Development and validation of an alpha fetoprotein immunoassay using Gyros technology.

Circulating alpha fetoprotein (AFP) is a diagnostic and prognostic biomarker for hepatocellular carcinoma (HCC) with potential utility as a pharmacodynamic endpoint in rodent tumor models. This application is limited, however, by low sample volumes, highlighting the need for sensitive, sample-sparing biomarker assay methods. In order to improve the utility of AFP as an oncology biomarker, we developed a method for AFP using the Gyrolab™, an automated microimmunoassay platform. Commercially available antibodies were screened to identify optimal combinations that were then used in a multi-factorial design of experiments (DOE) to optimize reaction conditions. Analytical validation included assessments of accuracy and precision (A&P), and dilutional linearity/hook effect, as well as reagent and sample stability. The method is reliable, with total error, a measure of accuracy and precision, less than 30% for all concentrations tested. AFP concentrations were measurable in diseased mice and undetectable in normal mice. Therefore, this novel, low volume AFP immunoassay is suitable for pre-clinical drug development, where its miniaturized format facilitates serial sampling in rodent models of cancer.

MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIalpha.

During nitric oxide signaling, type Ialpha cGMP-dependent protein kinase (PKGIalpha) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIalpha interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH(2)-terminal LZ of PKGIalpha (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIalpha interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIalpha is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772-14777, 2000), and there is an RK motif within the aa 888-928 sequence of MYPT1 in LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIalpha interaction, we designed four MYPT1 fragments that contained both the aa 888-928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888-928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888-928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888-928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGIalpha in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R(916)K(917)) to AA decreased binding of MYPT1 to PKGIalpha in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R(916)K(917) to E(916)E(917) eliminated binding, suggesting that one factor important for the PKGIalpha-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIalpha-MYPT1 interaction.

PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle.

In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle.

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.

Vascular reactivity in heart failure: role of myosin light chain phosphatase.

Congestive heart failure (CHF) is a clinical syndrome, which is the result of systolic or diastolic ventricular dysfunction. During CHF, vascular tone is regulated by the interplay of neurohormonal mechanisms and endothelial-dependent factors and is characterized by both central and peripheral vasoconstriction as well as a resistance to nitric oxide (NO)-mediated vasodilatation. At the molecular level, vascular tone depends on the level of regulatory myosin light chain phosphorylation, which is determined by the relative activities of myosin light chain kinase and myosin light chain phosphatase (MLCP). The MLCP is a trimeric enzyme with a catalytic, a 20-kDa and a myosin targeting (MYPT1) subunit. Alternative splicing of a 3' exon produces leucine zipper positive and negative (LZ+/-) MYPT1 isoforms. Expression of a LZ+ MYPT1 has been suggested to be required for NO-mediated smooth muscle relaxation. Thus, we hypothesized that the resistance to NO-mediated vasodilatation in CHF could be attributable to a change in the relative expression of LZ+/- MYPT1 isoforms. To test this hypothesis, left coronary artery ligation was used to induce CHF in rats, and both the dose response relationship of relaxation to 8-Br-cGMP in skinned smooth muscle and the relative expression of LZ+/- MYPT1 isoforms were determined. In control animals, the expression of the LZ+ MYPT1 isoform predominated in both the aorta and iliac artery. In CHF rats, LVEF was reduced to 30+/-5% and there was a significant decrease in both the sensitivity to 8-Br-cGMP and expression of the LZ+ MYPT1 isoform. These results indicate that CHF is associated with a decrease in the relative expression of the LZ+ MYPT1 isoform and the sensitivity to 8-Br-cGMP-mediated smooth muscle relaxation. The data suggest that the resistance to NO-mediated relaxation observed during CHF lies at least in part at the level of the smooth muscle and is a consequence of the decrease in the expression of the LZ+ MYPT1 isoform.