Inhibitor 2 of protein phosphatase 2A (I2PP2A), a biological inhibitor of the cellular serine/threonine protein phosphatase PP2A, is associated with numerous cellular processes that often lead to the formation and progression of cancer. In this study we hypothesized that targeting the inhibition of I2PP2A's multiple functions in prostate cancer cells might prevent cancer progression. We have investigated the effect of the small chain C6-ceramide, known to be a bioactive tumor suppressor lipid, on I2PP2A function, thereby affecting c-Myc signaling and histone acetylation in cells. Our data indicated that C6-ceramide treatment of prostate cancer cells induces cell death in PC-3, DU145, and LNCaP cells, but not normal prostate epithelial cells. C6-ceramide was able to disrupt the association between PP2A and I2PP2A. C6-ceramide inhibits I2PP2A's upregulation of c-Myc and downregulation of histone acetylation in prostate cancer cells. Our data indicated that targeting cancer related signaling pathways through I2PP2A using ceramide as an anti-I2PP2A agent could have beneficial effects as a therapeutic approach to prevent prostate cancer.

Successful treatment and diagnosis of neurological diseases depend on reliable delivery of molecules across the blood-brain barrier (BBB), which restricts penetration of pharmaceutical drugs and diagnostic agents into the brain. Thus, developing new non-invasive strategies to improve drug delivery across the BBB is critically needed. This study was aimed at evaluating the activity of HAV6 peptide (Ac-SHAVSS-NH2) in improving brain delivery of camptothecin-glutamate (CPT-Glu) conjugate and gadolinium-diethylenetriaminepentaacetate (Gd-DTPA) contrast agent in Sprague-Dawley rats. Brain delivery of both CPT-Glu and Gd-DTPA was evaluated in an in situ rat brain perfusion model in the presence and absence of HAV6 peptide (1.0 mM). Gd-DTPA (0.6 mmol/kg) was intravenously (i.v.) administered with and without HAV6 peptide (0.019 mmol/kg) in rats. The detection and quantification of CPT-Glu and Gd-DTPA in the brain were carried out by LC-MS/MS and quantitative magnetic resonance imaging (MRI), respectively. Rats perfused with CPT-Glu in combination with HAV6 had significantly higher deposition of drug in the brain compared to CPT-Glu alone. MRI results also showed that administration of Gd-DTPA in the presence of HAV6 peptide led to significant accumulation of Gd-DTPA in various regions of the brain in both the in situ rat brain perfusion and in vivo studies. All observations taken together indicate that HAV6 peptide can disrupt the BBB and enhance delivery of small molecules into the brain.

The transient-state kinetic approach reveals the formation and subsequent interconversions of intermediates in real-time. Its potential for the mechanistic resolution of enzymatic and other complex chemical mechanisms has been severely limited however by the lack of a rigorous and applicable theoretical basis in contrast to that of the less direct but soundly based algebraic algorithms of the steady-state approach. Having recently established three rigorously derived fundamental Rules of transient-state kinetics applicable to realistic multiple step reactions, we present here the successful application of the very counter-intuitive Second Rule to the resolution of the mechanism of the L-phenylalanine dehydrogenase catalyzed reaction.The steady-state kinetic approach has provided the overwhelming portion of what we now know about the mechanisms of enzymatic catalysis. While any such experiment provides only a single pair of parameters (V and V/K m ), their interpretation rests on a large body of rigorously derived algebraic formulations. Variation of pH, reactant and product concentrations, and kinetic isotope effects (KIEs) combined with X-ray crystallographic structures have produced many detailed mechanisms interpretable in terms of physical organic chemical theory(1). Yet, we are still a very long way from a thorough understanding of the phenomenon of enzymatic catalysis.In contrast to the steady-state approach, transient state kinetic experiments typically provide a wide variety of optical phenomena tracking the formation and interconversions of enzyme bound-intermediates in real time. The effectiveness of the transient (pre-steady state) approach, however, has been severely limited by the lack of a coherent body of rigorous analytical theorems applicable to the results of transient-state (stopped-flow) experimental data (2). The necessity of interpreting our experimental results has led us to develop such a theory.Kinetic isotope effects (KIEs) are an important tool for the steady-state kineticist (3). By definition, a steady state KIE is defined as a single value at zero-time. In contrast, transientstate KIEs (tKIEs) are strongly time dependent and may be presumed to contain significantly more extensive mechanistic information. To our knowledge, no analytic theory applicable to realistic enzyme mechanisms for tKIEs existed prior to this report. Experimental and theoretical developments described here have now provided a solid basis for the development of such a body of theory. † Transient State KineticsThe transient state absorbance versus wavelength and time array was obtained using an Applied Photophysics SX-18MV stopped flow apparatus by assembling a series of side-byside single-wavelength time traces, in which a solution containing enzyme and h-or d-L-Phe in one syringe is mixed with a solution containing enzyme and NAD in the other. The spectra were collected at 1 nm and 0.05 ms intervals. An individual experiment such as that portrayed in Figure 3a takes approximately 20 min to produce an ...

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