Over Expression of Plk1 Does Not Induce Cell Division in Rat Cardiac Myocytes In Vitro

Background

Mammalian cardiac myocytes withdraw from the cell cycle during post-natal development, resulting in a non-proliferating, fully differentiated adult phenotype that is unable to repair damage to the myocardium, such as occurs following a myocardial infarction. We and others previously have shown that forced expression of certain cell cycle molecules in adult cardiac myocytes can promote cell cycle progression and division in these cells. The mitotic serine/threonine kinase, Polo-like kinase-1 (Plk1), is known to phosphorylate and activate a number of mitotic targets, including Cdc2/Cyclin B1, and to promote cell division.

Principal Findings

The mammalian Plk family are all differentially regulated during the development of rat cardiac myocytes, with Plk1 showing the most dramatic decrease in both mRNA, protein and activity in the adult. We determined the potential of Plk1 to induce cell cycle progression and division in cultured rat cardiac myocytes. A persistent and progressive loss of Plk1 expression was observed during myocyte development that correlated with the withdrawal of adult rat cardiac myocytes from the cell cycle. Interestingly, when Plk1 was over-expressed in cardiac myocytes by adenovirus infection, it was not able to promote cell cycle progression, as determined by cell number and percent binucleation.

Conclusions

We conclude that, in contrast to Cdc2/Cyclin B1 over-expression, the forced expression of Plk1 in adult cardiac myocytes is not sufficient to induce cell division and myocardial repair.     

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

Background

Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

Methodology/Principal Findings

Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

Conclusions/Significance

Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.     

Ds transposition mediated by transient transposase expression in Heiracium aurantiacum

The feasibility of using transient transposase expression to mobilize Ds elements for gene tagging in Hieracium aurantiacum was evaluated. A T-DNA construct carrying the Ac transposase gene and either a visible marker gene (uidA) or the conditionally-lethal marker gene (codA) was transferred to H. aurantiacum leaf discs (previously transformed with a Ds element) by co-cultivation with Agrobacterium tumefaciens. Shoots were regenerated directly from the co-cultivated leaf discs under selection for antibiotic resistance resulting from Ds excision. Most regenerants carried unique transposition events. Of 84 regenerated plants, twenty one (25%) did not express the marker gene and the DNA coding sequence of the transposase could not be detected in seven (8.3%). Potential advantages of this method over conventional gene-tagging methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; and the transient nature of transposase expression should facilitate the stabilisation of the transposed element.     

Cryoenzymology of Bacillus cereus beta-lactamase II

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution conformation of endothelin, a potent vaso-constricting bicyclic peptide. A combined use of 1H NMR spectroscopy and distance geometry calculations

The solution structure of endothelin-1, a newly discovered potent bicyclic peptide vaso-constrictor agent, has been investigated using 1H NMR conformational constraints and distance geometry calculations. The conformation is constrained by two disulphide bridges between Cys1-Cys15 and Cys3-Cys11 but the NMR data and computed conformers show additional helical structure between residues Leu6 and Cys11. Our results are compared with previous conflicting reports on the solution conformation of this peptide.     

GPS tracking reveals rafting behaviour of Northern Gannets (Morus bassanus): implications for foraging ecology and conservation

Capsule Three quarters of tracked Northern Gannets (Morus bassanus) at Grassholm gathered in rafts around the colony, concentrated within a recently designated at-sea Special Protection Area (SPA), but rafting was not correlated with foraging effort.

Aims To investigate the incidence, distribution and foraging implications of Northern Gannet rafting behaviour in waters adjacent to a large colony.

Methods Using bird-borne global positioning system (GPS) loggers we reconstructed at-sea movement and used a speed filter to identify rafting behaviour within 10?km of the colony. We mapped the spatial distribution of rafting events from 160 breeding individuals over 5 years, and investigated the relationship between foraging effort (trip duration and total distance travelled) and the presence/absence of rafting.

Results On average, 74% of tracked birds engaged in rafting. Of the 381 foraging trips analysed, rafting was recorded on 237 (62%). Birds were more likely to raft on outbound (224 trips, 59%), than inbound journeys (38 trips, 10%). Presence/absence of rafting did not correlate significantly with foraging trip distance or duration nor with duration of nest attendance. The majority of rafting was concentrated in a 2-km radius around the colony within a recently designated seaward SPA extension. Birds showed low individual repeatability in rafting, although there was lower variation within, than among, individuals.

Conclusion Our results show that rafting is important for breeding gannets on Grassholm, and a recently designated at-sea SPA encapsulates the core distribution of rafting. Rafting did not appear to be correlated with foraging behaviour. Given the dearth of literature on rafting and the wealth of GPS tracking data for seabirds, we suggest that similar research be conducted elsewhere to further elucidate the ecological and applied significance of this behaviour.