We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation. We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR). The beta-lactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo. Three GPCRs were studied, including the chemokine receptor CXCR4 (Gi-coupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), and beta2 adrenergic receptor (Gs-coupled) that was both stably transfected. Agonists for each receptor activated transcription of the beta-lactamase tagged EGR-3 gene. Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase). Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3. Conversely, beta2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580. In addition, both beta2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation. This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation.
Culture Media ; DNA-Binding Proteins ; metabolism ; Early Growth Response Protein 3 ; Gene Expression Regulation ; Genes, Reporter ; Humans ; Jurkat Cells ; MAP Kinase Signaling System ; Receptors, CXCR4 ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; Transcription Factors ; metabolism ; beta-Lactamases ; metabolism

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.
Amino Acid Motifs ; genetics ; Amino Acid Sequence ; Antigens, Viral ; immunology ; Base Composition ; Base Sequence ; Cluster Analysis ; Computational Biology ; DNA Primers ; Enzyme-Linked Immunosorbent Assay ; Genetic Variation ; Molecular Sequence Data ; Nucleocapsid Proteins ; genetics ; immunology ; metabolism ; Phosphorylation ; SARS Virus ; genetics ; Sequence Analysis, DNA

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Base Sequence ; China ; Cluster Analysis ; Gene Components ; Genetic Variation ; Genome, Viral ; Genotype ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; genetics

Hydrocephalus is often treated with a cerebrospinal fluid shunt (CFS) for excessive amounts of cerebrospinal fluid in the brain. However, it is very difficult to distinguish whether the ventricular enlargement is due to hydrocephalus or other causes, such as brain atrophy after brain damage and surgery. The non-trivial evaluation of the consciousness level, along with a continuous drainage test of the lumbar cistern is thus clinically important before the decision for CFS is made. We studied 32 secondary mild hydrocephalus patients with different consciousness levels, who received T1 and diffusion tensor imaging magnetic resonance scans before and after lumbar cerebrospinal fluid drainage. We applied a novel machine-learning method to find the most discriminative features from the multi-modal neuroimages. Then, we built a regression model to regress the JFK Coma Recovery Scale-Revised (CRS-R) scores to quantify the level of consciousness. The experimental results showed that our method not only approximated the CRS-R scores but also tracked the temporal changes in individual patients. The regression model has high potential for the evaluation of consciousness in clinical practice.

OBJECTIVE To systematically review the background of bioequivalence assessment of nasal sprays and nasal aerosols and the guiding considerations for the bioequivalence assessment of these complex drug-device combination products by regulatory authorities in the United States, the European Union(EU) and China. METHODS This article provided detailed explanations on the innovative weight of evidence assessment approach adopted by the US Food and Drug Administration(FDA), and the statistical rationale, methods and considerations for the bioequivalence assessment of nasal sprays and nasal aerosols. Using the calculation methods described in the draft guidance for budesonide inhalation suspension and the draft guidance for fluticasone nasal spray propionate issued by FDA, the statistical parameters of two-sided and one-sided population bioequivalence calculation were realized through R language programming, and pseudo-code for the population bioequivalence (PBE) calculation programs was provided. This article also presented a comprehensive review of published guidelines and summaries review principles of the EU and China for nasal sprays and nasal aerosols equivalence assessment. RESULTS & CONCLUSION Nasal sprays/nasal aerosols is the focus of innovative and generic drug development in recent years. This paper provided valuable considerations references for the research and development, quality control and bioequivalence evaluation of generic preparations of nasal sprays/nasal aerosols.