Cardiomyocytes structure, function and associated pathologies

The heart is the first formed organ in the developing fetus. During fetal and postnatal development cardiomyocytes become terminally differentiated muscular cells that are connected end to end by gap junctions, allowing concerted contractile activity. The contraction-relaxation cycle of cardiomyocytes is orchestrated by cyclic increases and decreases in intracellular Ca(2+) initiated by depolarization of the sarcolemma and sustained by Ca(2+) release and re-uptake by the sarcoplasmic reticulum. When stressed, cardiomyocytes undergo hypertrophic growth and apoptotic responses in vivo as well as in cell culture models. Such changes predispose to heart failure in the longer term.     

Insect host location: a volatile situation

Locating a host plant is crucial for a phytophagous (herbivorous) insect to fulfill its nutritional requirements and to find suitable oviposition sites. Insects can locate their hosts even though the host plants are often hidden among an array of other plants. Plant volatiles play an important role in this host-location process. The recognition of a host plant by these olfactory signals could occur by using either species-specific compounds or specific ratios of ubiquitous compounds. Currently, most studies favor the second scenario, with strong evidence that plant discrimination is due to central processing of olfactory signals by the insect, rather than their initial detection. Furthermore, paired or clustered olfactory receptor neurons might enable fine-scale spatio-temporal resolution of the complex signals encountered when ubiquitous compounds are used.     

DNA damage responses at low radiation doses

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.     

Comparison of marker types and map assumptions using Markov chain Monte Carlo-based linkage analysis of COGA data

We performed multipoint linkage analysis of the electrophysiological trait ECB21 on chromosome 4 in the full pedigrees provided by the Collaborative Study on the Genetics of Alcoholism (COGA). Three Markov chain Monte Carlo (MCMC)-based approaches were applied to the provided and re-estimated genetic maps and to five different marker panels consisting of microsatellite (STRP) and/or SNP markers at various densities. We found evidence of linkage near the GABRB1 STRP using all methods, maps, and marker panels. Difficulties encountered with SNP panels included convergence problems and demanding computations.     

The functional basis of granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 receptor activation, basic and clinical implications

The cytokines granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 have overlapping activities on cells expressing their receptors. This is explained by their sharing a receptor signal transduction subunit, beta c. This communal signaling subunit is also required for high affinity binding of all three cytokines. Therapeutic approaches attempting to interfere or modulate haemopoietic cells using cytokines or their analogues can in some instances be limited due to functional redundancy amongst cytokines using shared receptor signaling subunits. Therefore, a better approach would be to develop therapeutics against the shared subunit. Studies examining the GM-CSF, IL-3 and IL-5 receptors have identified the key events leading to functional receptor activation. With this knowledge, it is now possible to identify new targets for the development of a new class of antagonist that blocks the biological activity of all the cytokines utilizing beta c. This approach may be extended to other receptor systems such as IL-4 and IL-13 where receptor activation is dependent on a common signaling and binding subunit.     

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.