The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. The enzyme RB binding protein 9 (RBBP9) is widely expressed in different tissues and upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. We previously reported a modestly potent, RBBP9 reversible inhibitor, ML081 (CID-6603320). However, ML081 exhibits high cytotoxicity. We, therefore, have now identified a newer probe, ML114 (CID-5934766), which is 10-fold more potent than ML081, exhibits no cytotoxicity, and is from an entirely different structural and mechanistic class of compounds that covalently inhibit RBBP9. This new probe will be useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
Due to its expression profile and biological action, neuropeptide Y (NPY)-Y2 receptor (Y2R) is an attractive G protein-coupled receptor (GPCR) target for anxiolytic research. Additionally, NPY-Y2R is predicted to be a therapeutic target in alcoholism. Four different compounds, ML075 (CID-2936384), ML074 (CID-2228302), ML073 (CID-3236979) and ML072 (CID-4460128) are claimed as novel antagonist probes to the NPY-Y2R, producing increased cAMP (3',5'-cyclic-AMP phosphodiesterase) levels. These probes demonstrate submicromolar affinity to the Y2 receptor, do not antagonize NPY-Y1R, do not present significant cytotoxicity, and are blood-brain barrier penetrant. Hence, these probes represent an improvement over previously described NPY-Y2R antagonists and offer greater promise to serve as valuable in vivo pharmacological probes for elucidating the Y2R signaling pathway.
Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for more than 90% of all serine/threonine phosphatase activity. Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications rather than number. Mutations in several of the PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor. Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated. Protein phosphatase methylesterase-1 (PME-1) is specifically responsible for demethylation of the carboxyl terminus. Methylesterification is thought to control the binding of different subunits to PP2A, but little is known about physiological significance of this post-translational modification in vivo. Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 knockout mice generated by targeted gene disruption result in perinatal lethality, underscoring the importance of PME-1 but hindering biological studies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a potent and selective PME-1 inhibitor probe, ML174, by high-throughput screening using fluorescence polarization-activity-based protein profiling (FluoPol-ABPP). ML174, with an IC50 of 10 nM, is based on the aza-beta-lactam scaffold and is selective for PME-1 among serine hydrolases in human cell line proteomes as assessed by gel-based competitive-activity-based protein profiling. Among more than 30 serine hydrolase anti-targets, ML174 is selective at 1 μM. Additionally, ML174 was shown in situ to be highly active against PME-1 and to result in 85% reduction of demethylated PP2A. We previously reported a modestly potent 500 nM inhibitor that was selective for PME-1, the first reported selective PME-1 inhibitor. ML174 is 50 times more potent and from an entirely different structural and mechanistic class of inhibitors. Due to its much higher potency, ML174 has greater potential for use in long time-course in situ studies, and is a much better candidate for in vivo applications.
Degenerate and specific PCR assays were developed for bovine leukaemia virus (BLV) and/or primate T cell leukaemia/lymphoma viruses (PTLV). The degenerate assays detected all major variants of the BLV/PTLV genus at a sensitivity of 10-100 copies of input DNA; the specific systems detected 1–10 copies of input target. Sensitivity was 100% in specific DNA-PCR assays done on peripheral blood from seropositive BLV-infected cattle and HTLV-I- or HTLV-II-infected humans, and 62% in RNA/DNA- PCR assays on sera from BLV seropositive cattle. The pol fragments from 21 different BLV strains, isolated from cattle in North and Central America, were cloned and sequenced, and compared to other published BLV and PTLV pol sequences. BLV and PTLV sequences differed by 42%. Sequence divergence was up to 6% among the BLV strains, and up to 36% among the PTLV strains (with PTLV-I and PTLV-II differing among themselves by 15% and 8%, respectively). Some cows were infected with several BLV strains. Among retroviruses, BLV and PTLV sequences formed a distinct clade. The data support the interpretation that BLV and PTLV evolved from a common ancestor many millennia ago, and some considerable time before the PTLV- I and PTLV-II strains diverged from each other. The dissemination of the BLV strains studied probably resulted from the export of European cattle throughout the world over the last 500 years. The relatively similar mutation rates of BLV and PTLV, after their various points of divergence, suggest that there could be a much wider genetic range of BLV than has currently been defined.
Abstract Background : Multiple organ transplants have become frequent. Combined heart‐and‐kidney grafting has been reported recently and we have pursued this in selected cases. Aims : To devise a protocol for simultaneous heart‐and‐kidney transplantation, review our clinical experience with the procedure and the causes of cardiac and renal disease in this group. Methods : Seven patients with advanced cardiac failure (LV ejection fraction < 0.29 units; five with IDCM), and chronic renal failure (serum creatinine > 375 μmol/L) due to a variety of causes, were accepted for combined heart‐and‐kidney transplantation. Four males, of mean age 33 years, underwent the procedure. Each received his organs from a single cadaveric donor with ABO blood group compatibility and a negative ‘current’ lymphocytotoxic cross‐match, but without regard to HLA‐antigen matching. Cardiac ischaemic time averaged 3 hours 40 minutes, the renal first warm time was 0 minutes in all cases, and renal cold and second warm ischaemic times averaged 5 hours 17 minutes and 52 minutes respectively. The heart was grafted first and the kidney second in a procedure which averaged seven hours. Immunosuppression was achieved by induction with antithymocyte globulin, thence steroids, azathioprine and cyclosporin A. Results : No patient required post‐operative dialysis. One patient had early urological complications requiring operative correction, but no serious opportunistic infections were observed. Early cardiac rejection on biopsy (ISHT grade 3a) was seen in three patients at four‐ten weeks and responded promptly to increased steroids, but severe steroid‐resistant rejection of both heart and kidney contemporaneously occurred in one of these three at 19 months and required a course of muromonab‐CD3. All four patients developed hypertension. Mean creatinine clearance was 1.23 ± 0.22 mL/second (74±13 mL/minute) at last follow‐up. All four recipients were alive, well and rehabilitated 5, 20, 28 and 35 months after grafting. Two patients died while waiting for the double procedure and another patient eventually died after being taken off the dual waiting list and receiving a renal transplant only. Conclusions : In experienced hands, combined heart‐and‐kidney transplantation is feasible and offers a compelling therapeutic solution in the treatment of advanced cardiac and renal failure. IDCM is a frequent cause of the heart failure in this group. (Aust NZ J Med 1994; 24: 554–560.)
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