Phosphorylation of protein kinase Cdelta on distinct tyrosine residues induces sustained activation of Erk1/2 via down-regulation of MKP-1: role in the apoptotic effect of etoposide

The mechanism underlying the important role of protein kinase Cdelta (PKCdelta) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCdelta in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCdelta since the phosphorylation of Erk1/2 was inhibited by a PKCdelta-KD mutant and PKCdelta small interfering RNA. We recently reported that phosphorylation of PKCdelta on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCdeltatyrosine mutants, we found that the phosphorylation of PKCdeltaon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCdelta was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCdelta. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCdelta and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCdeltaon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.     

Mycosporine-like amino acids in azooxanthellate and zooxanthellate early developmental stages of the soft coral Heteroxenia fuscescens

The ontogenetic changes of MAAs in the soft coral Heteroxenia fuscescens was studied in relation to their symbiotic state (azooxanthellate vs. zooxanthellate) under different temperature conditions in the Gulf of Eilat, northern Red Sea. The HPLC chromatograms for extracts of the planulae, azoo- and zooxanthellate primary polyps of H. fuscescens from all dates of collection yielded a single peak at 320 nm that has been identified as the compound palythine. Concentration of palythine in planulae at 23 °C was 7.57 ± 1 nmol mg^− 1 protein and at 28 °C reached 17.29 ± 1 nmol × mg^− 1 protein. Concentration of palythine in azooxanthellate primary polyps was 16.4 ± 3 nmol × mg^− 1 protein and 28.37 ± 2.8 nmol × mg^− 1 protein at 23 °C and 28 °C respectively. The palythine concentration for zooxanthellate primary polyps at 23 °C was 13 ± 3 nmol × mg^− 1 protein and at 28 °C 32.7 ± 2 nmol mg^− 1 protein. Palythine concentrations were significantly higher at 28 °C in the different animal groups and correlated linearly with the ambient collection temperature. This study shows for the first time that UVR and temperature act synergistically and affect the MAA levels of early life-history stages of soft corals.     

The dynamics of mammalian P body transport, assembly, and disassembly in vivo

Exported mRNAs are targeted for translation or can undergo degradation by several decay mechanisms. The 5′→3′ degradation machinery localizes to cytoplasmic P bodies (PBs). We followed the dynamic properties of PBs in vivo and investigated the mechanism by which PBs scan the cytoplasm. Using proteins of the decapping machinery, we asked whether PBs actively scan the cytoplasm or whether a diffusion-based mechanism is sufficient. Live-cell imaging showed that PBs were anchored mainly to microtubules. Quantitative single-particle tracking demonstrated that most PBs exhibited spatially confined motion dependent on microtubule motion, whereas stationary PB pairs were identified at the centrosome. Some PBs translocated in long-range movements on microtubules. PB mobility was compared with mitochondria, endoplasmic reticulum, peroxisomes, SMN bodies, and stress granules, and diffusion coefficients were calculated. Disruption of the microtubule network caused a significant reduction in PB mobility together with an induction of PB assembly. However, FRAP measurements showed that the dynamic flux of assembled PB components was not affected by such treatments. FRAP analysis showed that the decapping enzyme Dcp2 is a nondynamic PB core protein, whereas Dcp1 proteins continuously exchanged with the cytoplasm. This study reveals the mechanism of PB transport, and it demonstrates how PB assembly and disassembly integrate with the presence of an intact cytoskeleton.     

Parallel rate heterogeneity in chloroplast and mitochondrial genomes of Brazil nut trees (Lecythidaceae) is consistent with lineage effects

We investigated whether relative rates of divergence were correlated between the mitochondrial and chloroplast genomes as expected under lineage effects or were genome specific as expected with locus-specific effects. Five mitochondrial noncoding regions (nad1B_C, nad4exon1_2, nad7exon2_3, nad7exon3_4, and rps14-cob) for 21 samples from Lecythidaceae were sequenced. Three chloroplast regions (rpl20-5'rps12, trnS-trnG, and psbA-trnH) were sequenced to expand the taxa in an existing data set. Absolute rates of nucleotide and insertion and deletion (indel) changes were 13 times faster in the chloroplast genome than in the mitochondrial genome. Similar indel length frequency distributions for both organelles suggested that common mechanisms were responsible for generating indels. Molecular clock tests applied to phylogenetic trees estimated from mitochondrial and chloroplast sequences revealed global rate heterogeneity of nucleotide substitution. Maximum likelihood and Tajima's 1D relative rate tests show that Lecythis zabucajo exhibited a rate acceleration for both the mitochondrial and chloroplast sequences. Whereas Eschweilera romeu-cardosoi showed a significant rate slowdown for chloroplast sequences, the mitochondrial sequences for 3 Eschweilera taxa showed evidence for a rate slowdown only when compared with L. zabucajo. Significant rate heterogeneity was also observed for indel changes in the mitochondrial genome but not for the chloroplast. The lack of mitochondrial nucleotide changes for some taxa as well as chloroplast indel homoplasy may have limited the power of relative rate tests to detect rate variation. Relative ratio tests consistently indicated rate proportionality among branch lengths between the mitochondrial and chloroplast phylogenetic trees. The relative ratio tests showed that taxa possessing rate heterogeneity had parallel relative divergence rates in both mitochondrial and chloroplast sequences as expected under lineage effects. A neutral replication-dependent model of rate heterogeneity for both nucleotide and indel changes provides a simple explanation for common patterns of rate heterogeneity across the 2 organelle genomes in Lecythidaceae. The lineage effects observed here were uncoupled from annual/perennial habit because all the species from this study are perennial.     

Early nonsurgical correction of congenital auricular deformities

Congenital auricular deformation is not an uncommon phenomenon, and it may cause substantial annoyance to the growing child. Many parents of affected children will seek surgical correction of the deformed auricles. The variety of techniques that have been described for the correction of this anomaly suggests that none has been considered satisfying. The consequent possible surgical complications should also be considered, when the surgical procedure can be replaced by an effective conservative treatment. The authors describe their experience using early splinting for congenital auricular deformities. Fifty-two newborn infants with lop, prominent, Stahl's, and constricted ears referred to us by the neonatal department staff were enrolled in this study. Putty Soft, a vinyl polysiloxane impression material, has been used for early molding of the auricles. Surgical tapes were used for the fixation of the mold and to fix the auricle to the scalp. The results were evaluated by one of the parents and by a layperson (medical student) 6 months after completion of the procedure. The above-described early splinting procedure was applied onto 92 auricles of 52 newborn infants aged 1 to 10 days, mostly around day 3. The mean treatment time was 6.8 weeks. All treated auricles were improved, 87 percent were rated as excellent improvement, and there were no complications related to the treatment. The authors conclude that early splinting of deformed auricles should be offered to parents of affected children, and the awareness of this procedure by neonatologists, pediatricians, and nursery staff should be increased.     

YOS9, the putative yeast homolog of a gene amplified in osteosarcomas,is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins

The OS-9 gene maps to a region (q13-15) of chromosome 12 that is highly amplified in human osteosarcomas and encodes a protein of unknown function. Here we have characterized a homolog designated as YOS9 (YDR057w) from Saccharomyces cerevisiae. The yeast protein (Yos9) is a membrane-associated glycoprotein that localizes to the endoplasmic reticulum (ER). YOS9 interacts genetically with genes involved in ER-Golgi transport, particularly SEC34, whose temperature-sensitive mutant is rescued by YOS9 overexpression. Interestingly, Yos9 appears to play a direct role in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus. Yos9 binds directly to Gas1 and Mkc7 and accelerates Gas1 transport and processing in cells overexpressing YOS9. Correspondingly, Gas1 processing is slowed in cells bearing a deletion in YOS9. No effect upon the transport and processing of non-GPI-anchored proteins (e.g. invertase and carboxypeptidase Y) was detected in cells either lacking or overexpressing Yos9. As Yos9 is not a component of the Emp24 complex, it may act as a novel escort factor for GPI-anchored proteins in ER-Golgi transport in yeast and possibly in mammals.     

Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae

Insertions and deletions (indels) in chloroplast noncoding regions are common genetic markers to estimate population structure and gene flow, although relatively little is known about indel evolution among recently diverged lineages such as within plant families. Because indel events tend to occur nonrandomly along DNA sequences, recurrent mutations may generate homoplasy for indel haplotypes. This is a potential problem for population studies, because indel haplotypes may be shared among populations after recurrent mutation as well as gene flow. Furthermore, indel haplotypes may differ in fitness and therefore be subject to natural selection detectable as rate heterogeneity among lineages. Such selection could contribute to the spatial patterning of cpDNA haplotypes, greatly complicating the interpretation of cpDNA population structure. This study examined both nucleotide and indel cpDNA variation and divergence at six noncoding regions (psbB-psbH, atpB-rbcL, trnL-trnH, rpl20-5'rps12, trnS-trnG, and trnH-psbA) in 16 individuals from eight species in the Lecythidaceae and a Sapotaceae outgroup. We described patterns of cpDNA changes, assessed the level of indel homoplasy, and tested for rate heterogeneity among lineages and regions. Although regression analysis of branch lengths suggested some degree of indel homoplasy among the most divergent lineages, there was little evidence for indel homoplasy within the Lecythidaceae. Likelihood ratio tests applied to the entire phylogenetic tree revealed a consistent pattern rejecting a molecular clock. Tajima's 1D and 2D tests revealed two taxa with consistent rate heterogeneity, one showing relatively more and one relatively fewer changes than other taxa. In general, nucleotide changes showed more evidence of rate heterogeneity than did indel changes. The rate of evolution was highly variable among the six cpDNA regions examined, with the trnS-trnG and trnH-psbA regions showing as much as 10% and 15% divergence within the Lecythidaceae. Deviations from rate homogeneity in the two taxa were constant across cpDNA regions, consistent with lineage-specific rates of evolution rather than cpDNA region-specific natural selection. There is no evidence that indels are more likely than nucleotide changes to experience homoplasy within the Lecythidaceae. These results support a neutral interpretation of cpDNA indel and nucleotide variation in population studies within species such as Corythophora alta.     

Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells

We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.     

Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase

A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide alpha2, 3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.