Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin- stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca2+ mobilisation. in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1 alpha (D26A) (MIP-1 alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCLS), MIP-1 beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin -stimulated CAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCLS, CCL8 and CCL13 were able to stimulate Ca2+ mobilisation. through CCRS, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca2+ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca2+ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca2+ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCRS. (C) 2007 Elsevier Inc. All rights reserved.

Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier.

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

Analysis of wheat SAGE tags reveals evidence for widespread antisense transcription

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat. RESULTS: Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression. CONCLUSION: Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.

Proteornics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructurall protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Analysis of protein-protein interactions in the feline calicivirus replication complex

Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein-protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.

Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Calicivirus translation initiation requires an interaction between VPg and eIF4E

Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 50 end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF4E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF4E by 4E-BP1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF4E and the cap structure or 4E-BP1, suggesting that VPg binds to eIF4E at a different site from both cap and 4E-BP1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.

A novel method for the transport and analysis of genetic material from polyps and zooxanthellae of scleractinian corals

We have developed a new simple method for transport, storage, and analysis of genetic material from the corals Agaricia agaricites, Dendrogyra cylindrica, Eusmilia ancora, Meandrina meandrites, Montastrea annularis, Porites astreoides, Porites furcata, Porites porites, and Siderastrea siderea at room temperature. All species yielded sufficient DNA from a single FTA(R) card (19 mug-43 ng) for subsequent PCR amplification of both coral and zooxanthellar DNA. The D1 and D2 variable region of the large Subunit rRNA gene (LSUrDNA) was amplified from the DNA of P. furcata and S. siderea by PCR. Electrophoresis yielded two major DNA bands: an 800-base pair (bp) DNA, which represented the coral ribosomal RNA (rRNA) gene, and a 600-bp DNA, which represented the zooxanthellar srRNA gene. Extraction of DNA from the bands yielded between 290 mug total DNA (S. siderea coral DNA) and 9 mug total DNA (P. furcata zooxanthellar DNA). The ability to transport and store genetic material from scleractinian corals without resort to laboratory facilities in the field allows for the molecular Study of a far wider range and variety of coral sites than have been studied to date. (C) 2003 Elsevier Science B.V. All rights reserved.

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

Analysis of the sulfate-reducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments

The distribution and activity of communities of sulfate-reducing bacteria (SRB) and methanogenic archaea in two contrasting Antarctic sediments were investigated. Methanogenesis dominated in freshwater Lake Heywood, while sulfate reduction dominated in marine Shallow Bay. Slurry experiments indicated that 90% of the methanogenesis in Lake Heywood was acetoclastic. This finding was supported by the limited diversity of clones detected in a Lake Heywood archaeal clone library, in which most clones were closely related to the obligate acetate-utilizing Methanosaeta concilii. The Shallow Bay archaeal clone library contained clones related to the C-1-utilizing Methanolobus and Methanococcoides and the H-2-utilizing Methanogenium. Oligonucleotide probing of RNA extracted directly from sediment indicated that archaea represented 34% of the total prokaryotic signal in Lake Heywood and that Methanosaeta was a major component (13.2%) of this signal. Archaea represented only 0.2% of the total prokaryotic signal in RNA extracted from Shallow Bay sediments. In the Shallow Bay bacterial clone library, 10.3% of the clones were SRB-like, related to Desulfotalea/Desulforhopalus, Desulfofaba, Desulfosarcina, and Desulfobacter as well as to the sulfur and metal oxidizers comprising the Desulfuromonas cluster. Oligonucleotide probes for specific SRB clusters indicated that SRB represented 14.7% of the total prokaryotic signal, with Desulfotalea/Desulforhopalus being the dominant SRB group (10.7% of the total prokaryotic signal) in the Shallow Bay sediments; these results support previous results obtained for Arctic sediments. Methanosaeta and Desulfotalea/Desulforhopalus appear to be important in Lake Heywood and Shallow Bay, respectively, and may be globally important in permanently low-temperature sediments.

A structural analysis of M protein in coronavirus assembly and morphology

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.

Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca++ stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2α. Since TCTP has been characterized as an antiapoptotic and Ca++-binding protein, we asked whether it is involved in protecting cells from Ca++-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2α phosphorylation.

Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe- set, consisting of up to 16 probe-pairs. Signal intensities across probe- pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.

Global transcriptome analysis and identification of differentially expressed genes after infection of Fragaria vesca with powdery mildew (Podosphaera aphanis)

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry