Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs

The photospheric abundances of active binaries III. Abundance peculiarities at high activity level

We report the determination from high-resolution spectra of the atmospheric parameters and abundances of 13 chemical species (among which lithium) in 8 single-lined active binaries. These data are combined with our previous results for 6 other RS CVn systems to examine a possible relationship between the photospheric abundance patterns and the stellar activity level. The stars analyzed are generally found to exhibit peculiar abundance ratios compared to inactive, galactic disk stars of similar metallicities. We argue that this behaviour is unlikely an artefact of errors in the determination of the atmospheric parameters or non-standard mixing processes along the red giant branch, but diagnoses instead the combined action of various physical processes related to activity. The most promising candidates are cool spot groups covering a very substantial fraction of the stellar photosphere or NLTE effects arising from nonthermal excitation. However, we cannot exclude the possibility that more general shortcomings in our understanding of K-type stars (e.g. inadequacies in the atmospheric models) also play a significant role. Lastly, we call attention to the unreliability of the (V-R) and (V-I) colour indices as temperature indicators in chromospherically active stars.Comment: Accepted for publication in A&A, 17 pages, 7 figures (6 in colour

Multi-wavelength diagnostics of accretion in an X-ray selected sample of CTTSs

High resolution X-ray spectroscopy has revealed soft X-rays from high density plasma in Classical T-Tauri stars (CTTSs), probably arising from the accretion shock region. However, the mass accretion rates derived from the X-ray observations are consistently lower than those derived from UV/optical/NIR studies. We aim to test the hypothesis that the high density soft X-ray emission is from accretion by analysing optical accretion tracers from an X-ray selected sample of CTTSs in a homogeneous manner. We analyse optical spectra of a sample of CTTSs and calculate the accretion rates based on measuring optical emission lines. These are then compared to the accretion rates derived from the X-ray spectroscopy. We find that, for each CTTS in our sample, the different optical tracers predict mass accretion rates that agree within the errors, albeit with a spread of ~1 order of magnitude. Typically, mass accretion rates derived from Halpha and HeI 5876 Ang are larger than those derived from Hbeta, Hgamma and OI. When comparisons of the optical mass accretion rates are made to the X-ray derived mass accretion rates, we find that: a) the latter are always lower (but by varying amounts); b) the latter range within a factor of ~2 around 2x10^{-10} M_odot yr^{-1}, despite the fact that the former span a range of ~3 orders of magnitude. We suggest that the systematic underestimation of the X-ray derived mass accretion rates could depend on the density distribution inside the accretion streams, where the densest part of the stream is not visible in the X-ray band because of the absorption by the stellar atmosphere. We also suggest that a non-negligible optical depth of X-ray emission lines produced by post-shock accreting plasma may explain the almost constant mass accretion rates derived in X-rays if the effect is larger in stars with larger optical mass accretion rates.Comment: 12 pages, 4 figures. Accepted for publication by A&

Unstable high molecular weight inverted repetitive DNA in human lymphocytes.

About 1% of newly synthesized DNA from PHA-stimulated human lymphocytes can be isolated as large (up to 90 kilobase pairs) double stranded fragments that resist sequential alkali and heat denaturation steps but are not closed circular. By electron microscopy about 1% have single-strand hairpin loops at one end and therefore present inverted repetitive sequences (IR-DNA). Most of the remainder have a blunt-appearing double-strand terminus at both ends (78%) or one end (18%). Indirect evidence indicates that these also are inverted complementary structures with terminal hairpin loops too small to be visualized: (1) Treatment with either a 5' or 3' single-strand exonuclease generates essentially only fragments with a single strand at one end; (2) with partial denaturation, the number of fragments with identifiable single-strand hairpin loops increases (to about 20%); (3) after S1 nuclease digestion, greater than 95% can be fully heat denatured. Cot analysis indicates that these fragments are derived from dispersed sites throughout the genome. Up to 25% of DNA released from lymphocytes during growth similarly resists denaturation, and released DNA and IR-DNA are both enriched in the same set of repetitive sequences. Thus at least a portion of IR-DNA appears to be unstable