Exploitation of a non-apoptotic caspase to regulate the abundance of the cdkl p27KIP1 in lymphoid cells

Expression of the cyclin dependent kinase inhibitor p27KIP1 is intimately linked to the control of proliferation, and is itself regulated by transcription, translation, phosphorylation, protein stability or sequestration. p27KIP1 is also regulated during apoptosis; cleavage occurs at DPSD139S and ESQD108V, by a sub-set of Z-VAD-fmk-sensitive caspases. We have identified a related but distinct mechanism that regulates p27KIP1 in proliferating lymphoid cell lines. In a B-lymphoid cell line (BJAB), the abundance of p27KIP1 oscillates inversely to proliferation; loss of full-length p27KIP1 correlates with the appearance of a truncated version corresponding to cleavage at DPSD139S. A direct correlation exists between the appearance of truncated p27KIP1 and the presence of an activity able to cleave peptides representing DPSD139S and a caspase-8 substrate (Ac-IETD-AMC) in vitro. This activity is inhibited by Ac-IETD-CHO but not Z-VAD-fmk in vitro. Furthermore a requirement for caspase-8 has been excluded. The activity differs from the apoptosis related p27KIP1-cleaving activity; indeed few cells undergoing apoptosis are present in the population of proliferating cells. The activity is further distinguished by its inability to cleave a peptide based on ESQD108V in vitro, together with the lack of a corresponding cleavage product in vivo. Inhibition of the caspase activity in vivo promotes an accumulation of full length p27KIP1, as well as a decrease in cell proliferation. Together these studies highlight the importance of non-apoptotic caspases in regulating p27KIP1 in transformed lymphoid cells

The Zipper Region of Epstein-Barr Virus bZIP Transcription Factor Zta Is Necessary but Not Sufficient To Direct DNA Binding

The viral bZIP transcription factor Zta (BZLF1, EB1, ZEBRA) mediates the switch between the latent and lytic cycles of Epstein-Barr virus (EBV). In part, its activity requires the formation of homodimers and interaction with specific DNA sequence elements (ZREs). Zta has an atypical zipper motif that has a lower stability than do typical bZIP proteins. Here we show that a synthetic peptide directed against the zipper can disrupt the DNA-binding function of Zta. This highlights the relevance of this region for the function of Zta and demonstrates that the zipper region is a potential target for therapeutic agents. We also unmask the relevance of a region adjacent to the zipper (CT region), which is required to direct the interaction of Zta with DNA and to transactivate ZRE-dependent promoters in vivo

Automatic conformal prescription of very selective saturation bands for in vivo 1H-MRSI of the prostate

An important step in the implementation of three-dimensional in vivo proton magnetic resonance spectroscopic imaging (1H-MRSI) of the prostate is the placement of spatial saturation pulses around the region of interest (ROI) for the removal of unwanted contaminating signals from peripheral tissue. The present study demonstrates the use of a technique called conformal voxel magnetic resonance spectroscopy (CV-MRS). This method automates the placement, orientation, timing and flip angle of very selective saturation (VSS) pulses around an irregularly-shaped, user-defined ROI. The method employs a user adjustable number of automatically positioned VSS pulses (20 used in the present study) which null the signal from periprostatic lipids while closely conforming the shape of the excitation voxel to the shape of the prostate. A standard endorectal coil in combination with a torso-phased array coil was used for all in vivo prostate studies. Three-dimensional in vivo prostate 1H-MRSI data were obtained using the proposed semi-automated CV-MRS technique, and compared with a standard point resolved spectroscopy (PRESS) technique at TE\u2009=\u2009130\u2009ms using manual placement of saturation pulses. The in vivo prostate 1H-MRSI data collected from 12 healthy subjects using the CV-MRS method showed significantly reduced lipid contamination throughout the prostate, and reduced baseline distortions. On average there was a 50\u2009\ub1\u200917% (range 12% \u2013 68%) reduction in lipids throughout the prostate. A voxel-by-voxel benchmark test of over 850 voxels showed that there were 63% more peaks fitted using the LCModel when using a Cramer-Rao Lower Bound (CRLB) cut-off of 40% when using the optimized conformal voxel technique in comparison to the manual placement approach. The evaluation of this CV-MRS technique has demonstrated the potential for easy automation of the graphical prescription of saturation bands for use in 1H-MRSI.Peer reviewed: YesNRC publication: Ye

Short echo time in vivo prostate 1H-MRSI

Visualization of short echo time (TE) metabolites in prostate magnetic resonance spectroscopic imaging is difficult due to lipid contamination and pulse timing constraints. In this work, we present a modified pulse sequence to permit short echo time (TE=40ms) acquisitions with reduced lipid contamination for the detection of short TE metabolites. The modified pulse sequence employs the conformal voxel MRS (CV-MRS) technique, which automatically optimizes the placement of spatial saturation planes to adapt the excitation volume to the shape of the prostate, thus reducing lipid contamination in prostate magnetic resonance spectroscopic imaging (MRSI). Metabolites were measured and assessed using a modified version of LCModel for analysis of in vivo prostate spectra. We demonstrate the feasibility of acquiring high quality spectra at short TEs, and show the measurement of short TE metabolites, myo-inositol, scyllo-inositol, taurine and glutamine/glutamate for both single and multi-voxel acquisitions. In single voxels experiments, the reduction in TE resulted in 57% improvement in the signal-to-noise ratio (SNR). Additional 3D MRSI experiments comparing short (TE=40 ms), and long (TE=130 ms) TE acquisitions revealed a 35% improvement in the number of adequately fitted metabolite peaks (775 voxels over all subjects). This resulted in a 42\ub124% relative improvement in the number of voxels with detectable citrate that were well-fitted using LCmodel. In this study, we demonstrate that high quality prostate spectra can be obtained by reducing the TE to 40 ms to detect short T2 metabolites, while maintaining positive signal intensity of the spin-coupled citrate multiplet and managing lipid suppression.Peer reviewed: YesNRC publication: Ye