A novel chromosome region maintenance 1-independent nuclear export signal of the large form of hepatitis delta antigen that is required for the viral assembly

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus, as it requires hepatitis B virus for virion production and transmission. We have previously demonstrated that sequences within the C-terminal 19-amino acid domain flanking the isoprenylation motif of the large hepatitis delta antigen (HDAg-L) are important for virion assembly. In this study, site-directed mutagenesis and immunofluorescence staining demonstrated that in the absence of hepatitis B virus surface antigen (HBsAg), the wild-type HDAg-L was localized in the nuclei of transfected COS7 cells. Nevertheless, in the presence of HBsAg, the HDAg-L became both nuclei- and cytoplasm-distributed in about half of the cells. An HDAg-L mutant with a substitution of Pro-205 to alanine could neither form HDV-like particles nor shift the subcellular localization in the presence of HBsAg. In addition, nuclear trafficking of HDAg-L in heterokaryons indicated that HDAg-L is a nucleocytoplasmic shuttling protein. A proline-rich HDAg peptide spanning amino acid residues 198 to 210, designated NES(HDAg-L), can function as a nuclear export signal (NES) in Xenopus oocytes. Pro-205 is critical for the NES function. Furthermore, assembly of HDV is insensitive to leptomycin B, indicating that the NES(HDAg-L) directs nuclear export of HDAg-L to the cytoplasm via a chromosome region maintenance 1-independent pathway.     

Oxygen- and growth rate-dependent regulation of Escherichia coli fumarase (FumA, FumB, and FumC) activity

Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.     

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.     

G Protein beta subunit types differentially interact with a muscarinic receptor but not adenylyl cyclase type II or phospholipase C-beta 2/3

In comparison with the alpha subunit of G proteins, the role of the beta subunit in signaling is less well understood. During the regulation of effectors by the betagamma complex, it is known that the beta subunit contacts effectors directly, whereas the role of the beta subunit is undefined in receptor-G protein interaction. Among the five G protein beta subunits known, the beta(4) subunit type is the least studied. We compared the ability of betagamma complexes containing beta(4) and the well characterized beta(1) to stimulate three different effectors: phospholipase C-beta2, phospholipase C-beta3, and adenylyl cyclase type II. beta(4)gamma(2) and beta(1)gamma(2) activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting alpha(o)beta(4)gamma(2) was stimulated significantly more by the M2 muscarinic receptor compared with alpha(o)beta(1)gamma(2). Because alpha(o) forms heterotrimers with beta(4)gamma(2) and beta(1)gamma(2) equally well, these results show that the beta subunit type plays a direct role in the receptor activation of a G protein.     

Interferon gamma (IFNgamma ) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways

We investigated the molecular mechanism of the synergism between interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNgamma/TNFalpha synergistically induced apoptosis of ME-180 cervical cancer cells. IFNgamma induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNgamma/TNFalpha-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFalpha. Dominant-negative IkappaBalpha transfection sensitized ME-180 cells to TNFalpha. IFNgamma pretreatment attenuated TNFalpha- or p65-induced NF-kappaB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-kappaB. IRF-1 transfection alone inhibited TNFalpha-induced NF-kappaB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNgamma/TNFalpha combination; however, caspase inhibition did not abrogate IFNgamma/TNFalpha-induced cell death. Instead, caspase inhibitors directed IFNgamma/TNFalpha-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNgamma and TNFalpha synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNgamma play a critical role in IFNgamma/TNFalpha synergism by inhibiting cytoprotective NF-kappaB. IFNgamma/TNFalpha synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.     

Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.     

Differential regulation of interleukin-12 and interleukin-10 by heat shock response in murine peritoneal macrophages

Heat shock response is a conserved stress response and has been shown to have anti-inflammatory effects. We investigated the effect of heat shock response on LPS-induced production of IL-12 and IL-10, which are two important cytokines playing contradictory roles in regulation of immune response, by murine peritoneal macrophages. The data showed that induction of heat shock response strongly suppressed LPS-induced production of IL-12 while augmented that of IL-10, suggesting the pleiotropic effects of heat shock response on immune regulatory gene expression. Also, the novel observation on up-regulation of IL-10 by heat shock response adds to the mechanism by which heat shock response exerts its anti-inflammatory effects.     

Electron transfer in the substrate-dependent suicide inactivation of lysine 5,6-aminomutase

The lysine 5,6-aminomutase (5,6-LAM) purified from Clostridium sticklandii was found to undergo rapid inactivation in the absence of the activating enzyme E(2) and ATP. In the presence of substrate, inactivation was also seen for the recombinant 5,6-LAM. This adenosylcobalamin-dependent enzyme is postulated to generate cob(II)alamin and the 5'-deoxyadenosyl radical through enzyme-induced homolytic scission of the Co-C bond. However, the products cob(III)alamin and 5'-deoxyadenosine were observed upon inactivation of 5,6-LAM. Cob(III)alamin production, as monitored by the increase in A(358), proceeds at the same rate as the loss of enzyme activity, suggesting that the activity loss is related to the adventitious generation of cob(III)alamin during enzymatic turnover. The cleavage of adenosylcobalamin to cob(III)alamin is accompanied by the formation of 5'-deoxyadenosine at the same rate, and the generation of cob(III)alamin proceeds at the same rate both aerobically and anaerobically. Suicide inactivation requires the presence of substrate, adenosylcobalamin, and PLP. We have ruled out the involvement of either the putative 5'-deoxyadenosyl radical or dioxygen in suicide inactivation. We have shown that one or more reaction intermediates derived from the substrate or/and the product, presumably a radical, participate in suicide inactivation of 5,6-LAM through electron transfer from cob(II)alamin. Moreover, L-lysine is found to be a slowly reacting substrate, and it induces inactivation at a rate similar to that of D-lysine. The alternative substrate beta-lysine induces inactivation at least 25 times faster than DL-lysine. The inactivation mechanism is compatible with the radical isomerization mechanism proposed to explain the action of 5,6-LAM.