Occurrence of unusual molecular species of sphingomyelin containing 28-34-carbon polyenoic fatty acids in ram spermatozoa.

The high levels of very long chain fatty acids found in ram spermatozoa are located almost exclusively in one of two separable species of sphingomyelin. Mass spectral analysis, including fast atom bombardment of the purified sphingomyelin, has shown the fatty acids to have a carbon chain length of between 28 and 34, with between four and six double bonds, and to belong predominantly to the n-3 series.     

Exponentiated exponential model (Gompertz kinetics) of Na+ and K+ conductance changes in squid giant axon

The conductance changes, gK(t) and gNa(t), of squid giant axon under voltage clamp (Hodgkin and Huxley, 1952) may be modeled by exponentiated exponential functions (Gompertz kinetics) from any holding potential VO to any membrane clamp potential V. The equation constants are set by the membrane potential V, and include, for any voltage step in the case of gK, the initial conductance, gO, the asymptote conductance g, and rate constant k: gK = g exp(-be-kt) where b = 1n g/gO. Equations of similar form relate g and k to the voltage V, and govern the corresponding parameters of the gNa system. For the gNa, the fast phase y = y exp (-be-kt) is cut down in proportion to a slow process p = (1 - p)e-k't + p, and thus gNa = py. The expo-exponential functions involve fewer constants than the Hodgkin-Huxley model. In particular, the role of the n, m, h parameters appears to be filled largely by 1n (g/gO) in the case of gK and by 1n (y/yO) in the case of gNa. Membrane action potentials during current clamp may be computed from the conductances generated by use of the appropriate differential forms of the equations; diverse other membrane behaviors may be predicted.     

Metabolism of 7,12-dimethylbenz[a]anthracene and its methyl-hydroxylated metabolites: formation of phenolic metabolites at the 2-positions.

The 1- and 2-positions of 7,12-dimethylbenz[a]anthracene (DMBA) were thought not to be involved in biotransformation to 1,2-epoxide and 1,2-dihydrodiol because of steric hindrance from the 12-methyl group (Biochem. Biophys. Res. Commun. $\text{85}$: 357–362, 1978). However, we have identified four 2-phenols as rat liver microsomal metabolites of DMBA and its methyl-hydroxylated metabolites, 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]-anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. Our findings suggest that neither the 12-methyl group nor the 12-hydroxymethyl group blocks the microsomal oxygenations of the 1,2 positions of DMBA or its methyl-hydroxylated derivatives. The 2-phenols may be formed as nonenzymatic rearrangement products of the 1,2-epoxide intermediates, although their formations by a direct hydroxylation mechanism cannot be ruled out.     

Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 microM), whereas "inactive" derivatives were > or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.     

Signaling cascades activated upon antibody cross-linking of myelin oligodendrocyte glycoprotein: potential implications for multiple sclerosis

Antibody-induced demyelination is an important component of pathology in multiple sclerosis. In particular, antibodies to myelin oligodendrocyte glycoprotein (MOG) are elevated in multiple sclerosis patients, and they have been implicated as mediators of demyelination. We have shown previously that antibody cross-linking of MOG in oligodendrocytes results in the repartitioning of MOG into glycosphingolipid-cholesterol membrane microdomains ("lipid rafts"), followed by changes in the phosphorylation of specific proteins, including dephosphorylation of beta-tubulin and the beta subunit of the trimeric G protein and culminating in rapid and dramatic morphological alterations. In order to further elucidate the mechanism of anti-MOG-mediated demyelination, we have carried out a proteomic analysis to identify the set of proteins for which the phosphorylation states or expression levels are altered upon anti-MOG treatment. We demonstrate that treatment of oligodendrocytes with anti-MOG alone leads to an increase in calcium influx and activation of the MAPK/Akt pathways that is independent of MOG repartitioning. However, further cross-linking of anti-MOG.MOG complexes with a secondary anti-IgG results in the lipid raft-dependent phosphorylation of specific proteins related to cellular stress response and cytoskeletal stability. Oligodendrocyte survival is not compromised by these treatments. We discuss the possible significance of the anti-MOG-induced signaling cascade in relation to the initial steps of MOG-mediated demyelination.     

A combined genomewide linkage scan of 1,233 families for prostate cancer-susceptibility genes conducted by the international consortium for prostate cancer genetics

Evidence of the existence of major prostate cancer (PC)–susceptibility genes has been provided by multiple segregation analyses. Although genomewide screens have been performed in over a dozen independent studies, few chromosomal regions have been consistently identified as regions of interest. One of the major difficulties is genetic heterogeneity, possibly due to multiple, incompletely penetrant PC-susceptibility genes. In this study, we explored two approaches to overcome this difficulty, in an analysis of a large number of families with PC in the International Consortium for Prostate Cancer Genetics (ICPCG). One approach was to combine linkage data from a total of 1,233 families to increase the statistical power for detecting linkage. Using parametric (dominant and recessive) and nonparametric analyses, we identified five regions with “suggestive” linkage (LOD score >1.86): 5q12, 8p21, 15q11, 17q21, and 22q12. The second approach was to focus on subsets of families that are more likely to segregate highly penetrant mutations, including families with large numbers of affected individuals or early age at diagnosis. Stronger evidence of linkage in several regions was identified, including a “significant” linkage at 22q12, with a LOD score of 3.57, and five suggestive linkages (1q25, 8q13, 13q14, 16p13, and 17q21) in 269 families with at least five affected members. In addition, four additional suggestive linkages (3p24, 5q35, 11q22, and Xq12) were found in 606 families with mean age at diagnosis of 65 years. Although it is difficult to determine the true statistical significance of these findings, a conservative interpretation of these results would be that if major PC-susceptibility genes do exist, they are most likely located in the regions generating suggestive or significant linkage signals in this large study.     

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.