Faculty Bibliography

Complete brain fixation can be achieved with transthoracic cardiac infusion without thoracotomy. Light and electron microscopy tissue sections reveal preservation of cytoplasmic and nuclear structure at all magnification levels. Punched samples were obtained from the fixed tissue specimens in precisely localized areas for study using electron microscopy. This perfusion fixation technique provides both faster tissue harvesting capability and higher quality tissue preservation, without the artifacts of brain swelling and ventricular dilation observed in direct cardiac perfusion. Acute, discrete change in brain tissue can be studied

Disabled-2 (DAB2) is an adapter protein that is up-reg-ulated during megakaryocytic differentiation of hematopoietic cells and is abundantly expressed in platelets. In this study, the role of DAB2 in integrin alpha(IIb)beta(3)-mediated matrix protein fibrinogen adhesion and cell signaling was investigated. In K562 cells differentiating to the megakaryocytic lineage, down-regulation of DAB2 by DAB2 small interfering RNA augmented integrin alpha(IIb)beta(3) activation and resulted in an increase in cell adhesion to fibrinogen. Ectopic expression of DAB2 reversed the DAB2 small interfering RNA effect or, by itself, decreased fibrinogen adhesion of K562 cells. Mutational analysis revealed that a DAB2 Ser(24) phosphorylation mutant (S24A) abrogated the inhibitory function of DAB2. The spatial and temporal association/interaction of DAB2 and platelet integrin alpha(IIb)beta(3) (CD61) in both megakaryocytic cells and platelets led us to examine the effect of Ser(24) phosphorylation on the interaction between DAB2 and integrin beta(3). Through cellular localization and co-immunoprecipitation analysis, we demonstrate for the first time that Ser(24) phosphorylation promotes membrane translocation of DAB2 and its subsequent interaction with integrin beta(3), thereby defining a mechanism for DAB2 in regulating integrin alpha(IIb)beta(3) activation and inside-out signaling. Consistent with the effect on fibrinogen adhesion, Ser(24) phosphorylation of DAB2 was also involved in the negative regulation of alpha(IIb)beta(3)-induced T cell factor transcriptional activity. In contrast, the S24A mutant acted like wild-type DAB2 and inhibited both beta-catenin- and plakoglobin-mediated T cell factor transactivation. Hence, DAB2 elicits distinct regulatory mechanisms in alpha(IIb)beta(3) and beta-catenin/plakoglobin signaling in a Ser(24) phosphorylation-dependent and -independent manner, respectively. These findings indicate Ser(24) phosphorylation as a molecular basis for DAB2 acting as a negative regulator in alpha(IIb)beta(3) inside-out signaling and contribute to our understanding of DAB2 in megakaryocytic differentiation and platelet function.

Among the many initiating events that lead to apoptosis or programmed cell death, loss of contact between the cell and the extracellular matrix has been extensively studied. Adhesion-related apoptosis referred to as anoikis is initiated by the action of anti-adhesive substances. Nitric oxide is one of these anti-adhesive substances that have the capacity to signal and trigger pro-apoptotic events in a variety of cell types. Nitric oxide can inhibit cell adhesion, interfere with the assembly of focal adhesion complexes, and disrupt the cell-extracellular matrix interactions. These actions occur in cell that exhibit a dissociation of growth factor signals from alterations in the cytoskeleton, ultimately leading to apoptosis. Since this involves anti-adhesive events, nitric oxide can be considered as causing anoikis. This review article summarizes the available evidence of how nitric oxide participates in apoptosis induced by loss of anchorage (anoikis).

The free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. In the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT II, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p21Ras on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p21Ras, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. The above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p21Ras. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. In addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2.

Previous studies have shown that Disabled-2 (DAB2) is up-regulated during megakaryocytic differentiation of human K562 cells. To delineate the consequences of DAB2 induction, a DNA vector-based small interfering RNA (siRNA) was designed to intervene in DAB2 expression. We found that DAB2 siRNA specifically inhibited DAB2 induction, resulting in the modulation of cell-cell adhesion and mitogen-activated protein kinase (MAPK) phosphorylation. The morphological changes and beta3 integrin expression associated with megakaryocytic differentiation were not affected. Since the MAPK pathway has been shown to involve DAB2 induction [Tseng et al., Biochem. Biophys. Res. Commun. 285 (2001) 129-135], our results suggest a reciprocal regulation between DAB2 and MAPK in the differentiation of K562 cells. In addition, we have demonstrated for the first time that DAB2 siRNA is a valuable tool for unveiling the biological consequences of DAB2 expression.

Palladium (Pd(2+)) and platinum (Pt(2+)) ions were found to inhibit erythrocyte membrane-bound acetylcholinesterase (AChE) with Ki values of 6.0 and 6.5 microg/ml, respectively. Lineweaver-Burke plots revealed that the inhibition of erythrocyte AChE by both metal ions was competitive in nature. Binding studies using alkaline phosphatase as a reporting enzyme confirmed that both metal ions indeed did bind to the enzyme molecules. In the process of red cell vesiculation, membrane-bound AChE is shed along with vesicles. The measurement of AChE activities in the medium containing vesiculated RBC could potentially be served as an index of vesiculation. Inhibition of AChE activities by both metal ions can thus constitute a potential source of error in vesiculation measurement. To illustrate these effects, a simulated vesiculation system, using green tea polyphenol in the presence (25 microg/ml) or absence of Pd(2+) ion was simultaneously examined by the electronmicrography and the AChE method. We observed vesiculation under the experimental condition in Pd(2+)-free controls that was associated with a time-dependent increase in AChE activity were barely detected in the Pd(2+)-spiked specimen because of the masking effect exerted by the metal ions themselves.

INTRODUCTION: The neuronal cell death that occurs after ischemia-induced cerebral infarction (stroke) contains elements of apoptosis and necrosis, an intermediary form of the two, and a distinct excitotoxic process. We previously developed a photochemical model of stroke in the rat. We have now adapted this model for use in the mouse. The present manuscript describes the mouse model. METHODS: Minimal beam intensity (0.1 W/cm(2)) cold white light (8 min exposure) was used to evoke discrete infarcts in the parietal lobes of 11 mice sensitized by the administration of fresh Rose Bengal (10 mg/kg by rapid iv infusion). RESULTS: At 2 h, five out of five mice and at 6 h, six out of six mice demonstrated light microscopic histologic features like those in the rat model. These included a superior ischemic zone with shrunken and pyknotic nuclei, a middle transition zone of edematous vacuolated neuropil but normal neurons with open chromatin and retained Nissl granules, and an inferior zone with normal neurons. There was widespread nuclear terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) in the superior infarct zone in 11/11 mice. However, in the edematous vacuolated transition zone, 11/11 mice had TUNEL positive and negative nuclei randomly mixed. Light microscopic analysis of that same transition zone showed no pyknosis or chromatin bodies in the TUNEL positive or negative cells. DISCUSSION: In mice, photoactivation of Rose Bengal evoked similar infarct and transition zone patterns found previously in rats, with TUNEL evidence of apoptotic and nonapoptotic events. Thus, it will be possible to use this model for further quantitative study of apoptotic and excitotoxic events in wild and transgenic mice

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.