Construction of representative transcript and protein sets of human, mouse, and rat as a platform for their transcriptome and proteome analysis

The number of mammalian transcripts identified by full-length cDNA projects and genome sequencing projects is increasing remarkably. Clustering them into a strictly nonredundant and comprehensive set provides a platform for functional analysis of the transcriptome and proteome, but the quality of the clustering and predictive usefulness have previously required manual curation to identify truncated transcripts and inappropriate clustering of closely related sequences. A Representative Transcript and Protein Sets (RTPS) pipeline was previously designed to identify the nonredundant and comprehensive set of mouse transcripts based on clustering of a large mouse full-length cDNA set (FANTOM2). Here we propose an alternative method that is more robust, requires less manual curation, and is applicable to other organisms in addition to mouse. RTPSs of human, mouse, and rat have been produced by this method and used for validation. Their comprehensiveness and quality are discussed by comparison with other clustering approaches. The RTPSs are available at ftp://fantom2.gsc.riken.go.jp/RTPS/. (C). 2004 Elsevier Inc. All rights reserved.

Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis

Background/aims: Chronic infections such as those caused by Chlamydia pneumoniae and periodontopathic bacteria such as Porphyromonas gingivalis have been associated with atherosclerosis, possibly due to cross-reactivity of the immune response to bacterial GroEL with human heat shock protein (hHSP) 60. Methods: We examined the cross-reactivity of anti-GroEL and anti-P. gingivalis antibodies with hHSP60 in atherosclerosis patients and quantified a panel of six pathogens in atheromas. Results: After absorption of plasma samples with hHSP60, there were variable reductions in the levels of anti-GroEL and anti-P. gingivalis antibodies, suggesting that these antibodies cross-reacted with hHSP60. All of the artery specimens were positive for P. gingivalis. Fusobacterium nucleatum, Tannerella forsythia, C. pneumoniae, Helicobacter pylori, and Haemophilus influenzae were found in 84%, 48%, 28%, 4%, and 4% of arteries, respectively. The prevalence of the three periodontopathic microorganisms, P. gingivalis, F. nucleatum and T. forsythia, was significantly higher than that of the remaining three microorganisms. Conclusions: These results support the hypothesis that in some patients, cross-reactivity of the immune response to bacterial HSPs including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may be a possible mechanism for the association between atherosclerosis and periodontal infection.

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Expression of human neuronal protein 22, a novel cytoskeleton-associated protein, was decreased in the anterior cingulate cortex of schizophrenia

Human neuronal protein 22 (hNP22) is a novel neuron-specific protein featuring numerous motifs previously described in cytoskeleton-associating and signaling proteins. Because previous studies have supported abnormalities in neuronal cytoarchitecture and/or development in the schizophrenia brain, we examined the expression of hNP22 in the anterior cingulate cortex, the hippocampus and the prefrontal cortex of schizophrenic and normal control postmortem brains using high-sensitive immunohistochemistry. Seven schizophrenic and seven age- and sex-matched control brains were examined. The ratio of hNP22-immunopositive cells/total cells was significantly reduced in layer V (p = .020) and layer VI (p = .022) of the anterior cingulate cortex of schizophrenic brain compared with controls. In contrast, there were no significant changes observed in the hippocampus and the prefrontal cortex. These results suggest that altered expression of hNP22 may be associated with modifications in neuronal cytoarchitecture leading to dysregulation of neural signal transduction in the anterior cingulate cortex of the schizophrenia brain.

The human stress-activated protein kinase-interacting 1 gene encodes JNK-binding proteins

The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. In this study, we have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin 1 alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin 1 polyclonal antibody showed that full-length Sin I and several smaller isoforms are widely expressed. Sin 1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1 alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1 alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK. (c) 2004 Elsevier Inc. All rights reserved.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis

Objective: To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design: Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting: Investigational intensive care unit at a university hospital. Subjects: Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mu g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results., In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in PaO2/FiO(2) ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

The human papillomavirus type 16 E7 protein binds human interferon regulatory factor-9 via a novel PEST domain required for transformation

It is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction of the HPV-16 E7 protein with IFN regulatory factor-9 (IRF-9). Here, we investigated the details of the interaction using HPV-16 E7 peptide mapping to show that IRF-9 binds HPV-16 E7 in a domain encompassing amino acids 25-36. A closer examination of this region indicates this is a novel proline, glutamate, serine, and threonine-rich (PEST) domain, with a PEST score of 8.74. We have also mapped the region of interaction within IRF-9 and found that amino acids 354-393 play an important role in binding to HPV-16 E7. This region of IRF-9 encompasses the IRF association domain (IAD), a region important for protein-protein interaction central to IRF function. Finally, we used alanine-scanning mutagenesis to determine if E7-IRF-9 interaction was important for E7-mediated cellular transformation and found that the HPV-16 E7 mutants Y25A, E26A, S31A, S32A, and E35A, but not L28A and N29A, caused loss of transformation ability. Preliminary data suggest loss of IRF-9 interaction with E7 mutants correlated with transformation. Our work suggests E7- IRF- 9 interaction is important for the transforming ability of HPV-16 E7 and that HPV-16 E7 may interact with other IRF proteins that have IAD domains.

Interferon-induced, antiviral human MxA protein localizes to a distinct subcompartment of the smooth endoplasmic reticulum

Human MxA protein belongs to the superfamily of dynamin-like large GTPases that are involved in intracellular membrane trafficking. MxA is induced by interferons-alpha/beta (IFN-alpha/beta) and is a key component of the antiviral response against RNA viruses. Here, we show that MxA localizes to membranes that are positive for specific markers of the smooth endoplasmic reticulum, such as Syntaxin17, but is excluded from other membrane compartments. Overexpression of MxA leads to a characteristic reorganization of the associated membranes. Interestingly, Hook3, mannose-6-phosphate receptor, and Lamp-1, which normally accumulate in cis-Golgi, endosomes, and lysosomes, respectively, also colocalized with MxA, indicating that these markers were redistributed to the MxA-positive compartment. Functional assays, however, did not show any effect of MxA on endocytosis or the secretory pathway. The present results demonstrate that MxA is an IFN-induced antiviral effector protein that resembles the constitutively expressed large GTPase family members in its capacity to localize to and reorganize intracellular membranes.

Detection of elevated protein modification in human cerebellum in alcoholic cerebellar degeneration

Modification of proteins by reactive ethanol metabolites has been known for some time to occur in the liver, the main site of ethanol metabolism. In more recent studies of laboratory animals, similar modifications have been detected in organs with lesser ability to metabolize ethanol, such as skeletal and cardiac muscle and brain. Such modification may alter protein function or form a neoantigen, making it a target for immune attack. We now report an analysis of protein modification derived from ethanol metabolites in human brain tissue by ELISA using adduct-specific antibodies. We obtained autopsy cerebellum samples from 10 alcoholic cerebellar degeneration cases and 10 matched controls under informed written consent from the next of kin and clearance from the UQ Human Ethics Committee. Elevated levels of protein modifications derived from acetaldehyde (unreduced-acetaldehyde and acetaldehyde-advanced glycation end-product adducts), from malondialdehyde (malondialdehyde adducts) and from combined adducts (malondialdehydeacetaldehyde (MAA) adducts) were detected in alcoholic cerebellar degeneration samples when compared to controls. Other adduct types found in liver samples, such as reduced-acetaldehyde and those derived from hydroxyethyl radicals, were not detected in brain samples. This may reflect the different routes of ethanol metabolism in the two tissues. This is the first report of elevated protein modification in alcoholic cerebellar degeneration, and suggests that such modification may play a role in the pathogenesis of brain injury. Supported by NIAAA under grant NIH AA12404 and the NHMRC (Australia) under grant #981723.

Genomic and proteomic analysis of synaptic protein expression in the brains of human alcoholics

Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.