Heregulin promotes expression and subcellular redistribution of ADP-ribosylation factor 3

To identify genes whose expression is modulated by heregulin-beta1 (HRG), a regulatory polypeptide for mammary epithelial cells, we performed differential display screening of MCF-7 cell mRNA. One cDNA clone upregulated by HRG was identical to human ADP-ribosylation factor 3 (ARF3), a guanine nucleotide-binding protein functioning in vesicular trafficking, phospholipase D activation and intracellular transport. HRG treatment increased expression of ARF3 mRNA and protein. Also, HRG triggered a rapid redistribution of ARF3, first to cell membranes and then to the nuclear compartment, where ARF3 colocalized with acetylated histone H3 in discrete regions. In addition, the ARF3 protein was developmentally regulated in the mammary gland with the highest levels in virgin and post-weaning glands. Together, these findings suggest for the first time that stimulation of ARF3 expression, subcellular redistribution and interaction with acetylated histone H3 may play a role in the action of HRG in mammary epithelial cells.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can trigger apoptosis in some tumor cells but not other tumor cells. To explore the signal transduction events in TRAIL-triggered apoptosis and its modulation in nontransfected tumor cells, we analyzed TRAIL-induced death-inducing signaling complex (DISC) in TRAIL-sensitive and -resistant glioma cells. Caspase-8 and caspase-10 were recruited to the DISC, where they were proteolytically activated to initiate apoptosis in TRAIL-sensitive glioma cells. Caspase-8 and caspase-10 were also recruited to the DISC in TRAIL-resistant cells, but their further activation was inhibited by two antiapoptotic proteins termed cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15kDa (PED/PEA-15). Both long and short forms of c-FLIP were recruited to the DISC, where the long form c-FLIP was cleaved to produce intermediate fragments. Of the three isoforms of PED/PEA-15 proteins, only the doubly phosphorylated form was expressed and recruited to the DISC in TRAIL-resistant cells, indicating that the phosphorylation status of PED/PEA-15 determines its recruitment in the cells. Treatment with calcium/calmodulin-dependent protein kinase inhibitor rescued TRAIL sensitivity in TRAIL-resistant cells, providing a potential new approach to sensitize the cells to TRAIL-induced apoptosis.     

YfcX enables medium-chain-length poly(3-hydroxyalkanoate) formation from fatty acids in recombinant Escherichia coli fadB strains

Expression of Escherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional beta-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHA(MCL)s when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB(-) YfcX(-) PhaC(+) strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.     

Caspase proteolysis of the cohesin component RAD21 promotes apoptosis

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.     

Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients

We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.     

Identification of Taenia asiatica in China: molecular,morphological, and epidemiological analysis of a Luzhai isolate

Multiple analysis has characterized a recently described tapeworm of people, Taenia asiatica, in mainland China. Six adult tapeworms collected from people of the Zhuang minority residing in the southern part of China (Luzhai isolate) were comparatively analyzed with other tapeworms from people: T. asiatica (n = 2, South Korea), T. saginata (n = 1, Poland; n = 1, Korea), and T. solium (n = 1, People's Republic of China). Experimental infections with eggs from the Luzhai isolate in pigs and cattle produced cysticerci, each with a hookletless scolex and with wartlike formations on the external surface of the bladder wall. There were rostellar protrusions in the scolices of adult worms. Random amplified polymorphic DNA analysis using 3 arbitrary primers produced bands identical to those of the Korean T. asiatica. Conversely, T. saginata and T. solium exhibited different banding patterns. Phylogenetic relationships inferred from the complete nucleotide sequences of the internal transcribed spacer 2 placed the Chinese tapeworms consistently within the T. asiatica clade by 96% bootstrapping value in the maximum likelihood analysis, 96% in maximum parsimony, and 100% in neighbor joining. These collective data demonstrate that T. asiatica is sympatrically distributed with the other 2 species of Taenia in the human host in mainland China.     

Sepsis inhibits reduction of dehydroascorbic acid and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition

Sepsis is associated with oxidative stress and impaired glutamatergic transmission in brain. We investigated whether sepsis impairs accumulation of the antioxidant, ascorbate, and uptake of glutamate by astrocytes. Bacterial endotoxin (Escherichia coli lipopolysaccharide, LPS) and the inflammatory cytokine, interferon-gamma (IFNgamma), were applied to primary astrocyte cultures to model sepsis. In the absence of ascorbate, the combination of LPS and IFNgamma (LPS + IFNgammay) up-regulated inducible nitric oxide synthase (iNOS) and decreased the initial rate of glutamate uptake by 50% within 24 h. Cell viability and facilitated glucose transport activity were not affected at 24 h. Pre-treatment with ascorbate-2-O-phosphate increased intracellular ascorbate concentration and attenuated the induction of iNOS and inhibition of glutamate uptake caused by LPS + IFNgamma. Subsequent experiments examined the mechanisms by which cells accumulate ascorbate. LPS + IFNy decreased slightly the initial rate of uptake of ascorbate and inhibited markedly the rate with which intracellular dehydroascorbic acid (DHAA) was reduced to ascorbate. We conclude that septic insult impairs astrocytic clearance of DHAA from the extracellular fluid and decreases intracellular ascorbate concentration. Furthermore, sepsis induces iNOS and inhibits glutamate uptake by astrocytes through mechanisms that can be modulated by intracellular ascorbate. These results indicate treatments that increase intracellular ascorbate concentration may be beneficial for patients at risk for neurologic complication in sepsis.     

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.