Chloramphenicol-induced mitochondrial stress increases p21 expression and prevents cell apoptosis through a p21-dependent pathway

Pretreatment of HepG2 and H1299 cells with chloramphenicol rendered the cells resistant to mitomycin-induced apoptosis. Both mitomycin-induced caspase 3 activity and PARP activation were also inhibited. The mitochondrial DNA-encoded Cox I protein, but not nuclear-encoded proteins, was down-regulated in chloramphenicol-treated cells. Cellular levels of the p21(waf1/cip1) protein and p21(waf1/cip1) mRNA were increased through a p53-independent pathway, possibly because of the stabilization of p21(waf1/cip1) mRNA in chloramphenicol-treated cells. The p21(waf1/cip1) was redistributed from the perinuclear region to the cytoplasm and co-localized with mitochondrial marker protein. Several morphological changes and activation of the senescence-associated biomarker, SA beta-galactosidase, were observed in these cells. Both p21(waf1/cip1) antisense and small interfering RNA could restore apoptotic-associated caspase 3 activity, PARP activation, and sensitivity to mitomycin-induced apoptosis. Similar effects were seen with other antibiotics that inhibit mitochondrial translation, including minocycline, doxycycline, and clindamycin. These findings suggested that mitochondrial stress causes resistance to apoptosis through a p21-dependent pathway.     

The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway

The inhibitory Fc receptors function to regulate the antigen-driven activation and expansion of lymphocytes. In B cells, the Fc gammaRIIB1 is a potent inhibitor of B cell antigen receptor (BCR) signaling when coligated to the BCR by engagement of antigen-containing immune complexes. Inhibition is mediated by the recruitment of the inositol phosphatase, SHIP, to the Fc gammaRIIB1 phosphorylated tyrosine-based inhibitory motif (ITIM). Here we show that BCR-independent aggregation of the Fc gammaRIIB1 transduces an ITIM- and SHIP-independent proapoptotic signal that is dependent on members of the c-Abl tyrosine kinase family. These results define a novel Abl family kinase-dependent Fc gammaRIIB1 signaling pathway that functions independently of the BCR in controlling antigen-driven B cell responses.     

Photophysical properties, electronic and crystal structures of luminescent diphosphine digold(I)-pyridine-2-thiolate complexes

Treatment of diphosphine digold(I) complexes, PP(AuCl)₂ [PP=bis(diphenylphosphino)methane, dppm; 1,6-bis(diphenylphosphino)hexane, dpph], with two equivalents of pyridine-2-thiol (HNS) in the presence of NaOCH₃ affords two luminescent diphosphine digold(I)-pyridine-2-thiolate complexes, dppm(AuSN)₂ (1) and dpph(AuSN)₂ (2), respectively. Both crystal structures have been determined by crystallographic studies. The intramolecular aurophilic contact of 3.0478(3) Å is observed in the crystal structure of 1, whereas there is not any aurophilic interaction present in the crystal lattices of 2. At room temperature, 1 shows a low-energy emission at ca. 660 nm as well as a very weak high-energy emission at ca. 496 nm in the solid state, but 2 shows only a strong high-energy one at ca. 482 nm. Thus, the emission energy strongly dependent on the Au(I)?Au(I) interaction can be demonstrated in the diphosphine digold(I) thiolates studied herein.     

The C-terminal sequence of LMADS1 is essential for the formation of homodimers for B function proteins

LMADS1, a lily (Lilium longiflorum) AP3 orthologue, contains the complete consensus sequence of the paleoAP3 (YGSHDLRLA) and PI-derived (YEFRVQPSQPNLH) motifs in the C-terminal region of the protein. Interestingly, through yeast two-hybrid analysis, LMADS1 was found to be capable of forming homodimers. These results indicated that LMADS1 represents an ancestral form of the B function protein, which retains the ability to form homodimers in regulating petal and stamen development in lily. To explore the involvement of the conserved motifs in the C-terminal region of LMADS1 in forming homodimers, truncated forms of LMADS1 were generated, and their ability to form homodimers was analyzed using yeast two-hybrid and electrophoretic mobility shift assay. The ability of LMADS1 to form homodimers decreased once the C-terminal paleoAP3 motif was deleted. When both paleoAP3 and PI-derived motifs were deleted, the ability of LMADS1 to form homodimers was completely abolished. This result indicated that although the paleoAP3 motif promotes the formation of LMADS1 homodimers, the PI-derived motif is essential. Deletion analysis indicated that two amino acids, RV, of the 5 final amino acids, YEFRV, in the PI-derived motif are essential for the formation of homodimers. Further, point mutation analysis indicated that amino acid Val was absolutely necessary, whereas residue Arg played a less important role in the formation of homodimers. Furthermore, Arabidopsis AP3 was able to form homodimers once its C-terminal region was replaced by that of LMADS1. This result indicated that the C-terminal region of LMADS1 is responsible and essential for homodimer formation of the ancestral form of the B function protein.     

Synthesis and antiproliferative evaluations of certain 2-phenylvinylquinoline (2-styrylquinoline) and 2-furanylvinylquinoline derivatives

The present study describes the synthesis of 2-phenylvinylquinoline (styrylquinoline) and 2-furanylvinylquinoline derivatives and evaluation for their antiproliferative activities. (E)-2-Styrylquinolin-8-ol (14a) was inactive against a 3-cell line panel consisting of MCF-7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). Replacement of the phenyl ring with 5-nitrofuran-2-yl group significantly enhanced antiproliferative activity in which (E)-2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-8-ol (14i) and its 4-substituted derivatives 15–19 exhibited strong inhibitory effects against the growth of all three cancer cells. These compounds were further evaluated for their IC₅₀ against the growth of MCF-7, LNCaP, and PC3. Results indicated that a hydrogen bond donating oxime derivative 19a was more active than its hydrogen bond accepting methyloxime derivative 19b. For the inhibition of LNCaP, the potency decreased in an order 14i > 19a > 19b > 15 > 18 > 16. Compound 14i is the most active with an IC₅₀ value of 0.35 and 0.14 μM, respectively, against the growth of LNCaP and PC3 cancer cells. Therefore, compound 14i was evaluated by flow cytometric analysis for its effects on cell cycle distributions. Results indicated that 14i effectively induced cell cycle arrest at S phase for both cell lines, which consequently trigger late apoptosis for both LNCaP and PC3 cells.     

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca²⁺- and Zn²⁺-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca²⁺-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca²⁺/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152–1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca²⁺/CaM with a dissociation constant of 100–300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a “wrapping around” mode of interaction between RUBpep and Ca²⁺/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.     

Can Otolith Microchemistry Reveal Whether the Blind Cave Gudgeon, Milyeringa veritas (Eleotridae), is Diadromous within a Subterranean Estuary?

Synopsis The blind cavefish, Milyeringa veritas, inhabits an anchialine system, effectively a groundwater estuary, in which salinity varies between fresh and seawater at different locations and depths. Owing to the inaccessible habitat and the threatened status of the cavefish it is hard to obtain the biological information needed for their management. This paper explores the utility of otolith Sr:Ca ratio in elucidating cavefish biology. The mean Sr:Ca ratio of the water inhabited by the cavefish is correlated with both the TDS (total dissolved solids) of the habitat and with the Sr:Ca ratio of the sagittal otolith of the cavefish inhabiting that site. Mean values of Sr:Ca in otoliths suggest some cavefish inhabit sea or brackish waters while others remain in freshwater. Some individuals appear to move between waters of very different TDS at various stages but there is no consistency in the direction or apparent TDS range of the water bodies inhabited which indicates that the cavefish utilise the different water bodies opportunistically. Residual analyses indicate clear and routine changes in the TDS of the water occupied at various phases of growth, irrespective of the TDS at which the cavefish were sampled. Annular markings are present in some otoliths but they cannot be related to likely periodicities in the subterranean environment.     

Regulation of microglial activities by glial cell line derived neurotrophic factor

Much attention has been paid to the ability of glial cell line-derived neurotrophic factor (GDNF) to protect neurons from neurotoxic insults in the central nervous system (CNS). However, little is known about GDNF action on CNS glia that also can express GDNF receptor systems. In this study, we examined the effects of GDNF on primary rat microglia that function as resident macrophages in the CNS and as the source of proinflammatory mediators upon activation. We found that treatment of primary rat microglia with GDNF had no effect on the secretion of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), but it increased the nitric oxide (NO) production to some extent. In addition, GDNF increased the enzymatic activity of superoxide dismutase (SOD), the gene expression of surface antigen intercellular adhesion molecule-1 (ICAM-1), the production of the integrin alpha5 subunit, and the phagocytotic capability in primary rat microglia. Furthermore, inhibition of mitogen-activated protein kinase (Erk-MAPK) in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. In vivo evidence also showed that amoeboid cells with integrin alpha5 or with ED1 immunoreactivity appeared in GDNF-treated spinal cord tissues at the lesion site 1 week post spinal cord injury (SCI). Furthermore, inhibition of Erk-MAPK in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. Taken together, our results indicate that GDNF has a positive regulatory effect on microglial activities, such as phagocytosis and the upregulation of adhesion molecules.