Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines

The overexpression of urokinase (uPA), which plays a key role in tumour invasion and metastasis, is an established prognostic marker and potential therapeutic target. Plasminogen activator inhibitor type 2 (PAI-2), an efficient and specific inhibitor of uPA, has been shown to selectively deliver potent cytotoxins to tumour cells. However, a direct quantitative analysis of both the inhibition kinetics and subsequent fate of PAI-2 upon interaction with cell-surface uPA has not been previously undertaken. In this study, we analysed specific PAI-2 binding to receptor-bound uPA on human breast and prostate cancer cell lines to directly measure inhibition kinetics. Cell-surface uPA:PAI-2 complex formation, which is reflective of complete uPA inhibition, was found to be very efficient (inactivation constant [K(I)] = 60-80 pM, depending on cell line used) and rapid (inactivation rate constant [k(inact)] = 0.32-0.47 min(-1) at 37 degrees C, depending on cell line used). To directly quantify and visualise cellular internalisation and localisation, we developed a novel assay based on the use of PAI-2 labelled with Alexa(488) fluorochrome and a polyclonal antibody to quench Alexa(488) fluorescence. The efficient and rapid formation of uPA:PAI-2 complexes was thus shown to be associated with specific and rapid internalisation of PAI-2, which could be localised within endosomes and lysosomes. PAI-2 was subsequently degraded, presumably within lysosomes. This study is the first to provide definitive evidence for uPA/uPAR-mediated PAI-2 endocytosis.     

Respiratory metabolism in buckwheat seedlings

Young seedlings of buckwheat (Fagopyrum esculentum) respire in air with an RQ of unity. Analysis of respiratory substrates coupled with a study of the utilization of acetate-¹⁴C and glucose-¹⁴C suggest that both the Embden-Meyerhof-Parnas, tricarboxylic acid and pentose phosphate sequences participate in the total respiratory catabolism.

In anoxia CO₂ dropped to one third of the aerobic rate and ethanol accumulated to only about one half the rate of CO₂ output on a molar basis. Smaller amounts of lactate, succinate and free amino acids (particularly alanine and γ-aminobutyric acid) accumulated, carboxylic acids decreased and there were initial increased in pyruvate and α-ketoglutarate. The observed changes are consistent with residual tricarboxylic acid and pentose phosphate cycle activity in anoxia and may account for the excess CO₂ production over ethanol accumulation. CO₂, ethanol and lactate production did not account for all of the carbohydrate consumed in anoxia.

Relative rates of carbon loss were measured in air and in atmospheres containing 3.5%, 2.1%, 1.3% and 0.6% oxygen. The extinction point of anaerobic metabolism was 1.5%.

On return to air from anoxia the CO₂ output increased and the RQ rose from 0.8 to 1.0 over the first 2-hour period. Ethanol, lactate and succinate were consumed and other constituents returned to their previous aerobic level. Some of these changes suggest a rather slow resumption of tricarboxylic acid cycle activity on return to air.

Carbon loss as CO₂ in air was greater than the carbon loss as CO₂ at the extinction point. Carbon loss in anoxia as CO₂, ethanol and lactate was similar to carbon loss at the extinction point. Assessed in this orthodox manner buckwheat seedlings show no Pasteur effect but the complex nature of the changes in levels of metabolic substrates and intermediates do not allow firm conclusions to be drawn on the effects of oxygen on the rates of glycolysis and other respiratory processes.

     

Future research in weight bias: What next?

The 2015 Canadian Weight Bias Summit disseminated the newest research advances and brought together 40 experts, stakeholders, and policy makers in various disciplines in health, education, and public policy to identify future research directions in weight bias. In this paper we aim to share the results of the Summit as well as encourage international and interdisciplinary research collaborations in weight bias reduction. Consensus emerged on six research areas that warrant further investigation in weight bias: costs, causes, measurement, qualitative research and lived experience, interventions, and learning from other models of discrimination. These discussions highlighted three key lessons that were informed by the Summit, namely: language matters, the voices of people living with obesity should be incorporated, and interdisciplinary stakeholders should be included.     

Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1

Background

We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.

Methods

GC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.

Results

TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.

Conclusions

Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.     

Synthesis and pharmacological activity of metabolites of the 5-HT(4) receptor antagonist SB-207266

Three metabolites of N-[(1-butyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]-oxazino[3,2_a]indole-10-carboxamide (SB-207266) (1) were synthesised and their pharmacological activity determined.