The discovery of small molecules that bind to a specific target and disrupt the function of proteins is an important step in chemical biology, especially for poorly characterized proteins. Human pirin is a nuclear protein of unknown function that is widely expressed in punctate subnuclear structures in human tissues. Here, we report the discovery of a small molecule that binds to pirin. We determined how the small molecule bound to pirin by solving the cocrystal structure. Either knockdown of pirin or treatment with the small molecule inhibited melanoma cell migration. Thus, inhibition of pirin by the small molecule has led to a greater understanding of the function of pirin and represents a new method of studying pirin-mediated signaling pathways.

SummaryWe have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (vlL-10). This molecule is highly homologous to human (h)IL-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected vlL-10 expression within a few hours of EBV infection, followed, 20-30 h later by expression of hlL-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for vlL-10 mRNA (cytosolic halflife, '~6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)IL-10 rescued the transformation process in antisense-treated cells. Our observations establish vlL-10 as a new latency gene with a directly transformation-prerequisite function. EBV is a highly prevalent herpes virus associated with a growing number of lymphatic and epithelial malignancies (1-9). Lymphocytes (mainly of B cell lineage) that express the CD21 EBV receptor (10) are effidently growth transformed by the virus, and such cells develop into fatal lymphomas in susceptible hosts (11-13). EBV is maintained in its target cell as a nonintegrated, 180-kb episomal plasmid. In growth-transformed B cells the virus stays latent and transformed B cells enter lytic cycle with production of infectious virus progeny rarely at best (14). Latency of stable, growthtransformed B lymphoblasts is characterized by the expression of viral proteins, Epstein-Barr nuclear antigens (EBNAs) 1 -1, -2, -3a, b, and c, latent membrane protein (LMP-1), and terminal protein (TP)2a,b (LMP-2A/B) (15).Binding of EBV to the CD21 cell surface receptor starts two series of events that initially proceed independently although both are critical for subsequent growth transformation. A cellular activation cascade of postreceptor binding events includes calcium and proton currents and the induction of tyrosine kinase and stress proteins (16)(17)(18). Interference with any of these events prevents transformation, generating viable, principally transformable cells that harbor competent virus without undergoing growth transformation (16). I Abbreviations used in this paper: ds, double stranded; EBNA, Epstein-Barr nuclear antigen; h, human; RNA i~ EBV-induced B cell RNA; RNA c~ common B cell RNA; RT, reverse transcriptase; v, viral.The second series of events, viral latency gene expression, immediately follows circularization a...

In patients with suspected nasopharyngeal carcinoma, fine-needle aspiration can provide tissue for diagnosis by DNA amplification of EBV genomes. The presence of EBV in metastases from an occult primary tumor is predictive of the development of overt nasopharyngeal carcinoma.

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…with tolvaptan at present not being available for clinical care, other options that lower AVP activity should be considered. An alternative might be to lower AVP concentration by increasing water intake. In a rat model for polycystic kidney disease, a 3.5 fold increased water intake reduced urinary osmolality, renal expression of the AVP V2 receptor and reduced kidney weight compared to normal water intake…

Following Epstein-Barr virus (EBV) binding to its CD21 cell surface receptor, virus internalization, nuclear translocation, and circularization of the viral episome were found to occur within 30 min, immediately preceding the expression of EB nuclear antigen (EBNA)-1 and -2 and latent membrane protein (LMP)-1 and -2 genes. Early viral gene expression was unaffected by blockade of the virus induced, transformation-prerequisite cellular activation pathway (Ca2+ currents, tyrosine phosphorylation, induction of p56lck, hsp70, and hsp90). Despite life times of only 3 h, antisense (but not sense) oligonucleotides for the above latency genes prevented subsequent transformation. Any one antisense oligonucleotide dramatically depleted transcripts not only of the target gene, but of all other latency genes. The blocking effect of antisense oligonucleotides allowed us to identify a new transformation-prerequisite latency gene near the fused termini. The concerted regulation of EBV gene expression is highly unusual and unexplained but our results imply critical, perhaps regulatory roles for initial latency gene transcripts themselves.

Introduction: In the Mayo Imaging Classification (MIC) for autosomal dominant polycystic kidney disease (ADPKD), the height-adjusted total kidney volume (HtTKV) growth rate is estimated for classification. Estimated HtTKV slope, termed as eHTKV-a, is calculated by the equation [HtTKV at age t] ¼ K(1þa/100) (t-A) , where K ¼ 150 and A ¼ 0 are used in MIC. If eHTKV-a is nearly stable during a standard-ofcare period, the change in eHTKV-a from baseline can be used for estimation of the treatment effect on the HtTKV slope. Methods: The constancy of eHTKV-a (A ¼ 0 and K ¼ 150) was evaluated using 453 placebo-assigned subjects in the Tolvaptan Efficacy and Safety in Management of ADPKD and Its Outcomes (TEMPO) 3:4 trial. A and K were sought out respectively by a converged pattern of regression lines of log10(HtTKV) plotted against age for subgroups divided according to MIC, and by change in eHTKV-a from baseline. A total of 239 standard-of-care patients from the Kyorin University Cohort (KUC) served as validation. Changes in eHTKV-a from baseline were evaluated in 809 tolvaptan-treated subjects in TEMPO 3:4. Results: In placebo-assigned subjects, eHTKV-a (A ¼ 0 and K ¼ 150) changed significantly from baseline at the third year. As regression lines of placebo-assigned subgroups converged around age 0, A was set as 0, which was confirmed by KUC. K ¼ 130 was selected because of minimal change in eHTKV-a from baseline. The KUC validated the constancy of eHTKV-a (A ¼ 0 and K ¼ 130) but not that of eHTKV-a (A¼0 and K¼150). In tolvaptan-treated subjects, eHTKV-a remained significantly lower than baseline for 3 years. Conclusions: eHTKV-a (A ¼ 0 and K ¼ 130) was nearly stable from baseline through follow-up in standardof-care adults. Treatment effects on the HtTKV slope can be estimated by changes in eHTKV-a from baseline.

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.