Damage by the Russian wheat aphid (RWA), Diuraphis noxia, significantly reduces wheat and barley yields worldwide. In compatible interactions, virulent RWA populations flourish and susceptible plants suffer extensive leaf chlorophyll loss. In incompatible interactions, RWA reproduction and population growth are significantly reduced and RWA-related chlorophyll loss in resistant plants is minor. The objectives of this study were to develop an understanding of the molecular and phytochemical bases of RWA resistance in plants containing the Dnx resistance gene. Microarray, real-time polymerase chain reaction, and phytohormone assays were conducted to identify transcriptome components unique to RWA-infested Dnx plants and susceptible (Dn0) plants, and to identify and characterize putative genes involved in Dnx plant defense responses. We found that RWA-infested Dnx plants upregulated > 180 genes related to reactive oxygen species, signaling, pathogen defense, and arthropod allelochemical and physical defense. The expression of several of these genes in RWA-infested Dnx plants increased significantly from 6- to 24-h post infestation (hpi), but their expression in Dn0 plants, when present, was delayed until 48- to 96 hpi. Concentrations of 16- and 18-carbon fatty acids, trans-methyl-12-oxophytodienoic acid, and abscisic acid were significantly greater in Dnx foliage than in Dn0 foliage after RWA infestation, suggesting that Dnx RWA defense and resistance genes may be regulated via the oxylipin pathway. These findings provide a foundation for the elucidation of the molecular basis for compatible- and incompatible plant-aphid interactions.
Diuraphis noxia; Insect; Microarray; Northern blot; Oxylipin signaling; Plant defense; Real-time polymerase chain reaction (PCR); Russian wheat aphid; Triticum aestivum; Wheat
In recent pioneer experiment, a strong spin-orbit coupling, with equal Rashba and Dresselhaus strengths, has been created in a trapped Bose-Einstein condensate. Moreover, many exotic superfluid phenomena induced by this strong spin-orbit coupling have been predicted. In this report, we show that this novel spin-orbit coupling has important applications in quantum metrology, such as spin squeezing. We first demonstrate that an effective spin-spin interaction, which is the heart for producing spin squeezing, can be generated by controlling the orbital degree of freedom (i.e., the momentum) of the ultracold atoms. Compared with previous schemes, this realized spin-spin interaction has advantages of no dissipation, high tunability, and strong coupling. More importantly, a giant squeezing factor (lower than −30 dB) can be achieved by tuning a pair of Raman lasers in current experimental setup. Finally, we find numerically that the phase factor of the prepared initial state affects dramatically on spin squeezing.
Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death.
piperlongumine; autophagy; p38; reactive oxygen species
It may be possible to achieve insulin sensitivity through the recently identified mitochondrial target of thiazolidinediones (mTOT), thereby avoiding peroxisome proliferator–activated receptor-γ (PPAR-γ)-dependent side effects. In this phase IIb clinical trial, 258 patients with type 2 diabetes completed a 12-week protocol with 50, 100, or 150 mg of MSDC-0160 (an mTOT modulator), 45 mg pioglitazone HCl (a PPAR-γ agonist), or a placebo. The two active treatments lowered fasting glucose levels to the same extent. The decreases in glycated hemoglobin (HbA1c) observed with the two higher doses of MSDC-0160 were not different from those associated with pioglitazone. By contrast, fluid retention as evidenced by reduction in hematocrit, red blood cells, and total hemoglobin was 50% less in the MSDC-0160–treated groups. There was also a smaller increase in high-molecular-weight (HMW) adiponectin with MSDC-0160 than with pioglitazone (P < 0.0001), suggesting that MSDC-0160 produces less expansion of white adipose tissue. Thus, mTOT modulators may have glucose-lowering effects similar to those of pioglitazone but without the adverse effects associated with PPAR-γ agonists.
Chronic infection with hepatitis C, genotype 2/3, responds better than other genotypes to peginterferon and ribavirin treatment. We hypothesized that a lower dose of peginterferon would be as effective, but less toxic than standard doses.
30 patients were treated with low-dose peginterferon alfa-2a (90 μg/week) and 27 patients with standard doses (180 μg/week) for 24 weeks in combination with 800 mg/day of ribavirin. Patients who failed treatment were offered 48 weeks of standard-dose treatment. Viral and serum IP-10 levels were measured and early viral kinetic parameters were calculated.
Sustained virological response was achieved in 68% of the low-dose and 87% of the standard-dose patients (per-protocol, p=0.79 for non-inferiority). Retreatment was successful in all patients who tolerated full dose and duration. The standard-dose group had greater first phase declines of viral levels and faster time to negativity. The second phase slope was not dose-dependent. IP-10 induction was significantly greater with the standard dose. Although fatigue and general feeling during treatment were worse for standard dose, hematologic toxicity and depression did not differ between groups.
A lower dose of peginterferon is associated with some symptomatic benefit but the response is not equivalent to standard dosing.
Liver; Viral hepatitis
Non-alcoholic steatohepatitis (NASH) is a form of progressive fatty liver disease that is strongly associated with insulin resistance, which suggests that insulin sensitizing agents such as metformin may be beneficial for NASH.
To assess the effects of metformin on insulin sensitivity, body composition, serum alanine aminotransferase (ALT) levels and liver histology in patients with NASH.
Patients underwent liver biopsy, metabolic profiling and imaging studies before and at the end 48 weeks of metformin (2000 mg/day) therapy. The primary endpoint was a three-point improvement in the histological NASH activity index.
Of 28 patients enrolled, 26 (13 females; average age 44 years) completed 48 weeks of treatment and underwent repeat metabolic studies, imaging and liver biopsy. Thirty per cent achieved a histological response. Most patients lost weight, the average being 6 kg. There was a marked association between weight loss and improvements in NASH activity index and ALT levels (both, P < 0.01). Insulin sensitivity also improved, but the degree of change did not correlate with histological improvement.
Metformin leads to improvements in liver histology and ALT levels in 30% of patients with NASH, probably by its effects in causing weight loss.
We have monitored Epstein–Barr virus (EBV) IgA antibody levels of 39 nasopharyngeal carcinoma (NPC) cases for up to 15 years before clinical onset of NPC, and assessed preclinical serologic status of another 68 cases. Our results identify a serologic window preceding diagnosis when antibody levels are raised and sustained. This window can persist for as long as 10 years, with a mean duration estimated to as 37±28 months. Ninety-seven of these 107 NPC cases exhibited such a window. Cases that did not may reflect individual antibody response to EBV. Serologic screening at enrollment identified those cases who had already entered the window and became clinically manifested earlier (median=28 months) than those who entered the window after enrollment (median=90 months). The former account for 19 of 21 cases diagnosed within 2 years of screening. Nasopharyngeal carcinoma risk levels among seropositive subjects were also highest during this period. Both prediction rates and risk levels declined thereafter; cases detected at later times were composed of increasing proportions of individuals who entered the serological window after screening. Our findings establish EBV antibody as an early marker of NPC and suggest that repeated screening to monitor cases as they enter this window has considerable predictive value, with practical consequences for cancer treatment.
NPC; EBV; serology; cancer screening; tumour marker
Viral mutations have been implicated in alteration of the biological phenotype of hepatitis B virus (HBV). We recently cloned and sequenced the viral genome of an HBV strain associated with an outbreak of fulminant hepatitis (FH strain). The FH strain contained numerous mutations in all genomic regions and was functionally characterized by a more efficient encapsidation of pregenomic RNA leading to highly enhanced replication. To define the responsible mutation(s) for the enhanced replication, we introduced individual mutations of the FH strain into a wild-type construct by oligonucleotide-directed mutagenesis. Analysis of viral replication showed that two adjacent mutations in the HBV core promotor (C to T at nucleotide 1768 and T to A at nucleotide 1770) led to high level replication. Similar to the FH strain, this mutant displayed the phenotype of enhanced encapsidation of pregenomic RNA. Functional studies in an encapsidation assay demonstrated that the identified mutations resulted in a minor increase of pregenomic RNA transcription (two- to threefold) and a major transcription-independent enhancement (> 10-fold) of viral encapsidation. Our results demonstrate that the two adjacent mutations in the HBV core promotor region are responsible for the enhanced replication of the FH strain. These two mutations, outside the previously described encapsidation signal, core, and polymerase polypeptides, appeared to affect a novel genetic element involved in viral encapsidation.
Although the biological importance of hepatitis B virus X protein (HBX) in the life cycle of hepatitis B virus has been well established, the cellular and molecular basis of its function remains largely undefined. Despite the association of multiple activities with HBX, none of them appear to provide a unifying hypothesis regarding the true biological function of HBX. Identification and characterization of cellular targets of HBX remain an essential goal in the elucidation of the molecular mechanisms of HBX. Using the Saccharomyces cerevisiae two-hybrid system, we have identified and characterized a novel subunit of the proteasome complex (XAPC7) that interacts specifically with HBX. We also showed that HBX binds specifically to XAPC7 in vitro. Mutagenesis studies have defined the domains of interaction to be critical for the function of HBX. Furthermore, overexpression of XAPC7 appeared to activate transcription by itself and antisense expression of XAPC7 was able to block transactivation by HBX. Therefore, the proteasome complex is possibly a functional target of HBX in cells.
Receptor tyrosine kinases are important in cell signal transduction and proliferation. Abnormal expression of tyrosine kinases often leads to malignant transformation. C-met is a tyrosine kinase receptor and its ligand is hepatocyte growth factor (HGF). HGF/c-met plays diverse role in regulation of cell growth, shape and movement. Constitutively activated met, such as tpr-met, is a potent oncogene in vitro, but its carcinogenic role in vivo remains unclear. Our study demonstrates that expression of tpr-met leads to development of mammary tumors and other malignancies in transgenic mice, and suggests that deregulated met expression may be involved in mammary carcinogenesis.
BACKGROUND: An important issue in autoimmune diseases mediated by T cells, such as experimental allergic encephalomyelitis (EAE), is the affinity of the disease-inducing determinants for MHC class II proteins. Tolerance, either due to clonal deletion or anergy induction, is thought to require high-affinity interactions between peptides and MHC molecules. Low-affinity binding is compatible with the hypothesis that breaking tolerance to self proteins does not have to occur for onset of disease. In contrast, a high-affinity interaction implies that an event leading to a breakdown of tolerance is central to the autoimmune process. MATERIALS AND METHODS: Detergent-solubilized and affinity-purified I-Au was incubated with varying concentrations of a set of peptides from myelin basic protein and a biotinylated peptide agonist. The specific complexes were separated from excess peptide by capture on antibody-coated plates, and the affinity of the peptides was measured by adding europium-labeled streptavidin and measuring the resultant fluorescence. RESULTS: The immunodominant and encephalitogenic determinant, Ac 1-11, was shown to bind to I-Au relatively poorly (IC50 = 100 microM), demonstrating that in this protein, immunodominance did not correlate with high-affinity binding. In contrast with the natural sequence, the ability of shorter analogs to induce EAE did correlate with their apparent affinity. CONCLUSIONS: The dominance of the natural determinant does not arise from a high-affinity interaction with the MHC class II molecule. This suggests that other mechanisms are operative and that the specific T cell for this peptide/MHC ligand is of high affinity.
From 1989 through 1992, endoscopic ultrasonography (EUS) was undertaken preoperatively to evaluate the extent of primary tumor, involvement of regional lymph nodes, and
distant metastases in 22 patients with ampullary carcinoma and 18 patients with bile
duct carcinoma. The results were compared with histopathological findings according to
the TNM staging system. The accurate rate in assessing the extent of cancer invasion was
82% for ampullary carcinoma, 66% for common hepatic duct carcinoma, and 78% for
common bile duct carcinoma. The accuracy of EUS in predicting regional lymph node
metastasis was 59% for ampullary carcinoma, 56% for common hepatic duct carcinoma,
and 67% for common bile duct carcinoma. Invasion of the portal vein was correctly
predicted by EUS in 2 of 3 patients. None of the 3 patients with liver metastasis was
detected by EUS. Therefore, endoscopic ultrasonography is an effective method in the
evaluation of the extent of cancer invasion of ampullary and bile duct carcinoma as well
as the involvement of regional lymph nodes preoperatively. However, due to its limited
penetration depth, EUS is inadequate in the assessment of liver metastasis.
Hepatitis B virus (HBV) mutants unable to synthesize HBV e antigen have been described in association with fulminant hepatitis. We have cloned and sequenced the entire viral genome of an HBV strain associated with an epidemic of fulminant hepatitis. This strain contained, in addition to two G-to-A mutations in the precore region (nucleotides 1898 and 1901), numerous other mutations in conserved nucleotide positions resulting in significant amino acid substitutions in HBV gene products. We introduced either or both of the two G-to-A mutations into wild-type HBV by oligonucleotide-directed mutagenesis and generated replication-competent constructs of these mutants as well as the fulminant strain. Viral antigen synthesis, transcription, and replication were analyzed after transfection into human hepatoma cells. All viral constructs produced and secreted similar levels of envelope proteins (HBV surface antigen). Analysis of cellular lysate for core-specific immunoreactivity demonstrated a much higher level of core-associated antigens in cells transfected with the fulminant strain. While cells transfected with mutant and wild-type HBV DNAs synthesized similar levels of viral RNAs, the fulminant strain directed the synthesis of a much higher level of core-associated replicative intermediates (as well as virion particles) than the wild type and mutants with either or both of the precore mutations. Increase in the encapsidation of pregenomic RNA into core particles likely the basis for the enhanced replication associated with the fulminant strain. Our study suggests that an HBV mutant with enhanced viral replication may be important in the pathogenesis of fulminant hepatic failure, and mutations other than the precore mutations may be responsible for this variant behavior.
Hepatitis B virus (HBV) variants with precore mutation(s) resulting in the absence of HBeAg production have been associated with the occurrence of fulminant hepatitis in Japan, Israel, and southern Europe, where the prevalence of this HBV strain appears common. In areas such as United States, where HBV infection is not endemic, the role of this mutant virus in fulminant hepatitis is unknown. We developed an amplification refractory mutation detection system to detect specifically the presence of the G to A mutation at nucleotide position 1898, which is the most frequently observed mutation resulting in a precore stop codon. In addition, this method provided a quantitative measurement of the relative ratio of one strain to the other. Using this system, we tested HBV strains for the presence of the stop codon mutation in sera from 40 cases of fulminant hepatitis B occurring in the United States. Serum HBV DNAs from 28 patients were analyzed successfully. A mixture of wild-type and mutant strains in various ratios were observed in 15 patients, wild type exclusively in 11, and mutant exclusively in 2. Four of these patients had undergone liver transplantation for HBV-associated cirrhosis and developed fulminant HBV-associated hepatitis after transplantation. Pre- and posttransplant serum samples from one patient were analyzed: a mixture of wild-type and mutant HBV strains was detected in both samples. Our study demonstrated that both wild-type and mutant HBV strains are associated with fulminant hepatitis, and that in some patients in the United States, factors other than precore mutations contribute to the development of fulminant hepatitis.
Many viruses possess complex mechanisms involving multiple gene products and cis-regulatory elements in order to achieve a fine control of their gene expression at both transcriptional and posttranscriptional levels. Hepatitis B virus (HBV) and retroviruses share many structural and functional similarities. In this study, by genetic and biochemical analyses, we have demonstrated the existence of a novel genetic element within the HBV genome which is essential for high-level expression of viral gene products. This element is located 3' to the envelope coding region. We have shown that this genetic element is cis acting at the posttranscriptional level and that its function is exerted at the level of RNA processing as part of transcribed sequences. This RNA element is also functional in the context of a heterologous gene. Similar to the function of Rev-Rev response element interaction of human immunodeficiency virus type 1, this element appears to inhibit the splicing process and facilitate the transport and utilization of HBV transcripts.
A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions.
A mutant of Anabaena sp. strain PCC 7120, called PAT-2, that grows poorly under nitrogen-fixing conditions, has been isolated. The heterocysts of the mutant strain develop much more slowly than those of the wild type and are spaced more closely in an older culture of the mutant than in the wild type. The wild-type gene that complements the mutation in PAT-2, called patB, was isolated and characterized. The predicted 529-amino-acid PatB protein contains a region very similar to the Fe4S4 bacterial-type ferredoxins near its N terminus and a helix-turn-helix motif near its C terminus. This pattern of domains resembles those of transcriptional regulators in both prokaryotes and eukaryotes. The mutation in strain PAT-2 is the deletion of G at nucleotide 1342 in the patB gene, resulting in the loss of a 62-amino-acid fragment from the C terminus of the PatB protein, including the helix-turn-helix motif.
The hepatitis B x (HBx) gene is the smallest open reading frame of the hepatitis B virus (HBV) genome. It is conserved among all mammalian hepadnaviruses and is expressed during viral infection. While the HBx protein (pX) has been shown to trans-activate the transcription of a wide range of viral and cellular genes and to induce liver cancer in transgenic mice, the significance of pX for the life cycle of HBV itself has not been elucidated. To assess the function of pX in viral replication and virion export, we designed an X-minus mutant by introduction of a stop codon at the beginning of the HBx gene without affecting the viral polymerase gene product. Transient transfection analyses using different cell lines revealed that this X-minus mutant directs the synthesis of wild-type levels of viral proteins, replicative intermediates, and virion export. These data suggest that the expression of the highly conserved HBx gene is not central for the life cycle of HBV in vitro but may be involved in the pathogenicity of hepadnavirus infection, including liver cancer development.
Reversible ADP-ribosylation of dinitrogenase reductase forms the basis of posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. This report describes the physiological effects of mutations in the genes encoding the enzymes that add and remove the ADP-ribosyl moiety. Mutants lacking a functional draT gene had no dinitrogenase reductase ADP-ribosyltransferase (DRAT, the draT gene product) activity in vitro and were incapable of modifying dinitrogenase reductase with ADP-ribose in vivo. Mutants lacking a functional draG gene had no dinitrogenase reductase-activating glycohydrolase (DRAG, the draG gene product) activity in vitro and were unable to remove ADP-ribose from the modified dinitrogenase reductase in vivo. Strains containing polar mutations in draT had no detectable DRAG activity in vitro, suggesting likely cotranscription of draT and draG. In strains containing draT and lacking a functional draG, dinitrogenase reductase accumulated in the active form under derepressing conditions but was rapidly ADP-ribosylated in response to conditions that cause inactivation. Detection of DRAT in these cells in vitro demonstrated that DRAT is itself subject to posttranslational regulation in vivo. Mutants affected in an open reading frame immediately downstream of draTG showed regulation of dinitrogenase reductase by ADP-ribosylation, although differences in the rates of ADP-ribosylation were apparent.
A hepatitis B virus (HBV) genome was cloned from human liver. Numerous mutations in all viral genes define this HBV DNA as a mutant, divergent from all known HBV DNA sequences. Functional analyses of this mutant demonstrated a defect blocking viral DNA synthesis. The genetic basis of this defect was identified as a single missense mutation in the 5' region of the viral polymerase gene, resulting in the inability to package pregenomic RNA into core particles. The replication defect could be trans-complemented by a full-length wild-type, but not by a full-length mutant or 3'-truncated wild-type, polymerase gene construct. Our findings indicate a critical role of the 5' polymerase gene region in the life cycle of the virus and suggest that introducing missense mutations in this region can be a strategy to terminate viral replication in vivo.
A sensitive and specific method has been developed to detect hepatitis B virus (HBV) in serum. The method involves two steps: the capture of viral genome from serum using a high affinity IgM monoclonal antibody directed against a common a domain epitope found on the envelope, and the amplification of viral DNA by the polymerase chain reaction (PCR). The amplification is initiated using "generic" primers derived from the core and pre-core sequences which are highly conserved amongst the hepadnaviruses. This rapid technique detects less than 10 infectious virions and may be useful in the study of individuals with acute and chronic liver disease of unknown etiology.
Dinitrogenase from a nifV mutant of Klebsiella pneumoniae contains an altered form of iron-molybdenum cofactor (FeMoco) that lacks a biologically active homocitric acid molecule. Change in the composition of FeMoco led to substantial variation in the kinetics of nitrogenase action. The KmS of the mutant enzyme for N2 and N2O were 0.244 and 0.175 atm (24,714 and 17,726 kPa), respectively. The km for N2 was higher and the Km for N2O was lower than that for the wild-type enzyme. The mutant enzyme was ineffective in N2 fixation, in N2O reduction, and in HD formation, as indicated by the low Vmax of these reactions with saturating levels of substrate and under conditions of saturating electron flux. These observations provide further support for the concept that N2, N2O, and D2 interact with the same form of dinitrogenase. H2 evolution by the mutant enzyme is only partially inhibited by CO. Observation that different numbers of electrons are stored in CO-inhibited than in noninhibited dinitrogenase before H2 is released suggests that the mutant enzyme has more sites responsible for H2 evolution than the wild-type enzyme, whose H2 evolution is not inhibited by CO.
The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py808A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py808A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py808A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.
We performed electroretinograms (ERG) on both eyes of 11 diabetic patients before and one month after panretinal photocoagulation for diabetic retinopathy. Each patient had one eye treated with argon laser and the fellow eye treated with the xenon arc photocoagulator. After photocoagulation the ERG was symmetrically reduced unless the retinal area burned with xenon arc was greater than twice the retinal area burned in the fellow eye by the argon laser photocoagulator.
Chorioretinal neovascularisation was noted in the midperipheral fundus of a young black girl with sickle cell anaemia. Rapid-sequence fluorescein angiography showed choroidally fed neovascularisation in the centre of a large black sunburst (chorioretinal scar). We suspect that a massive midperipheral retinal haemorrhage from the sickle cell disease caused the sunburst and altered Bruch's membrane, thus allowing the ingrowth of choroidally fed vessels.