Mutagenicity of aliphatic epoxides.

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity, trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

Anestrus in cycling beef heifers experimentally inoculated with Anaplasmamarginale

Normal estrous cycles were established for twenty beef heifers. Ten heifers were inoculated with blood from a known $\text{Anaplasma}$$\text{marginale}$ carrier. The inoculated heifers experienced anemia, anorexia, and positive $\text{Anaplasma}$$\text{marginale}$ complement-fixing antibody titers, and $\text{A}$. $\text{marginale}$ was observed on the erythrocytes while uninoculated control heifers remained normal. Further observation following inoculation revealed that five of ten inoculated heifers experienced anestrus while controls continued to cycle normally. Anestrus coincided with clinical signs of acute anaplasmosis. Normal estrus patterns returned following treatment and recovery. This study provides evidence that acute anaplasmosis in beef heifers may cause anestrus and therefore lead to reproductive inefficiency.     

A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.     

Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum) and Dothistroma septosporum: New insights into how these fungal pathogens interact with their host plants

Fulvia fulva and Dothistroma septosporum are closely related apoplastic pathogens with similar lifestyles but different hosts: F. fulva is a pathogen of tomato, whilst D. septosporum is a pathogen of pine trees. In 2012, the first genome sequences of these pathogens were published, with F. fulva and D. septosporum having highly fragmented and near-complete assemblies, respectively. Since then, significant advances have been made in unravelling their genome architectures. For instance, the genome of F. fulva has now been assembled into 14 chromosomes, 13 of which have synteny with the 14 chromosomes of D. septosporum, suggesting these pathogens are even more closely related than originally thought. Considerable advances have also been made in the identification and functional characterization of virulence factors (e.g., effector proteins and secondary metabolites) from these pathogens, thereby providing new insights into how they promote host colonization or activate plant defence responses. For example, it has now been established that effector proteins from both F. fulva and D. septosporum interact with cell-surface immune receptors and co-receptors to activate the plant immune system. Progress has also been made in understanding how F. fulva and D. septosporum have evolved with their host plants, whilst intensive research into pandemics of Dothistroma needle blight in the Northern Hemisphere has shed light on the origins, migration, and genetic diversity of the global D. septosporum population. In this review, we specifically summarize advances made in our understanding of the F. fulva–tomato and D. septosporum–pine pathosystems over the last 10 years.     

Reaction of peptide thiobenzyl esters with mammalian chymotrypsinlike enzymes: A sensitive assay method

Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin A_α with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m^?1 cm^?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values for these compounds.     

Narrow range of temperature and irradiance supports optimal development of Lessonia corrugata (Ochrophyta) gametophytes: implications for kelp aquaculture and responses to climate change

The kelp Lessonia corrugata (Ochrophyta, Laminariales) is being developed for integrated multi-trophic aquaculture (IMTA) trials in the vicinity of salmon cages in Tasmania, Australia. Gametophytes are vegetally maintained before seeding on hatchery twine; however, the optimal temperature and light conditions for growth and sexual development are unknown. We measured vegetative size of female and male gametophytes and sexual development of females over a range of temperatures and irradiances using a temperature gradient table and neutral density light filters. Over a 4-week experiment, gametophytes were exposed to a combination of thermal (5.7–24.9 °C) and irradiance (10–100 μmol photons m^?2 s^?1) gradients, to assess biological performance. At the temperature extremes (hottest = 24.9 °C, coldest = 5.7 °C), we observed the critical thermal limits for this species and the results reveal a narrow optimal temperature range for growth and sexual development between 15.7 and 17.9 °C, with irradiances between 40 and 100 μmol photons m^?2 s^?1 resulting in fertile female gametophytes. Lessonia corrugata inhabits a small geographic range, found only around Tasmania, south of the Australian mainland, hence oceanic changes such as ongoing increases in sea surface temperatures (SSTs), and altered irradiance regimes may limit recruitment of the early microscopic life stages in the future. Our findings provide optimised culture conditions for aquaculture and information to predict the future geographic range of L. corrugata under ocean global change.

     

Open label study of escalating doses of oral treprostinil diethanolamine in patients with systemic sclerosis and digital ischemia: pharmacokinetics and correlation with digital perfusion

Introduction

Treprostinil diethanolamine is an innovative salt form of the prostacyclin analogue, treprostinil sodium, developed as an oral sustained release (SR) osmotic tablet. The availability of a formulation permitting convenient systemic delivery might have applicability to scleroderma vascular complications. We evaluated pharmacokinetics and perfusion in scleroderma patients with digital ischemia following escalating twice-daily doses of treprostinil diethanolamine SR.

Methods

Scleroderma patients with digital ulcers were enrolled in this dual-center, open-label, phase I pharmacokinetic study. Drug concentrations and perfusion, quantified by laser Doppler imaging, were measured over 12 hours at the 2 mg and 4 mg (or maximally tolerated) doses. Pharmacokinetic parameters were determined from individual plasma concentration versus time profiles using non-compartmental analysis methods. Digital perfusion and skin temperature were modeled as a function of log-transformed drug concentration and other covariates by performing repeated measures analyses using random effects models.

Results

Nineteen scleroderma patients (84% female, 53% limited scleroderma) received treprostinil diethanolamine SR with dose titration up to 4 mg twice daily as tolerated. Peak concentrations (mean maximum plasma concentration (C_max) = 1,176 and 2,107 pg/mL) occurred approximately 3.6 hours after dose administration, and overall exposure (under the plasma concentration-time curve from time 0 to 12 hours post dose (AUC_0-12) = 7,187 and 12,992 hr*pg/mL) was linear between the 2 mg and 4 mg doses. Perfusion and digital skin temperature were positively associated with log-transformed plasma concentration at the 4 mg dose (P = 0.015 and P = 0.013, respectively). The most frequent adverse events were similar to those seen with prostacyclin analogues.

Conclusions

Oral treprostinil diethanolamine was effectively absorbed in patients with scleroderma. Drug administration was temporally associated with improved cutaneous perfusion and temperature. Treprostinil diethanolamine may provide a new therapeutic option for Raynaud''s phenomenon and the peripheral vascular disease of scleroderma.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00848939","term_id":"NCT00848939"}}NCT00848939.

Electronic supplementary material

The online version of this article (doi:10.1186/ar4216) contains supplementary material, which is available to authorized users.