Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein-coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation-dependent signaling pathway, which plays an essential role in growth factor-induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II-induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT1 receptor-mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca2+ by the Ca2+ ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II-induced activation of MAP kinase and RSK, chelating intracellular Ca2+ by BAPTA-AM completely abolished Ang II-induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for Gq protein-coupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to Gs protein-coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through Gq protein-coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca2+, rather than PKC, seems to be critical for Ang II-induced activation of these protein kinases in cardiac myocytes.

Arachidonic acid release is induced in macrophages with diverse agonists including calcium ionophores, phorbol myristate acetate (PMA), okadaic acid, and the phagocytic particle, zymosan, and correlates with activation of cytosolic phospholipase A 2 (cPLA 2 ). The role of calcium and phosphorylation of cPLA 2 in regulating arachidonic acid release was investigated. Zymosan induced a rapid and transient increase in [Ca 2؉ ] i . This in itself is not sufficient to induce arachidonic acid release since ATP and platelet activating factor (PAF), agonists that induce transient calcium mobilization in macrophages, induced little arachidonic acid release. Unlike zymosan, which is a strong activator of mitogen-activated protein kinase (MAPK), ATP and PAF were weak MAPK activators and induced only a partial and transient increase in cPLA 2 phosphorylation (gel shift). However, ATP or PAF together with colony stimulating factor-1 (CSF-1) synergistically stimulated arachidonic acid release. CSF-1 is a strong MAPK activator that induces a rapid and complete cPLA 2 gel shift but not calcium mobilization or arachidonic acid release. Arachidonic acid release was more rapid in response to CSF-1 plus ATP or PAF than zymosan and correlated with the time course of the cPLA 2 gel shift. Although low concentrations of ionomycin induced a lower magnitude of calcium mobilization than ATP, the response was more sustained resulting in arachidonic acid release. A23187 and ionomycin induced weak MAPK activation, and a partial and transient cPLA 2 gel shift. The MAPK kinase inhibitor, PD 98059 suppressed A23187-induced MAPK activation and cPLA 2 gel shift but had little effect on arachidonic acid release. These results indicate that in macrophages a transient increase in [Ca 2؉ ] i and sustained phosphorylation of cPLA 2 can act together to promote arachidonic acid release but neither alone is sufficient. A sustained increase in calcium is sufficient for inducing arachidonic acid release. However, PMA and okadaic acid induce arachidonic acid release without increasing [Ca 2؉ ] i , although resting levels of calcium are required, suggesting alternative mechanisms of regulation.

Transcriptional dysregulation plays a major role in the pathology of Huntington's disease (HD). However, the mechanisms causing selective downregulation of genes remain unknown. Histones regulate chromatin structure and thereby control gene expression; recent studies have demonstrated a therapeutic role for histone deacetylase (HDAC) inhibitors in polyglutamine diseases. This study demonstrates that despite no change in overall acetylated histone levels, histone H3 is hypo-acetylated at promoters of downregulated genes in R6/2 mice, ST14a and STHdh cells, as demonstrated by in vivo chromatin immunoprecipitation. In addition, HDAC inhibitor treatment increases association of acetylated histones with downregulated genes and corrects mRNA abnormalities. In contrast, there is a decrease in mRNA levels in wild-type cells following treatment with a histone acetyltransferase inhibitor. Although changes in histone acetylation correlate with decreased gene expression, histone hypo-acetylation may be a late event, as no hypo-acetylation is observed in 4-week-old R6/2 mice. Nevertheless, treatment with HDAC inhibitors corrects mRNA abnormalities through modification of histone proteins and may prove to be of therapeutic value in HD.

(2012) Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration, mAbs, 4:2, 243-255,

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

Hypotonic stress causes rapid cell swelling and initiates various cellular adaptive processes. However, it is unknown how cells initially sense low osmolarity and convert it into intracellular signals. We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c‐fos expression as a nuclear marker. Treatment of myocytes with hypotonic culture media rapidly induced c‐fos expression, whereas hypertonic stress had no effect. Transfection of c‐fos reporter gene constructs suggested that the hypotonic stress response element maps to the serum response element of the c‐fos promoter. Hypotonic stress immediately (within 5 s) activated tyrosine kinase activity, while activation of ERK1/2 peaked at 5 min. Stress‐activated kinase (JNK1) was modestly activated at 15 min, whereas HOG1 like kinase (p38) was not activated by hypotonic stress. Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling‐induced c‐fos expression. The effect of hypotonic stress was mimicked by chlorpromazine, which is known to cause membrane deformation. These results suggest that the signaling mechanism of hypotonic stress is distinct from that of hyperosmolar stress in mammalian cells. Tyrosine kinase activation is the earliest detectable cell response and plays an essential role in hypotonic swelling‐induced ERK1/2 activation and c‐fos expression.

Several ligands of the endocytic low density lipoprotein receptor-related protein (LRP), such as apoE-containing lipoproteins and activated ␣2-macroglobulin (␣2M*), promote neurite outgrowth, suggesting that LRP may have signaling functions. In this study, we found that the treatment of neurons with ␣2M* significantly increased the individual length (by 71%) and numbers (by 139%) of neurites of primary mouse cortical neurons. These effects were blocked by the LRP antagonist, the receptor-associated protein. We found similar neurite outgrowth with purified apoE3 and a tandem apoE peptide containing only the receptor-binding domain. To investigate the intracellular pathway of the LRP signaling involved in neurite outgrowth, we tested the effects of ␣2M* on the phosphorylation of the mitogen-activated protein (MAP) extracellular signal-regulated kinases 1 and 2 (ERK1/2). We found that 1) phospho-MAP kinase levels were altered within 30 min after treatment with ␣2M*, 2) the MAP kinase inhibitor, PD98059, specifically blocked the ␣2M*-induced neurite outgrowth, 3) manipulating intracellular calcium by BayK or BAPTA altered the neurite outgrowth and associated changes in the phospho-MAP kinase levels, which were blunted by ␣2M*, 4) ␣2M* promoted the phosphorylation of the transcription factor CREB through MAP kinase, and 5) LRP-specific antibodies increased levels of phosphorylated MAP kinase and phosphorylated CREB. The effects of ␣2M*, apoE3, and apoE peptides increased LRP levels in the cortical neurons, whereas LRP receptor-associated protein reduced dendritic LRP expression. These results demonstrate that p44/42 MAP kinase plays an important role in LRP-mediated neurite outgrowth with activation involving the effects on calcium homeostasis and downstream effects involving the activation of gene transcription through CREB.

Apolipoprotein E and ␣ 2 -macroglobulin (␣2M) are genetic risk factors for late-onset Alzheimer's disease, and both bind a cell surface receptor, the lowdensity lipoprotein receptor-related protein (LRP). To investigate the role of LRP on preventing the accumulation of ␤-amyloid peptide (A␤), we examined the effects of ␣2M on the clearance of endogenous A␤. Studies were performed in primary Tg2576 transgenic mouse cortical neuronal cultures expressing human mutant amyloid precursor protein (APP) 695. This system allowed us to follow endogenous A␤ using immunoblots to detect monomeric forms of the peptide. A␤ and APP levels were measured in conditioned media. We found that activated ␣2M (␣2M*) substantially decreased soluble A␤ levels and had no effect on secreted or full-length APP levels. Native ␣2M, which is not a ligand for LRP, did not affect A␤ levels. The receptor-associated protein, which inhibits interaction of all ligands with LRP in vitro, prevented ␣2M*-induced decreases of soluble A␤ levels. These data suggest that ␣2M* affects soluble A␤ clearance rather than A␤ production. Further studies showed that similar A␤ clearance via an LRP-mediated pathway was observed after treatment with another LRP ligand, lactoferrin. Taken together, these data demonstrate that ␣2M* enhances the clearance of soluble A␤ via LRP in cortical neurons. Key Words: Alzheimer's disease -Apolipoprotein E-Lactoferrin-Immunoblot-Amyloid precursor protein.