ER translocon inhibitor ipomoeassin F inhibits triple-negative breast cancer growth via blocking ER molecular chaperones.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer where no effective therapy has been developed. Here, we report that the natural product ER translocon inhibitor ipomoeassin F is a selective inhibitor of TNBC cell growth. A proteomic analysis of TNBC cells revealed that ipomoeassin F significantly reduced the levels of ER molecular chaperones, including PDIA6 and PDIA4, and induced ER stress, unfolded protein response (UPR) and autophagy in TNBC cells. Mechanistically, ipomoeassin F, as an inhibitor of Sec61α-containing ER translocon, blocks ER translocation of PDIA6, inducing its proteasomal degradation. Silencing of PDIA6 or PDIA4 by RNA interferences or treatment with a small molecule inhibitor of the protein disulfide isomerases in TNBC cells successfully recapitulated the ipomoeassin F phenotypes, including the induction of ER stress, UPR and autophagy, suggesting that the reduction of PDIAs is the key mediator of the pharmacological effects of ipomoeassin F. Moreover, ipomoeassin F significantly suppressed TNBC growth in a mouse tumor xenograft model, with a marked reduction in PDIA6 and PDIA4 levels in the tumor samples. Our study demonstrates that Sec61α-containing ER translocon and PDIAs are potential drug targets for TNBC and suggests that ipomoeassin F could serve as a lead for developing ER translocon-targeted therapy for TNBC.

A comprehensive analysis of different types of databases reveals that CDH1 mRNA and E-cadherin protein are not downregulated in most carcinoma tissues and carcinoma cell lines.

BACKGROUND: The CDH1 gene codes for the epithelial-cadherin (E-cad) protein, which is embedded in the plasma membrane of epithelial cells to form adherens junctions. E-cad is known to be essential for maintaining the integrity of epithelial tissues, and the loss of E-cad has been widely considered a hallmark of metastatic cancers enabling carcinoma cells to acquire the ability to migrate and invade nearby tissues. However, this conclusion has come under scrutiny. METHODS: To assess how CDH1 and E-cad expression changes during cancer progression, we analyzed multiple large transcriptomics, proteomics, and immunohistochemistry datasets on clinical cancer samples and cancer cell lines to determine the CDH1 mRNA and E-cad protein expression profiles in tumor and normal cells. RESULTS: In contrast to the textbook knowledge of the loss of E-cad during tumor progression and metastasis, the levels of CDH1 mRNA and E-cad protein are either upregulated or remain unchanged in most carcinoma cells compared to normal cells. In addition, the CDH1 mRNA upregulation occurs in the early stages of tumor development and the levels remain elevated as tumors progress to later stages across most carcinoma types. Furthermore, E-cad protein levels are not downregulated in most metastatic tumor cells compared to primary tumor cells. The CDH1 mRNA and E-cad protein levels are positively correlated, and the CDH1 mRNA levels are positively correlated to cancer patient's survival. We have discussed potential mechanisms underlying the observed expression changes in CDH1 and E-cad during tumor progression. CONCLUSIONS: CDH1 mRNA and E-cadherin protein are not downregulated in most tumor tissues and cell lines derived from commonly occurring carcinomas. The role of E-cad in tumor progression and metastasis may have previously been oversimplified. CDH1 mRNA levels may serve as a reliable biomarker for the diagnosis of some tumors (such as colon and endometrial carcinomas) due to the marked upregulation of CDH1 mRNA in the early stages of tumor development of these carcinomas.

A Bio-Conjugated Chlorin-Based Metal-Organic Framework for Targeted Photodynamic Therapy of Triple Negative Breast and Pancreatic Cancers.

The field of photodynamic therapy (PDT) has continued to show promise as a potential method for treating tumors. In this work a photosensitizer (PS) has been delivered to cancer cell lines for PDT by incorporation into the metal-organic framework (MOF) as an organic linker. By functionalizing the surface of MOF nanoparticles with maltotriose the PS can efficiently target cancer cells with preferential uptake into pancreatic and breast cancer cell lines. Effective targeting overcomes some current problems with PDT including long-term photosensitivity and tumor specificity. Developing a PS with optimal absorption and stability is one of the foremost challenges in PDT and the synthesis of a chlorin which is activated by long-wavelength light and is resistant to photo-bleaching is described. This chlorin-based MOF shows anti-cancer ability several times higher than that of porphyrin-based MOFs with little toxicity to normal cell lines and no dark toxicity.

RuvB-Like Protein 2 Interacts with the NS1 Protein of Influenza A Virus and Affects Apoptosis That Is Counterbalanced by Type I Interferons.

The NS1 protein of influenza A virus (IAV) plays important roles in viral pathogenesis and host immune response. Through a proteomic approach, we have identified RuvB-like proteins 1 and 2 (RuvBL1 and RuvBL2) as interacting partners of the NS1 protein of IAVs. Infection of human lung A549 cells with A/PR/8/34 (PR8) virus resulted in reductions in the protein levels of RuvBL2 but not RuvBL1. Further studies with RuvBL2 demonstrated that the NS1-RuvBL2 interaction is RNA-independent, and RuvBL2 binds the RNA-binding domain of the NS1. Infection of interferon (IFN)-deficient Vero cells with wild-type or delNS1 PR8 virus reduced RuvBL2 protein levels and induced apoptosis; delNS1 virus caused more reductions in RuvBL2 protein levels and induced more apoptosis than did wild-type virus. Knockdown of RuvBL2 by siRNAs induced apoptosis and overexpression of RuvBL2 resulted in increased resistance to infection-induced apoptosis in Vero cells. These results suggest that a non-NS1 viral element or elements induce apoptosis by suppressing RuvBL2 protein levels, and the NS1 inhibits the non-NS1 viral element-induced apoptosis by maintaining RuvBL2 abundance in infected cells in the absence of IFN influence. In contrast to Vero cells, infection of IFN-competent A549 cells with PR8 virus caused reductions in RuvBL2 protein levels but did not induce apoptosis. Concomitantly, pretreatment of Vero cells with a recombinant IFN resulted in resistance to infection-induced apoptosis. These results demonstrate that the infection-induced, RuvBL2-regulated apoptosis in infected cells is counterbalanced by IFN survival signals. Our results reveal a novel mechanism underlying the infection-induced apoptosis that can be modulated by the NS1 and type I IFN signaling in IAV-infected cells.

Ring Expansion Leads to a More Potent Analogue of Ipomoeassin F.

Two new ring-size-varying analogues (2 and 3) of ipomoeassin F were synthesized and evaluated. Improved cytotoxicity (IC50: from 1.8 nM) and in vitro protein translocation inhibition (IC50: 35 nM) derived from ring expansion imply that the binding pocket of Sec61α (isoform 1) can accommodate further structural modifications, likely in the fatty acid portion. Streamlined preparation of the key diol intermediate 5 enabled gram-scale production, allowing us to establish that ipomoeassin F is biologically active in vivo (MTD: ∼3 mg/kg).

Maltotriose Conjugated Metal-Organic Frameworks for Selective Targeting and Photodynamic Therapy of Triple Negative Breast Cancer Cells and Tumor Associated Macrophages.

Herein, we report a nano-MOF conjugated to maltotriose as a new DDS. MA-PCN-224-0.1Mn/0.9Zn showed its ability to target cancer and TAM. This novel MOF is an effective PDT agent and shows little dark toxicity, MA-PCN-224-0.1Mn/0.9Zn uptakes selectively into cancer cells. A well-suited size control methodology was used so that the nano-scaled MOFs may take advantage of the EPR effect. This development of a nano-scale MOF for PDT that is conjugated to a cancer targeting ligand represents a meaningful development for the use of MOFs as drug delivery systems.

Upregulation of CD73 Confers Acquired Radioresistance and is Required for Maintaining Irradiation-selected Pancreatic Cancer Cells in a Mesenchymal State.

The molecular mechanisms underlying exceptional radioresistance in pancreatic cancer remain elusive. In the present study, we established a stable radioresistant pancreatic cancer cell line MIA PaCa-2-R by exposing the parental MIA PaCa-2 cells to fractionated ionizing radiation (IR). Systematic proteomics and bioinformatics analysis of protein expression in MIA PaCa-2 and MIA PaCa-2-R cells revealed that several growth factor-/cytokine-mediated pathways, including the OSM/STAT3, PI3K/AKT, and MAPK/ERK pathways, were activated in the radioresistant cells, leading to inhibition of apoptosis and increased epithelial-mesenchymal plasticity. In addition, the radioresistant cells exhibited enhanced capabilities of DNA repair and antioxidant defense compared with the parental cells. We focused functional analysis on one of the most up-regulated proteins in the radioresistant cells, ecto-5'-nucleotidase (CD73), which is a cell surface protein that is overexpressed in different types of cancer. Ectopic overexpression of CD73 in the parental cells resulted in radioresistance and conferred resistance to IR-induced apoptosis. Knockdown of CD73 re-sensitized the radioresistant cells to IR and IR-induced apoptosis. The effect of CD73 on radioresistance and apoptosis is independent of the enzymatic activity of CD73. Further studies demonstrate that CD73 up-regulation promotes Ser-136 phosphorylation of the proapoptotic protein BAD and is required for maintaining the radioresistant cells in a mesenchymal state. Our findings suggest that expression alterations in the IR-selected pancreatic cancer cells result in hyperactivation of the growth factor/cytokine signaling that promotes epithelial-mesenchymal plasticity and enhancement of DNA repair. Our results also suggest that CD73, potentially a novel downstream factor of the enhanced growth factor/cytokine signaling, confers acquired radioresistance by inactivating proapoptotic protein BAD via phosphorylation of BAD at Ser-136 and by maintaining the radioresistant pancreatic cancer cells in a mesenchymal state.

Model-free feature screening for categorical outcomes: Nonlinear effect detection and false discovery rate control.

Feature screening has become a real prerequisite for the analysis of high-dimensional genomic data, as it is effective in reducing dimensionality and removing redundant features. However, existing methods for feature screening have been mostly relying on the assumptions of linear effects and independence (or weak dependence) between features, which might be inappropriate in real practice. In this paper, we consider the problem of selecting continuous features for a categorical outcome from high-dimensional data. We propose a powerful statistical procedure that consists of two steps, a nonparametric significance test based on edge count and a multiple testing procedure with dependence adjustment for false discovery rate control. The new method presents two novelties. First, the edge-count test directly targets distributional difference between groups, therefore it is sensitive to nonlinear effects. Second, we relax the independence assumption and adapt Efron's procedure to adjust for the dependence between features. The performance of the proposed procedure, in terms of statistical power and false discovery rate, is illustrated by simulated data. We apply the new method to three genomic datasets to identify genes associated with colon, cervical and prostate cancers.

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90ß) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90ß in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17ß-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

Total Synthesis and Biological Evaluation of Ipomoeassin F and Its Unnatural 11R-Epimer.

Ipomoeassin F, a macrolide glycoresin containing an embedded disaccharide, possesses potent in vitro antitumor activity with an unknown mechanism of function. It inhibits tumor cell growth with single-digit nanomolar IC50 values, superior to many clinical chemotherapeutic drugs. To facilitate translation of its bioactivity into protein function for drug development, we report here a new synthesis for the gram-scale production of ipomoeassin F (3.8% over 17 linear steps) from commercially available starting materials. The conformation-controlled subtle reactivity differences of the hydroxyl groups in carbohydrates were utilized to quickly construct the disaccharide core, which, along with judicial selection of protecting groups, made the current synthesis very efficient. The same strategy was also applied to the smooth preparation of the 11R-epimer of ipomoeassin F for the first time. Cytotoxicity assays demonstrated the crucial role of the natural 11S configuration. In addition, cell cycle analyses and apoptosis assays on ipomoeassin F and/or its epimer were conducted. This work has laid a solid foundation for understanding the medicinal potential of the ipomoeassin family of glycolipids in the future.

hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export.

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation.

Estrogen receptor beta interacts and colocalizes with HADHB in mitochondria.

Estrogen receptors are localized in mitochondria, but their functions in this organelle remain unclear. We previously found that ERα interacted with mitochondrial protein HADHB and affected the thiolytic cleavage activity of HADHB in ß-oxidation. It is known that ERß binds to ERα. In addition, ERß is predominately located in mitochondria. These facts led us to speculate that ERß may also be associated with HADHB in mitochondria. In order to test this hypothesis, we performed co-immunoprecipitation and confocal microscopy analyses with human breast cancer MCF7 cells. The results demonstrated that ERß was indeed associated and colocalized with HADHB within mitochondria. Interestingly, in contrast to the stimulatory effect of ERα on HADHB enzyme activity observed in the previous study, silencing of ERß enhanced the enzyme activity of HADHB in the present study, suggesting that ERß plays an inhibitory role in HADHB enzyme activity in the breast cancer cells. Our results imply that ERα and ERß may differentially affect cellular oxidative stress through influencing the rate of ß-oxidation of fatty acids in breast cancer cells.

Proteome alterations in primary human alveolar macrophages in response to influenza A virus infection.

To obtain a global picture of how alveolar macrophages respond to influenza A virus (IAV) infection, we used a quantitative proteomics method to systematically examine protein expression in the IAV-infected primary human alveolar macrophages. Of the 1214 proteins identified, 43 were significantly up-regulated and 63 significantly down-regulated at >95% confidence. The expression of an array of interferon (IFN)-induced proteins was significantly increased in the IAV-infected macrophages. The protein with the greatest expression increase was ISG15, an IFN-induced protein that has been shown to play an important role in antiviral defense. Concomitantly, quantitative real-time PCR analysis revealed that the gene expression of type I IFNs increased substantially following virus infection. Our results are consistent with the notion that type I IFNs play a vital role in the response of human alveolar macrophages to IAV infection. In addition to the IFN-mediated responses, inflammatory response, apoptosis, and redox state rebalancing appeared also to be major pathways that were affected by IAV infection. Furthermore, our data suggest that alveolar macrophages may play a crucial role in regenerating alveolar epithelium during IAV infection.

Acquisition of resistance of pancreatic cancer cells to 2-methoxyestradiol is associated with the upregulation of manganese superoxide dismutase.

Acquired resistance of cancer cells to anticancer drugs or ionizing radiation (IR) is one of the major obstacles in cancer treatment. Pancreatic cancer is an exceptional aggressive cancer, and acquired drug resistance in this cancer is common. Reactive oxygen species (ROS) play an essential role in cell apoptosis, which is a key mechanism by which radio- or chemotherapy induce cell killing. Mitochondria are the major source of ROS in cells. Thus, alterations in the expression of mitochondrial proteins, involved in ROS production or scavenging, may be closely linked to the resistance of cancer cells to radio- or chemotherapy. In the present study, we generated a stable cell line by exposing pancreatic cancer cells to increasing concentrations of ROS-inducing, anticancer compound 2-methoxyestradiol (2-ME) over a 3-month period. The resulting cell line showed strong resistance to 2-ME and contained an elevated level of ROS. We then used a comparative proteomics method to profile the differential expression of mitochondrial proteins between the parental and the resistant cells. One protein identified to be upregulated in the resistant cells was manganese superoxide dismutase (SOD2), a mitochondrial protein that converts superoxide radicals to hydrogen peroxides. Silencing of SOD2 resensitized the resistant cells to 2-ME, and overexpression of SOD2 led the parental cells to 2-ME resistance. In addition, the 2-ME-resistant cells also showed resistance to IR. Our results suggest that upregulation of SOD2 expression is an important mechanism by which pancreatic cancer cells acquire resistance to ROS-inducing, anticancer drugs, and potentially also to IR.

Estrogen receptor alpha interacts with mitochondrial protein HADHB and affects beta-oxidation activity.

It is known that estrogen receptors can function as nuclear receptors and transcription factors in the nucleus and as signaling molecules in the plasma membrane. In addition, the localization of the receptors in mitochondria suggests that they may play important roles in mitochondria. In order to identify novel proteins that are involved in ERα-mediated actions of estrogens, we used a proteomic method that integrated affinity purification, two-dimensional gel electrophoresis, and mass spectrometry to isolate and identify cellular proteins that interact with ERα. One of the proteins identified was trifunctional protein ß-subunit (HADHB), a mitochondrial protein that is required for ß-oxidation of fatty acids in mitochondria. We have verified the interaction between ERα and HADHB by coimmunoprecipitation and established that ERα directly binds to HADHB by performing an in vitro binding assay. In addition, we have shown that ERα colocalizes with HADHB in the mitochondria by confocal microscopy, and the two proteins interact with each other within mitochondria by performing coimmunoprecipitation using purified mitochondria as starting materials. We have demonstrated that the expression of ERα affects HADHB activity, and a combination of 17ß-estrodiol and tamoxifen affects the activity of HADHB prepared from human breast cancer cells that express ERα but not from the cells that are ERα deficient. Furthermore, we have demonstrated that 17ß-estrodiol plus tamoxifen affects the association of ERα with HADHB in human cell extract. Our results suggest that HADHB is a functional molecular target of ERα in the mitochondria, and the interaction may play an important role in the estrogen-mediated lipid metabolism in animals and humans.

Inhibition of type I interferon production via suppressing IKK-gamma expression: a new strategy for counteracting host antiviral defense by influenza A viruses?

Blockage of the induction of type I interferons (IFNs) is essential for the success of influenza virus proliferation in host cells. Several molecular mechanisms by which influenza viruses inhibit IFN induction have been characterized. Here we report a potentially new strategy influenza viruses employ to inhibit IFN production during viral infection. Through a two-dimensional gel electrophoresis based proteomic approach, we found that the expression of IκB kinase-gamma (IKKγ) was suppressed by influenza A virus infection in human lung epithelial A549 cells. Silencing of cellular IKKγ by small interfering RNA led to enhanced replication of influenza viruses. Concomitantly, overexpression of IKKγ resulted in increased production of IFNα/ß, whereas influenza virus infection completely eliminated the IKKγ-overexpression-induced production of IFNα/ß. Our results suggest that IKKγ and influenza virus are mutually inhibitory, and influenza viruses may inhibit IFN production through suppressing the expression of IKKγ during viral infection.

Streamline proteomic approach for characterizing protein-protein interaction network in a RAD52 protein complex.

Large-scale identification of protein-protein interactions (PPIs) in functional complexes represents an efficient route to elucidate the regulatory rules of cellular functions. Whereas many methods have been developed to identify the PPIs associated with particular target/bait protein in complexes, little information is available about the interaction relationships among all components in a complex. Here, we have established a strategy of integrating proteomic identification of complex components with mammalian two-hybrid screening of their binary relationships to achieve information content of both breadth (i.e., identifying all potential interacting partners of the protein of interest) and depth (i.e., detailed mapping of the physical interactions of a subset of the identified and functionally related proteins) in characterizing protein complexes. In the initial phase of quantitative proteomic analysis of this streamline, the proteins that specifically complex with the target/bait protein were pulled down by immunoprecipitation and identified by mass spectrometry (MS)-based "dual-tagging" quantitative proteomic approach. In the second phase of in-depth characterizations of binary relationships, the physical interactions of a subset of functionally closely related complex components are mapped by mammalian two-hybrid assay. The screening for binary relationships of complex components not only serves as a validation of the first phase of proteomic identification, but also further deepens the understanding of the protein complex of interest. With this streamlined approach, we studied the protein complexes that are associated with a DNA recombination protein RAD52. In the initial phase, multiple proteins both known and unknown to interact with RAD52 were identified by the "dual-tagging" proteomic method. In the second phase, a complex protein-protein interaction network, which may play important roles in coordinating the activity of DNA repair with that of cell division, was defined by the mammalian two-hybrid assay.

Immunoadjuvant effects of bacterial genomic DNA and CpG oligodeoxynucleotides on avian influenza virus subtype H5N1 inactivated oil emulsion vaccine in chicken.

This study investigated the immunoadjuvant effects of three types of bacterial genomic DNA and CpG oligonucleotides (CpG ODN) on the avian influenza virus (AIV) subtype H5N1 inactivated oil emulsion vaccine under two immunization strategies. The genomic DNA extracted from Escherichia coli O(2), Staphylococcus aureus,Streptococcus faecalis FQ68, and synthetic CpG ODN were used as adjuvants, and their effects on the AIV oil emulsion vaccine were examined in chickens. The results indicated that when administered separately from the vaccine, adjuvants induced lower haemagglutination inhibition (HI) titres and serum IgG titres but resulted in higher concentrations of IFN-gamma and IL-10. In contrast, when combined with the oil emulsion vaccine prior to inoculation, CpG ODN induced higher HI, IgG titres and IFN-gamma concentration but resulted in lower IL-10 concentration. These data suggest that, depending on the immunization approaches, adjuvants may exert distinct immune effects in chickens receiving AIV H5N1 oil emulsion vaccine: the prior incorporation of CpG ODN into the vaccine may augment both the humoral and Th1 type immune responses, while separate inoculation of adjuvants has not shown better adjuvanticity.

Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase.

In the green alga Chlamydomonas reinhardtii, an L290F substitution in the chloroplast-encoded large-subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) causes decreases in carboxylation Vmax, CO2/O2 specificity, and thermal stability. Analysis of photosynthesis-competent revertants selected at the 35 degrees C restrictive temperature identified a rare C65S suppressor substitution in the nuclear-encoded small subunit. C65S enhances catalysis and CO2/O2 specificity in the absence of other wild-type small subunits, and restores thermal stability in vivo. C65S, C65A, and C65P mutant strains were created. C65S and C65A enzymes have normal catalysis, but C65P Rubisco, which contains land-plant Pro, has decreases in carboxylation Vmax/Km and CO2/O2 specificity. In contrast to other small-subunit substitutions that affect specificity, Cys-65 contacts the large subunit, and the C65P substitution does not cause a decrease in holoenzyme thermal stability in vivo or in vitro. Further analysis of the C65P protein may identify structural alterations that influence catalysis separate from those that affect stability.