Glioma invasion mediated by the p75 neurotrophin receptor (p75(NTR)/CD271) requires regulated interaction with PDLIM1.

The invasive nature of glioblastoma renders them incurable by current therapeutic interventions. Using a novel invasive human glioma model, we previously identified the neurotrophin receptor p75(NTR) (aka CD271) as a mediator of glioma invasion. Herein, we provide evidence that preventing phosphorylation of p75(NTR) on S303 by pharmacological inhibition of PKA, or by a mutational strategy (S303G), cripples p75(NTR)-mediated glioma invasion resulting in serine phosphorylation within the C-terminal PDZ-binding motif (SPV) of p75(NTR). Consistent with this, deletion (ΔSPV) or mutation (SPM) of the PDZ motif results in abrogation of p75(NTR)-mediated invasion. Using a peptide-based strategy, we identified PDLIM1 as a novel signaling adaptor for p75(NTR) and provide the first evidence for a regulated interaction via S425 phosphorylation. Importantly, PDLIM1 was shown to interact with p75(NTR) in highly invasive patient-derived glioma stem cells/tumor-initiating cells and shRNA knockdown of PDLIM1 in vitro and in vivo results in complete ablation of p75(NTR)-mediated invasion. Collectively, these data demonstrate a requirement for a regulated interaction of p75(NTR) with PDLIM1 and suggest that targeting either the PDZ domain interactions and/or the phosphorylation of p75(NTR) by PKA could provide therapeutic strategies for patients with glioblastoma.

ING1 induces apoptosis through direct effects at the mitochondria.

The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1,interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis [corrected].

S100A2 promoter-driven conditionally replicative adenovirus targets non-small-cell lung carcinoma.

S100A2, a member of the S100 family of calcium-binding proteins, has been implicated in carcinogenesis as both a tumor suppressor and stimulator. Here, we characterized promoter activity of S100A2, generated an S100A2 promoter-driven conditionally replicative adenovirus (Ad/SA), and evaluated its anti-tumor activity in vitro and in vivo. Promoter activity of S100A2 was greatly restricted to tumor cells, and the S100A2 promoter bound with typical nuclear targets of epidermal growth factor receptor (EGFR) signaling. EGF-stimulated EGFR phosphorylation induced S100A2 expression and further activated E1A expression of Ad/SA, which was restored by EGFR signal inhibition in a concentration-dependent manner in non-small-cell lung carcinoma (NSCLC). In two EGFR-activated tumor xenograft animal models, Ad/SA exhibited potent anti-tumor activity, whereas cetuximab, an EGFR-targeting anticancer drug, was active transiently or ineffective. Combined treatment with cetuximab or cisplatin plus Ad/SA resulted in enhanced anti-tumor activity. Immunohistochemical analysis of tumor sections showed moderate-to-high grade signals for EGFR and adenovirus, and a reduction in viable cells in Ad/SA-treated tumors. Collectively, these results demonstrate that the S100A2 promoter-driven adenovirus is a potent inhibitor of cancers, and further suggest that S100A2 is a target gene of EGFR signaling pathway in NSCLC.

The study of new vacuum pressure measurement technique using ultrasonic acoustic impedance transducers.

Ultrasonic sensors of 500 kHz frequency have been used to study the measuring of gas pressure in the range 1.33 × 10(3)-2.026 × 10(5) Pa. From the experimental data it was observed that amplitude of the transmitted ultrasound was dependent on gas pressure. The results confirm that by using appropriate instruments including sensors and electronic devices, pressure of the gas can be successfully measured by measuring the magnitude of the received signal. The proposed method is expected to be applied to develop as new vacuum pressure measurement instrument.

Tid1 is a new regulator of p53 mitochondrial translocation and apoptosis in cancer.

The p53 tumor suppressor protein induces apoptosis in response to genotoxic and environmental stresses. Recent studies have revealed the existence of a transcription-independent mitochondrial p53 apoptotic pathway; however, the mechanism that regulates its translocation to the mitochondria has been unknown. In this study, we show that the tumor suppressor Tid1 forms a complex with p53 under hypoxic conditions that directs p53 translocation to the mitochondria and the subsequent initiation of the mitochondrial apoptosis pathway. Loss of Tid1 expression abrogated p53 translocation to the mitochondria and inhibited apoptosis, whereas the over-expression of Tid1 promoted p53 mitochondrial localization and apoptosis. Tid1's mitochondrial signal sequence and DnaJ domain were both required for the movement of the p53-Tid1 complex from the cytosol to the mitochondria. When Tid is over-expressed in cancer cell lines expressing mutant p53 isoforms defective in transcriptional activity, mitochondrial localization and pro-apoptotic activities of the mutant p53 proteins was restored. Our results establish Tid1 as a novel regulator of p53-mediated apoptosis, and suggest that therapies designed to enhance Tid1's function in promoting mitochondrial localization of p53 and apoptosis could be an effective therapy in many cancers.

A mitochondrial DNA variant at position 16189 is associated with type 2 diabetes mellitus in Asians.

AIMS/HYPOTHESIS: This multinational study was conducted to investigate the association between a mitochondrial DNA (mtDNA) T16189C polymorphism and type 2 diabetes in Asians. The mtDNA 16189C variant has been reported to be associated with insulin resistance and type 2 diabetes. However, a recent meta-analysis concluded that it is negatively associated with type 2 diabetes in Europids. Since the phenotype of an mtDNA mutant may be influenced by environmental factors and ethnic differences in the nuclear and mitochondrial genomes, we investigated the association between the 16189C variant and type 2 diabetes in Asians. METHODS: The presence of the mtDNA 16189C variant was determined in 2,469 patients with type 2 diabetes and 1,205 non-diabetic individuals from Korea, Japan, Taiwan, Hong Kong and China. An additional meta-analysis including previously published Asian studies was performed. Since mtDNA nucleotide position 16189 is very close to the mtDNA origin of replication, we performed DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry and chromatin immunoprecipitation to identify protein bound to the 16189 region. RESULTS: Analysis of participants from five Asian countries confirmed the association between the 16189C variant and type 2 diabetes [odds ratio (OR) 1.256, 95% CI 1.08-1.46, p=0.003]. Inclusion of data from three previously published Asian studies (type 2 diabetes n=3,283, controls n=2,176) in a meta-analysis showed similar results (OR 1.335, 95% CI 1.18-1.51, p=0.000003). Mitochondrial single-stranded DNA-binding protein (mtSSB) was identified as a candidate protein bound to the 16189 region. Chromatin immunoprecipitation in cybrid cells showed that mtSSB has a lower binding affinity for the 16189C variant than the wild-type sequence. CONCLUSIONS/INTERPRETATION: The mtDNA 16189C variant is associated with an increased risk of type 2 diabetes in Asians.

Long-term egg-yolk adaptation of the Orientia tsutsugamushi for preparation of a formalinized immunogen.

To assess the feasibility of preparing a killed rickettsial vaccine against the scrub typhus, the Karp and Gilliam strains of the Orientia tsutsugamushi were adapted through sequential passages in eggs for more than 100 times over 9 years to produce approximately five times more antigen than the unadapted rickettsia. The egg-grown rickettsia was purified by differential centrifugation and batch-type ion-exchange chromatography, and inactivated by formalin treatment. Strong protective immunity was acquired against lethal challenges of the homologous strains and lasted fully for longer than 8 months in the C3H/He mice immunized with either of the single-strain immunogen or in combination of the two strain immuinogens. However, neither immunogen protected animals from the challenges with the two heterologous strains or Boryong, the prevalent strain in Korea, despite that three antigenic proteins of 47, 56, and 110 kDa were eminent in both preparations. IgM, IgG, and neutralizing antibody were induced in the immunized mice in a level and pattern comparable to that in animals infected with live rickettsiae.

Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells.

We have developed a cell culture-derived, inactivated vaccine against Hantaan virus for prevention of the hemorrhagic fever with renal syndrome (HFRS). Hantaan virus was purified from a microcarrier culture of Vero E6 cells by ultrafiltration and density gradient centrifugation. Viral infection was inactivated by treatment of the viral stock with formaldehyde. Immunogenic properties of the vaccine were characterized in comparison with Hantavax, a mouse brain-derived, formalin-inactivated vaccine that has been in human use for a decade in Korea. Compared to the Hantavax, immunization of Balb/c mice with the cell culture-based vaccine resulted in a moderate difference in antibody response to the viral nucleocapsid protein but more than five-fold increase in neutralizing activity. Moreover, all six mice immunized with 5 microg of the cell culture-based vaccine were fully protected from challenge with infectious virus, whereas virus was detected in lung and spleen of all animals immunized with the same dose of Hantavax. Four times higher dose of the latter vaccine was needed for complete protection. In the analysis of the humoral immune response to the vaccines, we found that all three viral structural proteins, N, G1 and G2 were immunoprecipitated by sera from animals immunized with the cell culture-based vaccine. In contrast, N and some G1 but no G2 were precipitated by the sera from animals immunized with Hantavax. These results suggest that the cell culture-based vaccine can provide more effective immunity than the Hantavax.

A dominant antigenic region of the hantaan virus nucleocapsid protein is located within a amino-terminal short stretch of hydrophilic residues.

The nucleocapsid (N) protein of the Hantaan virus (HTNV) is a major viral antigen that induces a strong antibody response during the acute phase of infection. By immunoblot analyses of the recombinant N proteins using human sera of the hemorrhagic fever with renal syndrome (HFRS), we have confirmed previous finding by other investigators of the presence of a highly antigenic region near the amino terminus of the HTNV N protein. We have further located the antigenic region within a short stretch of hydrophilic sequences between the 26 and the 46th amino acid residues. The recombinant glutathione S-transferase fusion proteins containing this region was expressed as a soluble form in a large quantity in Escherichia coli, and purified by a single-step affinity chromatography. The recombinant antigen also showed a similar, but a weaker reactivity with human antisera to Seoul virus (SEOV), the virus most closely related to HTNV.

Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability.

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify amino acid residues essential for the thermostability of the SOD from Aquifex pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T(1/2) value of K12A was 210 min and that of the wild type was 175 min at 95 degrees C. However, the thermostability of the mutant E41A, which has a T(1/2) value of 25 min at 95 degrees C, was significantly decreased compared to the wild type of ApSOD. To explain the enhanced thermostability of K12A and thermolabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general significance of hydrogen bonds and ion-pair network in the thermostability of proteins.

Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium.

A DNA ligase gene from the hyperthermophilic bacterium Aquifex pyrophilus (Ap) was cloned and sequenced. An open reading frame of 2,157 bp that codes for a 82-kDa protein showed 40%-60% homology with a series of NAD+-dependent DNA ligases from different organisms. The recombinant enzyme Ap DNA ligase expressed in Escherichia coli was purified to homogeneity and characterized. The activity of Ap DNA ligase gradually increased in proportion to the concentration of monovalent salt up to 200 mM NaCl, 150 mM KCl, 200 mM NH4Cl, and 350 mM potassium glutamate. The optimum temperature and pH of Ap DNA ligase were greater than 65 degrees C and 8.0-8.6, respectively, for nick-closing activity. More than 75% of the ligation activity was retained after incubation at 95 degrees C for 60 min, whereas the half-lives of Thermus aquaticus and Escherichia coli DNA ligases at 95 degrees C were < or =15 min and 5 min, respectively. Thermostable Ap DNA ligase was applied to repeat expansion detection (RED) and could be a useful enzyme in DNA diagnostics.

Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity.

We constructed nine deletion mutants of NAD+-dependent DNA ligase from Aquifex pyrophilus to characterize the functional domains. All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD+-dependent self-adenylation, DNA binding, and nick-closing activity. Although the mutant lsub1 (91-362) included the active site lysine (KxDG), self-adenylation was not shown. However, the mutants lsub6 (1-362), lsub7 (1-516), and lsub9 (1-635) showed the same adenylation activity as that of wild type. The lsub5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity. These results suggest that the presence of N-terminal 90 residues is essential for the formation of an enzyme-AMP complex, while C-terminal domain (487-719) appears to play a minimal role in adenylation. It was found that the presence of C-terminal domain (487-719) is indispensable for DNA binding activity of lsub5 (91-719). The mutant lsub9 (1-635) showed reduced DNA binding activity compared to that of wild type, suggesting the contribution of the domain (636-719) for the DNA binding activity. Thus, we concluded that the N-terminal 90 residues and C-terminal domain (487-719) of NAD+-dependent DNA ligase from A. pyrophilus are mutually indispensable for binding of DNA substrate.

Construction of recombinant swinepox viruses and expression of the classical swine fever virus E2 protein.

To explore the swinepox virus (SPV) as a potential live vector for immunization, a vector was developed for the construction of a recombinant SPV carrying foreign genes. In this system, a foreign gene placed under the strong vaccinia virus promoter P(11) can be inserted into the viral thymidine kinase (TK) gene, and the recombinant virus can be isolated in a non-selective medium by the co-expression of E. coli lacZ gene. Compared with the wild type virus, the TK(-)recombinant SPV showed a modest level of attenuation in porcine cells while more attenuation was observed in monkey or human cells. Using this system, a recombinant virus expressing the E2 glycoprotein of classical swine fever virus (CSFV) was produced. Engineered with the gX signal sequence of the pseudorabies virus, and transmembrane domain of E2, the E2 protein was expressed as a dimeric form in the cytoplasm of the infected cells.

N-butyl-2-cyanoacrylate pulmonary embolism after endoscopic injection sclerotherapy for gastric variceal bleeding.

PURPOSE: The purpose of this work was to describe the radiologic and clinical manifestations of n-butyl-2-cyanoacrylate pulmonary embolism (PE) after endoscopic injection sclerotherapy (EIS) for gastric variceal bleeding. METHOD: From 1992 to 1999, the medical records of 140 patients who had undergone EIS using n-butyl-2-cyanoacrylate were reviewed for identification of respiratory symptoms and amount of injection, and their pre- and postprocedure chest radiographs were reviewed to identify PE. In patients with PE, pre- and postprocedure chest radiographs (6/6), chest CT scans (3/6), lung perfusion scans (3/6), and follow-up chest radiographs (6/6) were analyzed retrospectively. RESULTS: Radiographically evident PE was observed in 6 (4.3%) of 140 patients. In comparison with patients without emboli, these patients received a higher mean volume of injection (4.2 vs. 1.8 ml) (p = 0.0011). Four of the six patients with pulmonary emboli had respiratory symptoms. Chest radiographs and CT scans showed unusual tubular or nodular, radiopaque pulmonary emboli along the pulmonary vessels. Multiple peripheral, wedge-shaped, subsegmental perfusion defects were seen on perfusion lung scans. In five of six patients, the radiographic abnormalities showed complete or partial resolution. There were no fatalities directly associated with PE. CONCLUSION: Radiographically evident PEs are uncommonly observed following EIS and appear to be more common in patients receiving a higher volume of liquid acrylate. Affected patients were either mildly symptomatic or asymptomatic, and there were no direct fatalities of this complication.

Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen.

Inactivation of the retinoblastoma (Rb) tumor suppressor by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N-terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N-terminal region (residues 7-117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four-helix bundle, and residues from helices alpha2 and alpha4 and from a loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ-1, suggesting that large T antigen may use a chaperone mechanism for its biological function. However, there are significant differences between large T antigen and the molecular chaperones in other regions and these differences are likely to provide the specificity needed for large T antigen to inactivate Rb.

Induction of CTL responses and identification of a novel epitope of hepatitis B virus surface antigens in C57BL/6 mice immunized with recombinant vaccinia viruses.

MHC class I-restricted cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV) surface antigens (HBsAg) has been suggested to play essential roles in viral clearance and pathogenesis of HBV-induced hepatitis. In the present study, we analyzed CTL responses to endogenously synthesized or exogenously introduced HBsAg in C57BL/6 mice (H-2(b)). We show that the endogenously synthesized surface antigens of adr-type HBV encoded by recombinant vaccinia virus efficiently elicit CTL responses in C57BL/6 mice previously defined as non-responders to vaccinia-HBV immunization. We also show that two peptides, S(179-186) (FVQWFVGL) and S(208-216) (ILSPFLPLL), serve as effective motifs for CTL response in H-2(b) system after in vitro restimulation of the primed T cells with either of the two synthetic peptides. S(208-215) has recently been identified as a CTL epitope which could be produced by exogenous pathway only, in contrast to the current result, while S(179-186) appeared a novel epitope for CTL response. In addition, we show that soluble HBsAg also elicits CTL responses in H-2(b) mice upon in vitro restimulation with the two peptides, although less efficiently compared with the recombinant vaccinia viruses. These findings may provide an efficient experimental system for studying H-2(b)-restricted immune responses against endogenously synthesized and exogenously introduced HBsAg.

Structural and functional dissection of human cytomegalovirus US3 in binding major histocompatibility complex class I molecules.

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.

Efficiency of combined colonoscopy and computed tomography for diagnosis of colonic actinomycosis: a retrospective evaluation of eight consecutive patients.

The preoperative diagnosis of colonic actinomycosis is accurate in fewer than 20% of patients. Colonoscopy and computed tomography (CT) findings were analyzed to ascertain their diagnostic value and their role in determining therapeutic modality and outcome. Before and after treatment we retrospectively evaluated eight consecutive patients with colonic actinomycosis, all of whom were women with a previous history of intrauterine contraceptive device use. Median follow-up period was 20 months (7-57). Localized nodules were found in all cases by colonoscopy; half of these nodules included umbilication at the apex. The colonic mucosa demonstrated normal to moderate inflammation as well as hyperemia and edema. Mucosal ulceration was found in only one case. All cases showed some degree of stenosis. These findings differ from those in other inflammatory or neoplastic diseases of the colon. Abdominopelvic CT revealed extramural involvement of heterogeneous lesions all cases. Contrast enhancement also indicated severe thickening of the colonic wall (7-20 mm) with focal and dense enhancement in six cases. Seven of eight patients required surgery for diagnostic or therapeutic purposes. Complete resolution was determined using both colonoscopy and CT. As more than half of these cases could have been safely managed using an adequate antibiotic treatment, diagnostic studies indicating a high likelihood of colonic actinomycosis should be evaluated to avoid unnecessary surgeries carried out for diagnostic purposes. A combination of colonoscopy and CT appears to be important for both diagnosis and management because of their compensatory findings of mucosal and extramucosal lesions, respectively.

A thioredoxin from the hyperthermophilic archaeon Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities.

A thioredoxin homologue (Mj0307) from the hyperthermophilic archaeon Methanococcus jannaschii (MjTRX) was cloned, produced in E. coli, and compared to the thioredoxin from E. coli (ETRX). The secondary structure profile of MjTRX obtained by NMR spectroscopy shows that it has four beta-sheets and three alpha-helices arranged in betaalphabetaalphabetabetaalpha, similar to that of glutaredoxin. However, MjTRX supports the growth of T7 bacteriophage in E. coli and is weakly reduced by the thioredoxin reductase from E. coli, indicating that MjTRX is functionally closer to a thioredoxin than a glutaredoxin. MjTRX has higher specific insulin reductase activity than ETRX and retained its full activity over 4 days at 95 degrees C, whereas ETRX lost its activity in 150 min. The standard state redox potential of MjTRX is about -277 mV, which is the lowest value thus far known among redox potentials of the thioredoxin superfamily. This indicates that the lower redox potential is necessary in keeping catalytic disulfide bonds reduced in the cytoplasm and in coping with oxidative stress in an anaerobic hyperthermophile.