Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense bcl-2 oligodeoxynucleotides enhanced the sensitivity of leukemia cells to arsenic trioxide. A combination of arsenic trioxide with antisense bcl-2 oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced bcl-2 protein expression in K562 and NB4 leukemic cells more significantly than either arsenic trioxide or the oligodeoxynucleotides on their own (P<0.01). Thus, bcl-2 antisense oligodeoxynucleotides increase the sensitivity of leukemic cells to arsenic trioxide. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.     

Layer-by-layer assembly to modify poly(l-lactic acid) surface toward improving its cytocompatibility to human endothelial cells

A novel technique to introduce free amino groups onto polyester scaffolds via aminolyzing the ester groups with diamine has been developed recently. Positively charged chitosan was then deposited onto the aminolyzed poly(l-lactic acid) (PLLA) membrane surface in a layer-by-layer assembly manner using poly(styrene sulfonate, sodium salt) (PSS) as a negatively charged polyelectrolyte. The layer-by-layer deposition process of PSS and chitosan was monitored by UV-vis absorbance spectroscopy, energy transfer by fluorescence spectroscopy, and advancing contact angle measurements. The existed chitosan obviously improved the cytocompatibility of PLLA to human endothelial cells. The cell attachment, activity, and proliferation on the PLLA membranes assembled with three or five bilayers of PSS/chitosan with chitosan as the outermost layer were better than those with one bilayer of PSS/chitosan or the control PLLA. The cells also showed morphology of an elongated shape with abundant cytoplasm, and a confluent cell layer was reached after being cultured for 4 days. Measurement of von Willebrand factor secreted by these endothelial cells (ECs) verified the endothelial function. Hence, better ECs compatible PLLA were produced.     

Site-selective lateral multilayer assembly of bienzyme with polyelectrolyte on ITO electrode based on electric field-induced directly layer-by-layer deposition

We describe here a new approach to construct a multilayer enzyme/polyelectrolyte film on a structured transparent indium-tin oxide (ITO) covered glass electrode surface as micropattern, on which two different types of enzyme distributed laterally on one common substrate without interference. The multilayer film was prepared by alternate electric field directed layer-by-layer assembly deposition and alternate deposition of different redox enzymes and polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA) onto the site-selective ITO glass electrode surface. The cyclic voltammogram, obtained from the ITO glass electrode modified with the glucose oxidase (GO(X))/PDDA and catalase (CA(T))/PDDA multilayers, revealed that the bioelectrocatalytic response is directly correlated to the number of deposition bilayers. From the analysis of cyclic voltammetric characterization, the coverage of catalytically active enzymes per enzyme/PDDA bilayer during the multilayer formation was homogeneous, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, from the atomic force microscopy and Brewster angle microscopy measurements, more information of the multilayer constructed by different methods on the modified electrode surface is obtained and compared. This fabrication technique is simple and would be applicable to the construction of a thickness- and area-controlled biopattern composed of multi-enzymes as well as multiple biomaterials.     

Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock

FREQUENCY (FRQ), a key component of the Neurospora circadian clock, is progressively phosphorylated after its synthesis. Previously, we identified casein kinase II (CKII) as a kinase that phosphorylates FRQ. Disruption of the catalytic subunit of CKII abolishes the clock function; it also causes severe defects in growth and development. To further establish the role of CKII in clock function, one of the CKII regulatory subunit genes, ckb1, was disrupted in Neurospora. In the ckb1 mutant strain, FRQ proteins are hypophosphorylated and more stable than in the wild-type strain, and circadian rhythms of conidiation and FRQ protein oscillation were observed to have long periods but low amplitudes. These data suggest that phosphorylation of FRQ by CKII regulates FRQ stability and the function of the circadian feedback loop. In addition, mutations of several putative CKII phosphorylation sites of FRQ led to hypophosphorylation of FRQ and long-period rhythms. Both CKA and CKB1 proteins are found in the cytoplasm and in the nucleus, but their expressions and localization are not controlled by the clock. Finally, disruption of a Neurospora casein kinase I (CKI) gene, ck-1b, showed that it is not required for clock function despite its important role in growth and developmental processes. Together, these data indicate that CKII is an important component of the Neurospora circadian clock.     

Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer

The mechanism of DNA hypermethylation-associated tumor suppressor gene silencing in cancer remains incompletely understood. Here, we show by chromatin immunoprecipitation that for three genes (P16, MLH1, and the O(6)-methylguanine-DNA methyltransferase gene, MGMT), histone H3 Lys-9 methylation directly correlates and histone H3 Lys-9 acetylation inversely correlates with DNA methylation in three neoplastic cell lines. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in moderately increased Lys-9 acetylation at silenced loci with no effect on Lys-9 methylation and minimal effects on gene expression. By contrast, treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC) rapidly reduced Lys-9 methylation at silenced loci and resulted in reactivation for all three genes. Combined treatment with 5Aza-dC and TSA was synergistic in reactivating gene expression through simultaneous effects on Lys-9 methylation and acetylation, which resulted in a robust increase in the ratio of Lys-9 acetylated and methylated histones at loci showing dense DNA methylation. By contrast to Lys-9, histone H3 Lys-4 methylation inversely correlated with promoter DNA methylation, was not affected by TSA, and was increased moderately at silenced loci by 5Aza-dC. Our results suggest that reduced H3 Lys-4 methylation and increased H3 Lys-9 methylation play a critical role in the maintenance of promoter DNA methylation-associated gene silencing in colorectal cancer.     

Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis

Numb and Numblike, conserved homologs of Drosophila Numb, have been implicated in cortical neurogenesis; however, analysis of their involvement in later stages of cortical development has been hampered by early lethality of double mutants in previous studies. Using Emx1(IREScre) to induce more restricted inactivation of Numb in the dorsal forebrain of numblike null mice beginning at E9.5, we have generated viable double mutants that displayed striking brain defects. It was thus possible to examine neurogenesis during the later peak phase (E12.5-E16.5). Loss of Numb and Numblike in dorsal forebrain resulted in neural progenitor hyperproliferation, delayed cell cycle exit, impaired neuronal differentiation, and concomitant defects in cortical morphogenesis. These findings reveal novel and essential function of Numb and Numblike during the peak period of cortical neurogenesis. Further, these double mutant mice provide an unprecedented viable animal model for severe brain malformations due to defects in neural progenitor cells.     

Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228)

Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.     

Microbial transformation of baccatin VI and 1beta-hydroxy baccatin I by Aspergillus niger

The biotransformation of baccatin VI (1) and 1β-hydroxybaccatin I (2) with the filamentous fungus Aspergillus niger produced four new taxane diterpenoids taxumairol S₁ (3), taxumairol T₁ (4) and taxumairol S (5), taxumairol T (6), respectively. 1β-Dehydroxybaccatin VI (7) remained unreacted under the same condition.