Abstract. Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (S, e, e', and~) as well as the calcium-dependent isoforms (a, 01, ßn, and -y).These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised . Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide . PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography . Peak I reacted exclusively with antisera to PKCy, peak II with PKCß, and -ßu, and peak III with PKCa. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinc-P ROTEIN kinase C (PKC)' plays a major role in transmembrane signal transduction (35). PKC is activated by diacylglycerol, which is generated from membrane phospholipids upon stimulation of cells with various agonists (4) . PKC also serves as the receptor for phorbol esters and related tumor promoters (34), which activate the kinase by substituting for endogenous diacylglycerol (8) . Because of the pleiotropic actions of diacylglycerol and phorbol esters, PKC has been implicated in the regulation ofa variety of cellular processes, including proliferation, differentiation, and release of hormones and neurotransmitters (35,36).Molecular cloning studies have revealed that PKC consists of a large family of at least eight different isoenzymes (36, 37) that can be divided into two major groups. Initially, four isoforms of PKC were described . This group consists of PKCca, -01, -ß, 1, and -y, with PKCß, and -ß arising via alternate splicing of the same gene transcript and resulting in distinct carboxy-terminal regions . All four of these isoen-1. Abbreviations used in this paper: PKC, protein kinase C.O The Rockefeller University Press,

storage before transplantation correlates clinically with inThe injury resulting from cold ischemia and warm recreased primary graft nonfunction, graft rejection, and rate perfusion during liver transplantation is a major clinical of re-transplantation. 4,5 problem that limits graft success. Kupffer cell activation Reperfusion injury is difficult to study clinically, but has plays a pivotal role in reperfusion injury, and Kupffer been investigated in animal models, including orthotopic cell products, including free radicals and tumor necrosis liver transplantation in rats. These studies have shown that factor a (TNF-a), are implicated as damaging agents.hepatic reperfusion following ischemia induces Kupffer cell However, the second messengers and signaling pathactivation, superoxide formation, and elevated plasma levels ways that are activated by the stress of hepatic ischemia/ of tumor necrosis factor a (TNF-a). 6,7 Pretreatment with reperfusion remain unknown. The purpose of this study methyl palmitate inhibits Kupffer cell activation and imis to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the acti-proves transplant survival threefold, thereby supporting a vating protein 1 (AP-1) transcription factor in response role for Kupffer cells in reperfusion injury. 8 In addition, treatto ischemia and reperfusion in the transplanted rat ment with agents that suppress TNF-a release from activated liver. There was a potent, sustained induction of c-jun Kupffer cells decrease transplant failure.9 Nisoldipine, a Ca 2/ N-terminal kinase (JNK), but not of the related MAPKs channel blocker, reduces plasma levels of TNF-a and inextracellular signal-regulated kinases (ERK) or p38, creases transplant survival time. 10,11 Similarly, pentoxifylupon reperfusion after transplantation. TNF-a messen-line, a methylxanthine which suppresses TNF-a messenger ger RNA (mRNA) levels and transcription factors AP-1 RNA (mRNA) accumulation in response to lipopolysacchaand nuclear factor-kB (NF-kB) were induced in the liver ride, has a protective effect on liver grafts.12 Taken together, after 60 minutes of reperfusion. Finally, there was an these data suggest an important role for TNF-a in mediating elevation of ceramide, but not diacylglycerol or sphingo-reperfusion injury. sine, in the transplanted liver. Ceramide is a second mes-TNF-a is a pleiotropic cytokine that induces cellular effects senger generated by TNF-a treatment and is an activator ranging from proliferation to apoptosis. TNF-a is a potent of JNK. Because JNK activation preceded the elevations activator of activating protein 1 (AP-1) and NF-kB transcripin ceramide and TNF-a mRNA, these results suggest that tion factors and of the c-jun N-terminal kinase (JNK, also increased hepatic TNF-a and ceramide may perpetuate known as stress-activated protein kinase, SAPK).13-15 JNK is JNK induction, but that they are not the initiating sig-a member of the vertebrate mitogen activated protein kinase nals of JNK activation during reperfus...

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activationinduced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-DL-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.

One of the most intriguing enzymes of sphingolipid biology is acid sphingomyelinase (ASMase). In a phospholipase C reaction, ASMase catalyzes the cleavage of the phosphocholine head group of sphingomyelin to generate ceramide. Cumulative efforts of various laboratories over the past 40 years have placed ASMase and its product ceramide at the forefront of lipid research. Activation of the ASMase/ceramide pathway is a shared response to an ever-growing list of receptor and non-receptor mediated forms of cellular stress including: death ligands (TNFalpha, TRAIL, Fas ligand), cytokines (IL-1, IFNgamma), radiation, pathogenic infections, cytotoxic agents and others. The strategic role of ASMase in lipid metabolism and cellular stress response has sparked interest in investigatig the molecular mechanisms underlying ASMase activation. In this article, we review the translational role of the ASMase/ceramide pathway and recent advances on its mechanisms of regulation.

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose-and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was doselimiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.