Cardiac alternans in embryonic mouse ventricles

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.     

A propeptide toolbox for secretion optimization of <Emphasis Type="Italic">Flavobacterium meningosepticum</Emphasis> endopeptidase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background

Lactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.

Results

To expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4–2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.

Conclusions

Overall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

     

Xenobiotic-mediated production of superoxide by primary cultures of rat cerebral endothelial cells, astrocytes, and neurones

Previous works of our group demonstrated that xenobiotic metabolism by brain microsomes or cultured cerebral cells may promote the formation of reactive oxygen species. In order to characterise the risk of oxidative stress to both the central nervous system and the blood-brain barrier, we measured in the present work the release of superoxide in the culture medium of rat cerebrovascular endothelial cells during the metabolism of menadione, anthraquinone, diquat or nitrofurazone. Assays were run in the same experimental conditions on primary cultures of rat neurones and astrocytes. Quinone metabolism efficiently produced superoxide, but the production of radicals during the metabolism of diquat or nitrofurazone was very low, as a probable result of their reduced transport inside the cells. In all cell types assayed, superoxide production was time- and concentration-dependent, and cultured astrocytes always produced the highest amounts of radicals. Superoxide formation by microsomes prepared from the cultured cells was decreased by immunoinhibition of NADPH-cytochrome P450 reductase or by its irreversible inhibition by diphenyliodonium chloride, suggesting the involvement of this flavoprotein in radical production. Cerebrovascular endothelial cells cultured on collagen-coated filters produced equivalent amounts of superoxide both at their luminal side and through the artificial basement membrane, suggesting that in vivo, endothelial superoxide production may endanger adjacent astrocytes and neurones.     

Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.     

Use of a Taqman PCR to determine the response of Mycoplasma haemofelis infection to antibiotic treatment

A quantitative Taqman polymerase chain reaction (PCR) assay was used to evaluate the response of Mycoplasma haemofelis experimentally infected cats to three antibiotic treatment regimes. Sixteen cats were intravenously inoculated with M. haemofelis from a chronically infected donor. The cats were randomly assigned to one of four treatment groups each containing four cats: oral doxycycline at 10 mg/kg/day for 14 days, oral enrofloxacin at 5 mg/kg/day for 14 days, oral enrofloxacin at 10 mg/kg/day for 14 days, and an untreated control group. DNA, extracted from blood samples collected on days 0, 7, 14, 21, 25, 28, 32, 35, 42 and 54 post-inoculation (PI), was subjected to quantitative Taqman PCR. The M. haemofelis copy number was significantly lower in the doxycycline group (P=0.008), the 5 mg/kg/day enrofloxacin group (P=0.006) and the 10 mg/kg/day enrofloxacin group (P=0.005) compared to the untreated control group. No significant differences were found between any of the three antibiotic treated treatment groups. All three antibiotic treatment regimes evaluated in this study were effective at reducing M. haemofelis copy number.     

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10⁶ M⁻¹ s⁻¹ and 6.7 ± 2.5 × 10⁻² s⁻¹, respectively, which corresponded to an average equilibrium dissociation constant (K_D) of 2.6 ± 1.4 × 10⁻⁸ M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.     

Pyridine-containing 6-hydrazinonicotinamide derivatives as potential bifunctional chelators for 99mTc-labeling of small biomolecules

As a continuation of our interest in novel 99mTc chelating systems, several pyridine-containing HYNIC (6-hydrazinonicotinamide) derivatives (L1-L5) have been synthesized and characterized by NMR (1H and 13C) and LC-MS. 99mTc complexes of L1-L5 were prepared by the reaction of the HYNIC derivative with 99mTcO4- in the presence of excess tricine and stannous chloride. Results from this study show that the attachment site of the linker is critical for the formation of macrocyclic 99mTc complexes. For example, the pyridine-N in L3 is not able to bond to the Tc, because the lysine linker is attached to the 4-position. When the linker is at the 2-position, L1 forms the macrocyclic complex [99mTc(L1)(tricine)], but the radiochemical purity is relatively low. If the linker is attached to the 3-position of the pyridine ring, the HYNIC derivatives form macrocyclic complexes [99mTc(L)(tricine)] (L2, L4, and L5) in high yield (>95%). The HPLC data suggest that the macrocyclic complex [(99m)Tc(L2)(tricine)] exists in solution as four isomers: two diastereomers and two conformational isomers. Diastereomers are due to a combination of the chirality of the lysine linker and of the Tc chelate. Replacing lysine with a pentamethylenediamine linker results in the macrocyclic complex [99mTc(L4)(tricine)] with two conformational isomers, which interconvert rapidly at room temperature. Changing the linker from pentamethylenediamine to hexamethylenediamine did not eliminate the minor isomer; but the percentage of the minor isomer was reduced from approximately 10% for [99mTc(L4)(tricine)] to only 6% for [99mTc(L5)(tricine)]. The linker length is an important parameter to minimize the minor isomer. LC-MS data of complexes [99mTc(L)(tricine)] (L2, L4, and L5) are completely consistent with their proposed compositions. On the basis of these data, it is concluded that pyridine-containing HYNIC derivatives have the potential as bifunctional chelators for 99mTc-labeling of small biomolecules if the linker is attached to the 3-position of the pyridine ring.     

A structure-based strategy to identify new molecular scaffolds targeting the bacterial ribosomal A-site

The need for novel antibiotics is widely recognized. A well validated target of antibiotics is the bacterial ribosome. Recent X-ray structures of the ribosome bound to antibiotics have shed new light on the binding sites of these antibiotics, providing fresh impetus for structure-based strategies aiming at identifying new ribosomal ligands. In that respect, the ribosomal decoding region of the aminoacyl-tRNA acceptor site (A-site) is of particular interest because oligonucleotide model systems of this site are available for crystallography, NMR and compound binding assays. This work presents how these different resources can be combined in a hierarchical screening strategy which has led to the identification of new A-site ligands. The approach exploits an X-ray structure of the A-site against which large and diverse libraries of compounds were computationally docked. The complementarity of the compounds to the A-site was assessed using a scoring function specifically calibrated for RNA targets. Starting from approximately 1 million compounds, the computational selection of candidate ligands allowed us to focus the experimental work on 129 compounds, 34 of which showed affinity for the A-site in a FRET-based binding assay. NMR experiments confirmed binding to the A-site for some compounds. For the most potent compound in the FRET assay, a tentative binding mode is suggested, which is compatible with the NMR data and the limited SAR in this series. Overall, the results validate the screening strategy.