Distribution of neuronal apoptosis inhibitory protein in human tissues.

The neuronal apoptosis inhibitory protein (NAIP) gene, also known as the baculovirus inhibitor of apoptosis repeat-containing protein 1 (BIRC1) gene, is a member of the inhibitors of apoptosis (IAP) family and was first characterized as a candidate gene for spinal muscular atrophy (SMA). The expression of NAIP has been thoroughly studied in the central nervous system and overlaps the pattern of neurodegeneration in SMA. Recent studies have pointed to a role for NAIP in non-neuronal cells. We report here the production of a specific anti-NAIP antibody and the profile of NAIP expression in human adult tissues by Western blot and immunohistochemical detection methods. NAIP was detected in a number of tissues by Western blot analysis, but immunohistochemistry revealed that NAIP's presence in certain tissues, such as liver, lung, and spleen, is most likely due to macrophage infiltration. In the small intestine, the expression of NAIP coincides with the expression of p21(WAF1). This observation, coupled with findings from other groups, suggests a role for NAIP in increasing the survival of cells undergoing terminal differentiation as well as the possibility that the protein serves as an intestinal pathogen recognition protein. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The Ste20-like kinase SLK is required for cell cycle progression through G2.

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G2 upstream of H1 kinase activation.

A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells.

In platelets, PGHS-1-dependant formation of thromboxane A(2) is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01 which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10(-8) M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, which shows a consistent increase over the entire course of differentiation, can be used as an additional and better index by which to monitor megakaryocyte differentiation.