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1.
J Gen Virol ; 101(10): 1103-1118, 2020 10.
Article in English | MEDLINE | ID: mdl-32720890

ABSTRACT

Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.


Subject(s)
Infectious bronchitis virus/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription, Genetic/genetics , 5' Untranslated Regions/genetics , Animals , Base Sequence , Cell Line , Chickens , Chlorocebus aethiops , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Open Reading Frames/genetics , Poultry Diseases/virology , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/genetics
2.
mBio ; 7(6)2016 11 22.
Article in English | MEDLINE | ID: mdl-27879334

ABSTRACT

Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread. IMPORTANCE: Benzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.


Subject(s)
Anti-Infective Agents, Local/pharmacology , Benzalkonium Compounds/pharmacology , Burkholderia cepacia complex/drug effects , Drug Resistance, Bacterial , Anti-Infective Agents, Local/metabolism , Bacterial Proteins/analysis , Benzalkonium Compounds/metabolism , Biotransformation , Burkholderia cepacia complex/chemistry , Burkholderia cepacia complex/metabolism , Burkholderia cepacia complex/physiology , Gene Expression Profiling , Microbial Viability/drug effects , Proteome/analysis
3.
Nucleic Acids Res ; 43(18): 8830-8, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26250113

ABSTRACT

Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.


Subject(s)
DNA Polymerase II/metabolism , Replication Protein C/metabolism , S Phase Cell Cycle Checkpoints , Saccharomyces cerevisiae Proteins/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Multienzyme Complexes/metabolism , Mutation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
4.
Appl Environ Microbiol ; 81(13): 4263-76, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25888169

ABSTRACT

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications.


Subject(s)
Alkenes/metabolism , Bacterial Proteins/biosynthesis , Gene Expression Regulation, Bacterial/drug effects , Mycobacterium/drug effects , Mycobacterium/metabolism , Petroleum/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Gas Chromatography-Mass Spectrometry , Gene Expression Profiling , Mycobacterium/genetics , Petroleum Pollution , Proteome/analysis
5.
Trop Anim Health Prod ; 46(6): 1051-8, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24817479

ABSTRACT

Twenty-two flocks of turkeys affected by enteric problems, with ages between 10 and 104 days and located in the Southern region of Brazil, were surveyed for turkey by PCR for turkey astrovirus type 2 (TAstV-2), turkey coronavirus (TCoV), hemorrhagic enteritis virus (HEV), rotavirus, reovirus, Salmonella spp., and Lawsonia intracellularis (Li) infections. Eleven profiles of pathogen combination were observed. The most frequently encountered pathogen combinations were TCoV-Li, followed by TCoV-TAstV-2-Li, TCoV-TastV-2. Only TCoV was detected as the sole pathogen in three flocks. Eight and 19 flocks of the 22 were positive for TAstV-2 and TCoV, respectively. Six were positive for Salmonella spp. and L. intracellularis was detected in 12 turkey flocks. Reovirus and HEV were not detected in this survey. These results throw new light on the multiple etiology of enteritis in turkeys. The implications of these findings and their correlation with the clinical signs are comprehensively discussed, illustrating the complexity of the enteric diseases.


Subject(s)
Disease Outbreaks/veterinary , Enteritis/veterinary , Poultry Diseases/epidemiology , Poultry Diseases/microbiology , Turkeys , Animals , Avastrovirus/genetics , Avastrovirus/isolation & purification , Brazil/epidemiology , Coronavirus, Turkey/genetics , Coronavirus, Turkey/isolation & purification , DNA Primers/genetics , Enteritis/epidemiology , Enteritis/microbiology , Lawsonia Bacteria/genetics , Lawsonia Bacteria/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Rotavirus/genetics , Rotavirus/isolation & purification , Salmonella/genetics , Salmonella/isolation & purification
6.
ScientificWorldJournal ; 2014: 450423, 2014.
Article in English | MEDLINE | ID: mdl-24578633

ABSTRACT

Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease.


Subject(s)
Chickens , Poultry Diseases , RNA, Viral , Turkeys , Viruses , Animals , Brazil/epidemiology , Poultry Diseases/epidemiology , Poultry Diseases/genetics , Poultry Diseases/virology , RNA, Viral/genetics , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Viruses/genetics , Viruses/metabolism
7.
Carcinogenesis ; 35(6): 1292-300, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24510113

ABSTRACT

In cancer cells, the process of epithelial-mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival. Utilizing a transforming growth factor-ß-induced cell culture model of EMT, we quantitatively profiled differentially secreted proteins, by GeLC-tandem mass spectrometry. Integrating with the corresponding transcriptome, we derived an EMT-associated secretory phenotype (EASP) comprising of proteins that were differentially upregulated both at protein and mRNA levels. Four independent primary tumor-derived gene expression data sets of lung cancers were used for survival analysis by the random survival forests (RSF) method. Analysis of 97-gene EASP expression in human lung adenocarcinoma tumors revealed strong positive correlations with lymph node metastasis, advanced tumor stage and histological grade. RSF analysis built on a training set (n = 442), including age, sex and stage as variables, stratified three independent lung cancer data sets into low-, medium- and high-risk groups with significant differences in overall survival. We further refined EASP to a 20 gene signature (rEASP) based on variable importance scores from RSF analysis. Similar to EASP, rEASP predicted survival of both adenocarcinoma and squamous carcinoma patients. More importantly, it predicted survival in the early-stage cancers. These results demonstrate that integrative analysis of the critical biological process of EMT provides mechanism-based and clinically relevant biomarkers with significant prognostic value.


Subject(s)
Epithelial-Mesenchymal Transition , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Phenotype , Adult , Aged , Cell Line, Tumor , Cluster Analysis , Computational Biology , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression , Gene Expression Profiling , Humans , Lung Neoplasms/mortality , Lung Neoplasms/therapy , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Prognosis , Proteomics
8.
Avian Pathol ; 43(2): 125-9, 2014.
Article in English | MEDLINE | ID: mdl-24437352

ABSTRACT

The feasibility of using Flinders Technology Associates (FTA) cards for the molecular detection of avian metapneumovirus (aMPV) by reverse transcriptase-polymerase chain reaction (RT-PCR) was investigated. Findings showed that no virus isolation was possible from aMPV-inoculated FTA cards, confirming viral inactivation upon contact with the cards. The detection limits of aMPV from the FTA card and tracheal organ culture medium were 10(1.5) median ciliostatic doses/ml and 10(0.75) median ciliostatic doses/ml respectively. It was possible to perform molecular characterization of both subtypes A and B aMPV using inoculated FTA cards stored for up to 60 days at 4 to 6°C. Tissues of the turbinate, trachea and lung of aMPV-infected chicks sampled either by direct impression smears or by inoculation of the tissue homogenate supernatants onto the FTA cards were positive by RT-PCR. However, the latter yielded more detections. FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization.


Subject(s)
Chickens/virology , Metapneumovirus/isolation & purification , Paramyxoviridae Infections/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Specimen Handling/veterinary , Animals , Metapneumovirus/genetics , Micropore Filters , Paramyxoviridae Infections/diagnosis , Paramyxoviridae Infections/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Virus Inactivation
9.
Cell Rep ; 3(3): 892-904, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23499444

ABSTRACT

DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Histones/metabolism , Multienzyme Complexes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , G1 Phase , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Histone Chaperones/metabolism , Molecular Sequence Data , Multienzyme Complexes/genetics , Protein Binding , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/metabolism , Replication Origin , Replication Protein C/genetics , Replication Protein C/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolism
10.
Microbiology (Reading) ; 159(Pt 4): 782-791, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23393148

ABSTRACT

In a previous study, two proteins identified as hyaluronidases were detected in spent media by MS and found to be in greater quantity in the sarA and sarA agr mutant strains when compared with the parent and agr mutant strains of Staphylococcus aureus UAMS-1. In the present study, spent media and total RNA were isolated from UAMS-1 and its regulatory mutants and analysed for hyaluronidase activity and steady-state hyaluronidase (hysA) RNA message levels. Hyaluronidase activity was observed throughout all time points examined regardless of the regulatory effects of sarA and agr but activity was always substantially higher in the sarA and sarA agr mutant strains than in the UAMS-1 parent and agr mutant strains. Northern analysis did not detect hysA message for either the UAMS-1 parent or the agr mutant strains at any time point examined, while steady-state hysA message levels were detected throughout growth for the sarA mutant strain, but only at exponential and early post-exponential growth for the sarA agr mutant strain. An in vitro biofilm plate assay, pre-coated with human plasma as a source of hyaluronic acid, demonstrated no significant increase in biofilm for a sarA mutant strain of S. aureus UAMS-1 defective in hyaluronidase activity when compared with the sarA mutant strain. These data indicate that, while hysA message levels and hyaluronidase activity are elevated in the sarA mutant strains of S. aureus UAMS-1, the increase in activity did not contribute to the biofilm-negative phenotype observed in the sarA mutant strain of S. aureus UAMS-1.


Subject(s)
Bacterial Proteins/genetics , Biofilms/growth & development , Gene Expression Regulation, Bacterial , Hyaluronoglucosaminidase/metabolism , Mutation , Staphylococcus aureus/growth & development , Trans-Activators/genetics , Bacterial Proteins/metabolism , Humans , Hyaluronoglucosaminidase/genetics , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Trans-Activators/metabolism
11.
Nat Cell Biol ; 14(11): 1169-80, 2012 Nov.
Article in English | MEDLINE | ID: mdl-23103911

ABSTRACT

Although Rac and its activator Tiam1 are known to stimulate cell-cell adhesion, the mechanisms regulating their activity in cell-cell junction formation are poorly understood. Here, we identify ß2-syntrophin as a Tiam1 interactor required for optimal cell-cell adhesion. We show that during tight-junction (TJ) assembly ß2-syntrophin promotes Tiam1-Rac activity, in contrast to the function of the apical determinant Par-3 whose inhibition of Tiam1-Rac activity is necessary for TJ assembly. We further demonstrate that ß2-syntrophin localizes more basally than Par-3 at cell-cell junctions, thus generating an apicobasal Rac activity gradient at developing cell-cell junctions. Targeting active Rac to TJs shows that this gradient is required for optimal TJ assembly and apical lumen formation. Consistently, ß2-syntrophin depletion perturbs Tiam1 and Rac localization at cell-cell junctions and causes defects in apical lumen formation. We conclude that ß2-syntrophin and Par-3 fine-tune Rac activity along cell-cell junctions controlling TJ assembly and the establishment of apicobasal polarity.


Subject(s)
Cell Cycle Proteins/metabolism , Dystrophin-Associated Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Membrane Proteins/metabolism , Tight Junctions/metabolism , rac GTP-Binding Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Line , Cell Polarity/drug effects , Dogs , Doxycycline/pharmacology , Dystrophin-Associated Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Humans , Immunohistochemistry , Mass Spectrometry , Membrane Proteins/genetics , Microscopy, Fluorescence , Tight Junctions/drug effects , rac GTP-Binding Proteins/genetics
12.
J Neurol Sci ; 312(1-2): 146-57, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-21996270

ABSTRACT

INTRODUCTION: Embryo-derived PIF modulates systemic maternal immunity without suppression. Synthetic analog (sPIF) prevents juvenile diabetes, preserves islet function, reducing oxidative stress/protein misfolding. We investigate sPIF effectiveness in controlling neuroinflammation/MS. METHODS: Examine sPIF-induced protection against harsh, clinical-relevant murine EAE-PLP acute and chronic models. Evaluate clinical indices: circulating cytokines, spinal cord histology, genome, canonical global proteome, cultured PLP-activated splenocytes cytokines, and immunophenotype. RESULTS: Short-term, low-dose sPIF prevented paralysis development and lowered mortality (P<0.05). Episodic sPIF reversed chronic paralysis (P<0.0001) completely in >50%, by day 82. Prevention model: 12days post-therapy, sPIF reduced circulating IL12 ten-fold and inflammatory cells access to spinal cord. Regression model: sPIF blocked PLP-induced IL17 and IL6 secretions. Long-term chronic model: sPIF reduced spinal cord pro-inflammatory cytokines/chemokines, (ALCAM, CF1, CCL8), apoptosis-promoters, inflammatory cells access (JAM3, OPA1), solute channels (ATPases), aberrant coagulation factors (Serpins), and pro-antigenic MOG. Canonical proteomic analysis demonstrated reduced oxidative phosphorylation, vesicle traffic, cytoskeleton remodeling involved in neuro-cytoskeleton breakdown (tubulins), associated with axon re-assembly by (MTAPs)/improved synaptic transmission. CONCLUSION: sPIF--through coordinated central and systemic multi-targeted action--reverses neuroinflammation/MS and imparts significant neuroprotective effects up to total paralysis resolution. Clinical testing is warranted and planned.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/pathology , Inflammation Mediators/pharmacology , Nerve Regeneration/drug effects , Peptides/pharmacology , Animals , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Inflammation Mediators/therapeutic use , Mice , Mice, Inbred Strains , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Nerve Regeneration/immunology , Peptides/therapeutic use , Random Allocation
13.
Avian Dis ; 56(4): 720-4, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23397844

ABSTRACT

The current information on the prevalence of avian metapneumovirus (aMPV) infection in layers is fragmentary and its true impact on egg production often remains unknown or unclear. In order to draw an epidemiologic picture of aMPV presence in layer flocks in Italy, a survey was performed on 19 flocks of pullets and layers based on longitudinal studies or sporadic samplings. aMPV was detected by reverse transcription (RT)-PCR, and blood samples were collected for serology by aMPV ELISA. Occurrences of respiratory signs and a drop in egg production were recorded. Possible involvement of infectious bronchitis (IB) and egg drop syndrome (EDS) viruses that could have caused loss of egg production we ruled out for IB virus by RT-PCR, and EDS virus was ruled out by hemagglutination-inhibition (HI). Only subtype B of aMPV was found in both pullet and layer farms. Surveys of pullets showed that most groups became infected prior to the onset of lay without showing clear respiratory signs. At the point of lay, these groups were serologically positive to aMPV. In two layer flocks, egg drops were observed and could be strongly linked to the presence of aMPV infection. Results were correlated with aMPV vaccination programs applied to the birds in three flocks on the same farm. Only a vaccination program which included two live and one killed vaccines gave complete protection from aMPV infection to the birds, while a single live vaccine application was not efficacious. The current study gives an inside view of field aMPV diffusion in Italy and its control in layers.


Subject(s)
Chickens , Metapneumovirus/isolation & purification , Paramyxoviridae Infections/veterinary , Poultry Diseases/epidemiology , Poultry Diseases/prevention & control , Vaccination/methods , Adenoviridae Infections/diagnosis , Adenoviridae Infections/epidemiology , Adenoviridae Infections/veterinary , Animals , Atadenovirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Hemagglutination Inhibition Tests/veterinary , Infectious bronchitis virus/isolation & purification , Italy/epidemiology , Longitudinal Studies , Metapneumovirus/classification , Paramyxoviridae Infections/epidemiology , Paramyxoviridae Infections/prevention & control , Poultry Diseases/virology , Reproduction , Respiratory Tract Infections/epidemiology , Respiratory Tract Infections/prevention & control , Respiratory Tract Infections/virology , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Serologic Tests/veterinary , Vaccination/veterinary
14.
Appl Environ Microbiol ; 77(24): 8500-8, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21984245

ABSTRACT

Lactobacillus species are a predominant member of the vaginal microflora and are critical in maintaining an acidic vaginal environment thought to contribute to the prevention of a number of urogenital diseases. However, during menstruation the pH of the vaginal environment increases to neutrality, a pH conducive for Staphylococcus aureus proliferation and the production of toxic shock syndrome toxin 1 (TSST-1) in susceptible women. In order to generate Lactobacillus species capable of expressing lysostaphin (an endopeptidase that cleaves the cell wall of S. aureus) in a modified genital tract secretion medium (mGTS) under neutral-pH conditions, six prominent proteins from Lactobacillus plantarum WCFS1 spent medium were identified by mass spectrometry. Sequences for promoters, signal peptides, and mature lysostaphin were used to construct plasmids that were subsequently transformed into L. plantarum WCFS1. The promoter and signal sequences of Lp_3014 (putatively identified as a transglycosylase) or the promoter sequence of Lp_0789 (putatively identified as glyceraldehyde 3-phosphate dehydrogenase) with the signal sequence of Lp_3014 exhibited lysostaphin activity on buffered medium containing heat-killed S. aureus. The cassettes were integrated into the chromosome of L. plantarum WCFS1, but only the cassette containing the promoter and signal sequence from Lp_3014 had integrated into the appropriate site. Coculture assays using buffered mGTS showed that lysostaphin expressed from L. plantarum WCFS1 reduced the growth of TSST-1-producing strains of S. aureus under neutral-pH conditions. This study provides the basis for determining whether lysostaphin-producing Lactobacillus strains could potentially be used as a means to inhibit the growth of S. aureus during menstruation.


Subject(s)
Antibiosis , Lactobacillus plantarum/enzymology , Lysostaphin/metabolism , Staphylococcus aureus/growth & development , Culture Media/chemistry , Gene Expression , Humans , Lactobacillus plantarum/genetics , Organisms, Genetically Modified , Plasmids , Promoter Regions, Genetic , Protein Sorting Signals/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staphylococcus aureus/drug effects
15.
J Biol Chem ; 286(13): 11855-64, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21282109

ABSTRACT

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to license them for initiation in the upcoming S phase. After initiation, Mcm2-7 provide helicase activity to unwind DNA at the replication fork. Here we examine the structure of Mcm2-7 on chromatin in Xenopus egg extracts. We show that prior to replication initiation, Mcm2-7 is present at licensed replication origins in a complex with a molecular mass close to double that of the Mcm2-7 hexamer. This complex has approximately stoichiometric quantities of the 6 Mcm2-7 proteins and we conclude that it consists of a double heterohexamer. This provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase. We also show that after initiation, Mcm2-7 associate with Cdc45 and GINS to form a relatively stable CMG (Cdc45-MCM-GINS) complex. The CMG proteins also associate less strongly with other replication proteins, consistent with the idea that a single CMG complex forms the core of the replisome.


Subject(s)
Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , DNA Replication/physiology , Multiprotein Complexes/metabolism , Oocytes/metabolism , Xenopus Proteins/metabolism , Adenosine Triphosphatases/genetics , Animals , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell-Free System , Chromatin/metabolism , G1 Phase/physiology , Minichromosome Maintenance Complex Component 2 , Minichromosome Maintenance Complex Component 7 , Mitosis/physiology , Multiprotein Complexes/genetics , Protein Structure, Quaternary , Xenopus Proteins/genetics , Xenopus laevis
16.
Nat Cell Biol ; 12(11): 1078-85, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20935639

ABSTRACT

The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1-GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.


Subject(s)
Cell Movement , Sumoylation , rac1 GTP-Binding Protein/metabolism , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Dogs , HeLa Cells , Hepatocyte Growth Factor/metabolism , Humans , Molecular Chaperones/metabolism , Protein Inhibitors of Activated STAT/metabolism , Signal Transduction
17.
Vaccine ; 28(23): 3944-8, 2010 May 21.
Article in English | MEDLINE | ID: mdl-20392431

ABSTRACT

Avian metapneumovirus (aMPV) has become an important cause of viral respiratory infections in turkey and chickens. Live and inactivated vaccinations are available worldwide for prevention of disease and economic losses caused by this pathogen. The efficacy of these vaccines is vigorously tested under laboratory conditions prior to use in the field. In this study, a live subtype B aMPV vaccine was administered by spray, drinking water or oculo-oral methods to separate groups of broiler chicks under field conditions. Following this, the chicks were immediately transferred to separate rooms in an experimental isolation house, monitored and challenged with virulent subtype B aMPV. No clinical signs were recorded following the vaccination methods. In the oculo-oral vaccinated chicks, 40-60% of the birds were vaccine virus positive by RT-PCR. In addition, in comparison to other groups, statistically higher levels of aMPV ELISA antibodies were detected. After spray vaccination, the number of chicks positive for the vaccine virus increased gradually from 10% at one week to 30% by 3 weeks post vaccination. Following drinking water vaccination, 30% of chicks were aMPV positive at 1 week but negative by 3 weeks post vaccination. In both, spray and drinking water vaccinated groups, no ELISA antibodies were detected, but when challenged all chicks were protected against disease. At 5 days post challenge, 100% of chicks in the unvaccinated and those vaccinated by spray or drinking water routes but only 20% of the oculo-oral-vaccinated chicks were aMPV positive by RT-PCR. At 10 days post challenge, 10% of chicks in each group were aMPV RT-PCR positive. On challenge, all vaccinated chicks were protected against disease. It appears that when aMPV vaccine is accurately applied to chicks by spray or drinking water routes, both are capable of giving protection against clinical disease equal to that induced in those chicks vaccinated individually by the oculo-oral route.


Subject(s)
Paramyxoviridae Infections/veterinary , Poultry Diseases/prevention & control , Vaccination/methods , Vaccination/veterinary , Viral Vaccines/administration & dosage , Administration, Inhalation , Administration, Oral , Animals , Antibodies, Viral/blood , Chickens/immunology , Metapneumovirus/immunology , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/prevention & control , Poultry Diseases/immunology , RNA, Viral/isolation & purification , Viral Vaccines/immunology
18.
Br Poult Sci ; 51(1): 1-11, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20390564

ABSTRACT

1. The use of vaccines is the main approach to control of the economically important poultry viral respiratory diseases infectious laryngotracheitis (ILT), avian metapneumovirus (aMPV) infections and infectious bronchitis (IB). This paper appraises the current methods of vaccine control in the light of the nature of each virus and epidemiological factors associated with each disease. 2. Infectious laryngotracheitis virus (ILTV) exists as a single type with a wide range of disease severity. It is a serious disease in certain regions of the world. Recent work has distinguished molecular differences between vaccine and field strains and vaccine virus can be a cause of disease. Vaccines have remained unaltered for many years but new ones are being developed to counter vaccine side effects and reversion and reactivation of latent virus. 3. Avian metapneumoviruses, the cause of turkey rhinotracheitis and respiratory disease in chickens exists as 4 subtypes, A, B, C and D. A and B are widespread and vaccines work well provided that accurate doses are given. Newer vaccine developments are designed to eliminate reversion and possibly counter the appearance of newer field strains which may break through established vaccine coverage. 4. IB presents the biggest problem of the three. Being an unstable RNA virus, part of the viral genome that codes for the S1 spike gene can undergo mutation and recombination so that important antigenic variants can appear irregularly which may evade existing vaccine protection. While conventional vaccines work well against homologous types, new strategies are needed to counter this instability. Molecular approaches involving tailoring viruses to suit field challenges are in progress. However, the simple use of two genetically different vaccines to protect against a wide range of heterologous types is now a widespread practice that is very effective. 5. None of the three diseases described can claim to be satisfactorily controlled and it remains to be seen whether the newer generations of vaccines will be more efficacious and cost effective. The importance of constant surveillance is emphasised and the testing of novel vaccines cannot be achieved without the use of vaccine-challenge experiments in poultry.


Subject(s)
Poultry Diseases/virology , Respiratory Tract Diseases/veterinary , Viral Vaccines/immunology , Virus Diseases/veterinary , Animals , Herpesvirus 1, Gallid/immunology , Infectious bronchitis virus/immunology , Metapneumovirus/immunology , Poultry , Poultry Diseases/epidemiology , Poultry Diseases/immunology , Poultry Diseases/prevention & control , Respiratory Tract Diseases/epidemiology , Respiratory Tract Diseases/immunology , Respiratory Tract Diseases/prevention & control , Respiratory Tract Diseases/virology , Viral Vaccines/standards , Virus Diseases/epidemiology , Virus Diseases/immunology , Virus Diseases/prevention & control , Virus Diseases/virology
19.
J Proteome Res ; 8(12): 5629-41, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19899799

ABSTRACT

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.


Subject(s)
14-3-3 Proteins/chemistry , Chromatography, Affinity/methods , Guanine Nucleotide Exchange Factors/chemistry , 14-3-3 Proteins/metabolism , Animals , Binding Sites , Cell Line , Chromatography, Affinity/instrumentation , Guanine Nucleotide Exchange Factors/isolation & purification , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/isolation & purification , Phosphorylation , Protein Binding , Protein Stability , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , Tandem Mass Spectrometry
20.
Avian Dis ; 53(3): 356-62, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19848072

ABSTRACT

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3' UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3' UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.


Subject(s)
Coronavirus, Turkey/genetics , Enteritis, Transmissible, of Turkeys/virology , Genes, Viral , Turkeys , Animals , Brazil/epidemiology , DNA, Viral/genetics , Disease Outbreaks/veterinary , Enteritis, Transmissible, of Turkeys/epidemiology , Gene Expression Regulation, Viral/physiology , Phylogeny
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