Formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus 40 Vp1 in the cytoplasm

Pentamer formation by Vp1, the major capsid protein of simian virus 40, requires an interdigitation of structural elements from the Vp1 monomers [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. (1991) Nature (London) 354, 278-284]. Our analyses reveal that disulfide-linked Vp1 homooligomers are present in the simian virus 40-infected cytoplasm and that they are derived from a 41-kDa monomeric intermediate containing an intrachain disulfide bond(s). The 41-kDa species, emerging within 5 min of pulse labeling with [(35)S]methionine, is converted into a 45-kDa, disulfide-free Vp1 monomer and disulfide-bonded dimers through pentamers. The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryl-disulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm.     

Analysis of malaria endemic areas on the Indochina Peninsula using remote sensing

We applied remote sensing using satellite images capable of obtaining data over a broad range, transcending national borders, as a method of rapidly, precisely, and safely increasing our understanding of the potential distribution of malaria. Our target region was the so-called Mekong malaria region on the Indochina Peninsula. As a malaria index, we used existing distribution maps of total reported malaria cases, malaria mortality, vivax malaria and falciparum malaria incidences, and so forth for 1997 and 1998. We produced monthly distribution maps of a normalized difference vegetation index (NDVI) with values of 0.2+, 0.3+, 0.35+, and 0.4+ using the geographical information system/remote sensing software based on the East Asia monthly NDVI maps of 1997. These maps were overlaid with various malaria index distribution maps, and cross-tabulations were carried out. The resulting maps with NDVI values of 0.3+ and 0.4+ matched the falciparum malaria distribution well, and we realized, in particular, that falciparum malaria is prevalent in regions in which NDVI values of 0.4+ continue for 6 months or more, while cases are fewer in regions with NDVI values of 0.4+ that continue for 5 months or less. It will be necessary in the future to examine the relationship between NDVI values and the habitats of the various vector mosquitoes using high-resolution satellite images and to implement detailed forecasts for malaria endemic areas by means of NDVI.     

Potential roles of extracellular signal-regulated kinase but not p38 during myeloid differentiation of U937 cells stimulated by cytokines: augmentation of differentiation via prolonged activation of extracellular signal-regulated kinase

OBJECTIVE: To clarify the signaling mechanism of human myeloid differentiation by hematopoietic growth factors and cytokines, we investigated the role of extracellular signal-regulated kinase (ERK) during the differentiation of human monoblastic U937 cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF). MATERIALS AND METHODS: Myeloid differentiation was evaluated by morphology, function (respiratory burst activity), and cell surface expression of adhesion molecule (CD11b), and activation of ERK and/or p38 was determined by Western blotting and/or in vitro kinase assay. Inhibition of the ERK pathway was performed using PD98059, a specific inhibitor of this pathway. RESULTS: U937 cells were induced to be differentiated by the combination of GM-CSF and TNF, but only minimally by either cytokine alone. Transient phosphorylation and activation of ERK was induced by both GM-CSF alone and combination of the two cytokines, whereas sustained phosphorylation and activation was induced only by the combination. In addition, PD98059, a specific inhibitor of ERK pathway, almost completely abolished this prolonged phosphorylation of ERK and completely blocked differentiation. In contrast, both TNF alone and cytokine combination equivalently phosphorylated p38 in U937 cells, which was dissociated from differentiation, and a specific inhibitor of p38 (SB203580) did not inhibit differentiation. CONCLUSIONS: The results indicate potential roles of sustained activation of ERK but not of p38 in the signaling pathways for human myeloid differentiation in U937 cells synergistically stimulated by the two physiologic cytokines GM-CSF and TNF.     

Confirmation by FRET in individual living cells of the absence of significant amyloid beta -mediated caspase 8 activation

When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.     

Diverse p53 gene aberration in hepatocellular carcinoma detected by dual-color fluorescence in situ hybridization

BACKGROUND AND AIM: In order to evaluate loss of the p53 gene more precisely, we performed dual-color fluorescence in situ hybridization (dual-color FISH) for chromosome 17 and p53 gene together with DNA polymorphism analysis of the p53 gene in hepatocellular carcinoma (HCC). METHODS: Dual-color FISH using probes specific for the centromere of chromosome 17 and the p53 gene was performed for 41 HCC and DNA polymorphism analysis was also performed for them. RESULTS: Of the 34 HCC tested by dual-color FISH, 20 had loss of at least one p53 gene (58.8%). In contrast, of the 32 HCC tested by DNA polymorphism analysis, 23 gave informative results, among which only eight had loss of heterozygosity (LOH) of the p53 gene (34.8%). Notably, among 14 cases positive for loss of the p53 gene by dual-color FISH, seven cases were negative for LOH of the p53 gene. Moreover, dual-color FISH revealed that the percentage of cells that lost at least one p53 gene increased as the HCC became less differentiated (P < 0.01), whereas LOH did not reveal any such correlation. CONCLUSIONS: These data suggest that loss of the p53 gene was present in a considerable number of HCC, and diversity of the p53 gene aberration increases with progression of HCC. Dual-color FISH is an effective method for detection of p53 gene aberration, and it can provide new insight into oncogenesis in HCC.     

Targeted gene expression without a tissue-specific promoter: Creating mosaic embryos using laser-induced single-cell heat shock

We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of β-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.     

Loss of PTEN expression is associated with colorectal cancer liver metastasis and poor patient survival

Background  

The tumour suppressor phosphatase and tensin homolog (PTEN) is an important negative regulator of cell-survival signaling. To evaluate the correlation between PTEN expression and clinicopathological characteristics of colorectal cancer patients with and without liver metastases, we investigated PTEN expression in primary colorectal cancer and colorectal cancer liver metastases.     

New tumor necrosis factor-α-inducing protein released from<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> for gastric cancer progression

Purpose To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor- (TNF-)-inducing protein of H. pylori was studied.Methods The HP0596 gene of H. pylori was identified as the TNF--inducing protein (Tip) gene from genome sequence of H. pylori strain 26695. Using recombinant Tip (rTip) and deleted Tip (rdel-Tip) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF- gene expression and NF-B activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells.Results Tip protein as a homodimer form (38 kDa) was found in both extracts and culture medium of various H. pylori strains. rTip significantly induced TNF- gene expression and NF-B activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tip did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-B p65, and abrogated TNF- induction induced by Tip protein.Conclusion Tip is a new carcinogenic factor released from H. pylori mediated through NF-B activation.     

Molecular mimicry of mitochondrial and nuclear autoantigens in primary biliary cirrhosis

BACKGROUND & AIMS: The mechanism for development of primary biliary cirrhosis (PBC) remains enigmatic, but molecular mimicry has been implicated because of well-known cross-reactivity of human mitochondrial autoantigens and equivalent bacterial antigens. Virtually all patients with PBC have antimitochondrial autoantibodies (AMA), but, interestingly, approximately 50% also manifest antinuclear antibodies (ANA). METHODS: To determine whether generation of ANA are due to molecular mimicry of mitochondrial peptides, we established 6 T-cell clones selected by a peptide corresponding to the E2 subunit of mitochondrial pyruvate dehydrogenase complex and analyzed for reactivity to mimicry peptides derived from mitochondrial and nuclear autoantigens, including control sequences. RESULTS: For mitochondrial autoantigens, 1 peptide from the E2 subunit of the pyruvate dehydrogenase complex, 1 peptide from the E2 subunit of the oxo-glutarate dehydrogenase complex, 1 peptide from the E2 subunit of the branched-chain 2-oxoacid dehydrogenase complex, and 1 peptide from the E3-binding protein cross-reacted with these T-cell clones. For the nuclear autoantigens, 5 peptides from gp210 and 1 from Sp100 cross-reacted with these clones. Furthermore, 1 of 3 T-cell clones selected by recombinant gp210 protein reacted with a mimicry peptide corresponding to amino acids 188-201 of gp210, indicating that this part of the protein is a naturally processed immunodominant T-cell epitope. CONCLUSIONS: These results demonstrate molecular mimicry between mitochondrial and nuclear autoantigens in PBC and that a mimicry peptide may become an immunodominant T-cell epitope. These data have significance not only for PBC but also for the production of ANA in other disease processes.