with forward‐projecting (prognathous) and downward‐projecting (orthognathous) mouthparts. Sensitivity analyses show that the presence of eyes has a negligible influence on head capsule strain if a circumocular ridge is present. In contrast, the connection of the dorsal endoskeletal arms to the head capsule especially affects overall head loading in species with downward‐projecting mouthparts. Analysis of the relative strains between species for each head region reveals that concerted changes in head substructures such as the subgenal area, the endoskeleton and the epistomal area lead to a consistent relative loading for the whole head capsule and vulnerable structures such as the eyes. It appears that biting‐chewing loads are managed by a system of strengthening ridges on the head capsule irrespective of the general mouthpart and head orientation. Concerted changes in ridge and endoskeleton configuration might allow for more radical anatomical changes such as the general mouthpart orientation, which could be an explanation for the variability of this trait among insects. In an evolutionary context, many‐to‐one mapping of strain patterns onto a relatively similar overall head loading indeed could have fostered the dynamic diversification processes seen in insects.     

The Drosophila ets-2 gene: molecular structure, chromosomal localization, and developmental expression

The cellular homologs of the ets gene from the avian erythroblastosis retrovirus E26 have been studied in chickens, humans, mice, and cats. In this report a further evolutionary step is taken by isolating and characterizing a Drosophila ets-related genomic clone. Sequence analysis of this clone has shown it to contain the 3' end of the v-ets gene, called ets-2, corresponding to the last two exons of chicken ets. The predicted amino acid sequence was found to have over 90% homology when compared to that of v-ets. This is the highest level of conservation observed for any previously characterized Drosophila oncogene homolog. Expression of the ets-2 gene occurs throughout development, but is highest during the embryonic and pupal stages. By in situ hybridization, the ets-2 chromosomal position was determined to be 58A/B which corresponds to no known phenotypic mutant. As this is a highly conserved gene, the Drosophila model system should prove useful for the determination of the ets gene function.     

Analysis of a diffusion-limited hollow fiber reactor for the measurement of effective substrate diffusivities

Mathematical analyses of a diffusion-limited hollow fiber reactor for the measurement of effective substrate diffusivities are presented. An analytical solution to the mathematical model with a first order substrate consumption rate is used to show that the procedure of Webster and Shuler(1) is incorrect. A rigorous analysis that requires numerical solution is also outlined for any form of the substrate consumption rate. These analyses allow for more accurate estimations of effective substrate diffusivities since they should be used in conjunction with integral reactor behavior.