Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24-48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50 of 20 +/- 2 nM. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50 of CEP-1347 for JNK1 activation was the same as the EC50 for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

We have described a monoclonat antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin-coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies wffh differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 105 sites per myoblast with an apparent Kd in the range of 2-5 x 10 -9 molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of ~120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an ovbious candidate.

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl ␤-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, ␤-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl ␤-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation.

Treatment of chick embryos in ovo with IGF-I during the period of normal, developmentally regulated neuronal death (embryonic days 5-10) resulted in a dose-dependent rescue of a significant number of lumbar motoneurons from degeneration and death. IGF-II and two variants of IGF-I with reduced affinity for IGF binding proteins, des(1-3) IGF-I and long R3 IGF-I, also elicited enhanced survival of motoneurons equal to that seen in IGF-I-treated embryos. IGF-I did not enhance mitogenic activity in motoneuronal populations when applied to embryos during the period of normal neuronal proliferation (E2-5). Treatment of embryos with IGF-I also reduced two types of injury-induced neuronal death. Following either deafferentation or axotomy, treatment of embryos with IGF-I rescued approximately 75% and 50%, respectively, of the motoneurons that die in control embryos as a result of these procedures. Consistent with the survival-promoting activity on motoneurons in ovo, IGF-I, -II, and des(1-3) IGF-I elevated choline acetyltransferase activity in embryonic rat spinal cord cultures, with des(1-3) IGF-I demonstrating 2.5 times greater potency than did IGF-I. A single addition of IGF-I at culture initiation resulted in the maintenance of 80% of the initial ChAT activity for up to 5 days, during which time ChAT activity in untreated control cultures fell to 9%. In summary, these results demonstrate clear motoneuronal trophic activity for the IGFs. These findings, together with previous reports that IGFs are synthesized in muscle and may participate in motoneuron axonal regeneration and sprouting, indicate that these growth factors may have an important role in motoneuron development, maintenance, and recovery from injury.

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

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Erythrocyte ghosts loaded with ' 25 1-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol . Erythrocyte-mediated microinjection of ' 25 1-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum.A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins . Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of shortlived proteins . Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h . Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone .The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination .Intracellular protein degradation is a fundamentally important process occurring in all organisms from bacteria to humans (5,20,23,24,53) . The continued breakdown and replacement of proteins allows the cell to regulate concentrations of specific enzymes as well as to alter overall protein content in response to changing physiological demands. The major areas of current study in protein degradation can be divided into three broad topics : (a) the influence of polypeptide structure on protein half-lives (20, 23), (b) the physiological regulation of protein degradative rates (5,20,24), and (c) the mechanisms by which proteins are degraded within cells (5,24,53) . Despite considerable recent progress in each ofthese areas, many ofthe major questions in this field of research remain unanswered.Microinjection offers several advantages for analysis of protein degradation over more conventional approaches in which endogenous cellular proteins are radiolabeled and their deg- radative rates determined (I1, 34, 51). Microinjection of mammalian cells in culture has been achieved using microneedles (6,55) or by inducing fusion of the recipient cell with erythrocyte ghosts containing the protein to be microinjected (6,34,51) . Most studies of protein degradation have used erythrocytemediated microinjection because sufficient numbers of cells can be microinject...

Leupeptin, chymostatin and antipain inhibited the degradation of long-lived proteins in cultured rat hepatocytes by 20-30%, probably by inhibiting lysosomal proteases: (1) Leupeptin and chymostatin decreased to a similar extent the degradation of an exogenous protein 125I-asialo fetuin, a process known to occur within lysosomes. (2) In extracts of cells treated with leupeptin, cathepsin B activity was inhibited by 35-50%. (3) Leupeptin, chymostatin and antipain inhibited proteolysis by homogenates of liver lysosomes but not by the supernatant fraction. These agents, however, do not appear to rapidly permeate the membrane of isolated lysosomes. Leupeptin, chymostatin and antipain did not inhibit the breakdown of short-lived normal cell proteins, and ones containing amino acid analogs. Even when the amount of abnormal proteins was increased, such that it comprised a large fraction of cell protein, the degradation of these polypeptides was still very rapid and not affected by these inhibitors. The pathway for the degradation of short-lived cell proteins thus appears distinct from that responsible for degradation of long-lived cell proteins. In accord with this conclusion, reduction of the temperature of cultures inhibited the breakdown of long-lived proteins to a much greater extent than it affected the breakdown of short-lived ones. Treatment of cultured hepatocytes with glucagon, or deprivation for serum or amino acids stimulated the degradation of the more stable cell proteins but did not affect the breakdown of 125I-asialo-fetuin. Under these conditions leupeptin and chymostatin inhibited the breakdown of long-lived cell proteins to the same extent as in control cultures. Thus, lysosomal enzymes seem to play an important role in protein breakdown both in fed hepatocytes and in cells where proteolysis is accelerated.