1: Mol Cell Biol. 2004 Aug;24(15):6871-86. A triad of subunits from the Gal11/tail domain of Srb mediator is an in vivo target of transcriptional activator Gcn4p. Zhang F, Sumibcay L, Hinnebusch AG, Swanson MJ. Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Building 6A/Room B1A13, Bethesda, MD 20892, USA. The Srb mediator is an important transcriptional coactivator for Gcn4p in the yeast Saccharomyces cerevisiae. We show that three subunits of the Gal11/tail domain of mediator, Gal11p, Pgd1p, and Med2p, and the head domain subunit Srb2p make overlapping contributions to the interaction of mediator with recombinant Gcn4p in vitro. Each of these proteins, along with the tail subunit Sin4p, also contributes to the recruitment of mediator by Gcn4p to target promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a stable subcomplex in sin4Delta cells that interacts with Gcn4p in vitro and that is recruited independently of the rest of mediator by Gcn4p in vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for recruitment of intact mediator and appears to be sufficient for recruitment by Gcn4p as a free subcomplex. The med2Delta mutation impairs the recruitment of TATA binding protein (TBP) and RNA polymerase II to the promoter and the induction of transcription at ARG1, demonstrating the importance of the tail domain for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p triad is the only portion of Srb mediator recruited efficiently to the promoter in the sin4Delta strain, this mutant shows high-level TBP recruitment and wild-type transcriptional induction at ARG1. Hence, the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently of the rest of mediator. PMID: 15254252 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Nucleic Acids Res. 2004 Jun 14;32(10):3180-9. Print 2004. Direct interaction of NRSF with TBP: chromatin reorganization and core promoter repression for neuron-specific gene transcription. Murai K, Naruse Y, Shaul Y, Agata Y, Mori N. Department of Molecular Genetics, National Institute for Longevity Sciences, Gengo 36-3, Morioka, Oobu, Aichi 474-8522, Japan. Neural restrictive silencer factor, NRSF (also known as REST) binds a neuronal cell type selective silencer element to mediate transcriptional repression of neuron-specific genes in non-neuronal cells and neuronal progenitors. Two repression domains (RD-1 and RD-2) occur in its N-terminal and C-terminal regions, respectively. RD-1 recruits mSin3 and HDAC, thereby inhibiting transcription by inducing reorganization of the chromatin structure. However, little is known about how such global repression becomes promoter-specific repression or whether the NRSF-HDAC complex can interact with transcriptional core factors at each specific promoter. Here we show evidence that NRSF interacts with core promoter factors, including TATA-binding protein (TBP). The NRSF-TBP interaction occurred between the linear segments of the N- and C-terminal-most portions of NRSF and the C-terminal half of TBP. A RD-2 mutant of NRSF lost the TBP-binding activity and was unable to repress transcription at an exogenously introduced TGTA promoter. These results indicate that the direct interaction between the NRSF C-terminal domain and TBP is essential for the C-terminal repression mechanism of NRSF. Thus, the RD-1 and RD-2 repression domains of NRSF utilize both chromatin-dependent and chromatin-independent mechanisms, which may be segregated at various stages of neural development and modulation. PMID: 15197246 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Hepatol. 2004 Feb;40(2):228-33. Weight gain after transjugular intrahepatic portosystemic shunt is associated with improvement in body composition in malnourished patients with cirrhosis and hypermetabolism. Plauth M, Schutz T, Buckendahl DP, Kreymann G, Pirlich M, Grungreiff S, Romaniuk P, Ertl S, Weiss ML, Lochs H. Medizinische Klinik mit Schwerpunkt Gastroenterologie, Hepatologie und Endokrinologie, Charite Universitatsmedizin Berlin, Berlin, Germany. mathias.plauth@klinikum-dessau.de BACKGROUND/AIMS: To search for changes in body composition and energy metabolism associated with the repeatedly observed weight gain of cirrhotic patients after portosystemic shunting. METHODS: Twenty-one patients were studied prospectively before and 6 and 12 months after transjugular intrahepatic portosystemic shunt (TIPS) to assess body cell mass by two independent methods (total body potassium counting: body cell mass determined by TBP, BCMTBP, bioelectric impedance analysis: body cell mass determined by BIA, BCMBIA), muscle mass (anthropometry), resting energy expenditure (REECALO) by indirect calorimetry, and nutritional intake by dietary recall analysis. RESULTS: Prior to TIPS patients were hypermetabolic in terms of measured vs. predicted REE (REECALO median 1423 (range 1164-1838) vs. REEPRED 1279 (1067-1687) kcal; P<0.05) and their body cell mass was lower (19.1 (10.9-33.4) vs. 31.7 (16.8-47.1) kg; P=0.001). After TIPS body cell mass (BCMBIA) increased to 23.5 (12.7-44.3) (P<0.025) and 25.7 (14.2-39.7) kg (P=0.05) at 6 and 12 months after TIPS and this was confirmed by total potassium counting (BCMTBP before TIPS: 18.8 (10.6-26.7) vs. 22.4 (12.9-28.5) kg at 6 months; P<0.01). Hypermetabolism persisted throughout the study period. Energy and protein intake increased significantly by 26 and 33%. CONCLUSIONS: An increase of prognostically relevant variables body cell and muscle mass contributes to the weight gain after TIPS in malnourished patients with cirrhosis and hypermetabolism. PMID: 14739092 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Reg Anesth Pain Med. 2002 Jan-Feb;27(1):15-22. Comment in: Reg Anesth Pain Med. 2002 Jan-Feb;27(1):6-8. Reg Anesth Pain Med. 2002 Sep-Oct;27(5):535; author reply 535-6. Multimodal analgesia and intravenous nutrition preserves total body protein following major upper gastrointestinal surgery. Barratt SM, Smith RC, Kee AJ, Mather LE, Cousins MJ. Department of Anaesthesia and Pain Management, University of Sydney at Royal North Shore Hospital, Sydney, NSW, Australia. sbarratt@mail.usyd.edu.au BACKGROUND AND OBJECTIVES: This study examined whether perioperative multimodal analgesia (MMA) improves the effectiveness of intravenous nutrition (IVN) as a means of preventing protein wasting following major upper abdominal surgery (UAS). The MMA regimen utilized combined epidural opioid/local anesthetic and the systemic nonsteroidal anti-inflammatory drug (NSAID) ketorolac for 48 hours. METHODS: In a prospective, randomized, nonblinded study, 47 patients scheduled for major UAS were allocated to receive the following: MMA +/- intravenous lipid-based nutrition (IVN) or patient-controlled analgesia with opioids (PCA) +/- IVN. Pain scores, nitrogen balance, total body protein (TBP), arterial blood gases, and various hormones were measured. RESULTS: Pain control was significantly better in the MMA patients at rest and coughing. Only the MMA + IVN group maintained TBP, mean (+/-95% confidence interval) preoperative day 1, 10.5 (+/-1.0) kg; day 14, 10.7 (+/-1.2) kg, whereas TBP decreased in the other groups (P =.04). Nitrogen balance was significantly greater in patients receiving IVN on day 7 (P =.01), but there was no effect related to the analgetic regimen. Decreased PaO(2) seen on postoperative day 2 was not prevented by MMA. The hormonal response to surgery was not influenced by treatment modality, apart from a return to postprandial insulin levels on postoperative day 7 in those patients receiving IVN (P =.002). CONCLUSIONS: In conclusion, we have shown that the combination of MMA and IVN prevents protein loss and improves pain control after major UAS. Our results suggest that after UAS, MMA significantly reduced pain and, in combination with IVN, preserves total body protein and fat. This is the first direct evidence of such effects associated with a commonly used multimodal regimen. Publication Types: Clinical Trial Randomized Controlled Trial PMID: 11799500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Gene. 2000 Jan 25;242(1-2):1-13. TAFs revisited: more data reveal new twists and confirm old ideas. Albright SR, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA. Synthesis of messenger RNA by RNA polymerase II requires the combined activities of more than 70 polypeptides. Coordinating the interaction of these proteins is the basal transcription factor TFIID, which recognizes the core promoter and supplies a scaffolding upon which the rest of the transcriptional machinery can assemble. A multisubunit complex, TFIID consists of the TATA-binding protein (TBP) and several TBP-associated factors (TAFs), whose primary sequences are well-conserved from yeast to humans. Data from reconstituted cell-free transcription systems and binary interaction assays suggest that the TAF subunits can function as promoter-recognition factors, as coactivators capable of transducing signals from enhancer-bound activators to the basal machinery, and even as enzymatic modifiers of other proteins. Whether TAFs function similarly in vivo, however, has been an open question. Initial characterization of yeast bearing mutations in particular TAFs seemingly indicated that, unlike the situation in vitro, TAFs played only a minor role in transcriptional regulation in vivo. However, reconsideration of this data in light of more recent results from yeast and other organisms reveals considerable convergence between the models derived from in vitro experiments and those derived from in vivo studies. In particular, there is an emerging consensus that TAFs represent one of several classes of coactivators that participate in transcriptional activation in vivo. Publication Types: Review Review, Tutorial PMID: 10721692 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Biophys J. 1998 Nov;75(5):2411-21. Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors. Pardo L, Pastor N, Weinstein H. Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029, USA. We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there. PMID: 9788936 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Biol Chem. 1997 Dec 5;272(49):30651-61. The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter. Leveillard T, Wasylyk B. Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS, INSERM, ULP, 1 Rue Laurent Fries, BP 163, 67404 Illkirch cedex, France. MDM2 proto-oncogene expression is aberrant in many human tumors. Its normal role is to modulate the functions of p53. The N terminus of MDM2 interacts with p53, whereas the properties of the rest of the molecule are poorly understood. We show that MDM2 binds to the general transcription factor TFIID in vivo. The C-terminal Ring finger interacts with TAFII250/CCG1, and the central acidic domain interacts with TBP. Expression of MDM2 activates the cyclin A gene promoter but not c-fos, showing that the effects of MDM2 are specific. Deletion of the C-terminal region of MDM2 abolishes activation, showing that the C-terminal domain of MDM2 is functionally important. We found that increasing MDM2 expression to higher levels inhibits the cyclin A promoter. Inhibition appears to result from titration of general transcription factors because MDM2 overexpression inhibits c-fos as well as other promoters in vivo and basal transcription in vitro. The mechanisms of repression of the cyclin A and fos promoters appear to be different. Cyclin A repression is lost by deleting the C terminus, whereas that of c-fos is lost by removal of the acidic domain. These results reinforce the conclusion that the C terminus of MDM2 mediates effects on the cyclin A promoter. MDM2 transformed cells contain elevated levels of cyclin A mRNA, showing that activation occurs under physiological conditions. There is a positive correlation between MDM2 binding to TAFII250 and MDM2 activation of the cyclin A promoter. The C-terminal region of MDM2, which contains the Ring finger, interacts with TAFII250 and is required for regulation of the cyclin A promoter by MDM2. Our results link the activity of MDM2, a transforming protein implicated in many human tumors, with cyclin A, a regulator of the cell cycle. PMID: 9388200 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Biochemistry. 1995 Jun 27;34(25):8005-17. Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics. Perez-Howard GM, Weil PA, Beechem JM. Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA. A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation of basal level transcription, and an amino terminal domain (residues 1-60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino terminal domain of TBP undergoes a huge (30-40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. This "fluorescent" DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly described by a single second-order rate constant of 1.66 x 10(5) M(-1) s(-1). These measurements represent the very first stopped-flow anisotropy study of a protein/DNA interaction. Stopped-flow anisotropy off-rate experiments reveal a single exponential k(off) of 4.3 x 10(-2) min-1 (1/k(off) = 23 min) From the ratio of on-rate to off-rate, a predicted Kd of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino terminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior. PMID: 7794913 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Plant Mol Biol. 1993 Dec;23(5):995-1003. DNA sequence requirement of a TATA element-binding protein from Arabidopsis for transcription in vitro. Mukumoto F, Hirose S, Imaseki H, Yamazaki K. School of Agricultural Sciences, Nagoya University, Japan. We have analyzed the DNA sequence requirements for the functioning of TATA elements by examining the transcriptional activities associated with 24 promoters, including representatives of each of the 21 point mutations in the consensus sequence from plants, TATATATA, in a HeLa in vitro system and in a chimeric in vitro system in which human TATA-binding protein (hTBP) was replaced by purified TBP of Arabidopsis (aTBP-1). Although the relative transcriptional activities varied among these promoters, both systems gave virtually identical results. Among the mutant TATA elements, those with the sequences TAGAGATA and GAGAGAGA had undetectable activity. The rest had activities that ranged from 7% to 130% of the activity associated with the consensus element. These results suggest the functional conservation of TBP between plants and animals. PMID: 8260636 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Beitr Infusionsther. 1991;28:92-109. Results of a quality-control study of lyophilized pooled plasmas which have been 'virally inactivated' using a solvent detergent method (modified Horowitz procedure). Trobisch H. Institut fur Laboratoriumsmedizin, Duisburg. The 'virus-free' lyophilized pooled plasmas supplied to our institute by the German Red Cross in Hagen did not meet the quality norms found in standard products. Indeed, with respect to all the major parameters, they deviated greatly from standard coagulative fresh frozen plasmas. In order to achieve a suitable substitution effect and approximate the properties of fresh plasma, it would be necessary to administer two to three times the amount of 'virus-free' plasma. At the same time it should be noted that by contrast to coagulative fresh plasma, the new product neither compensates for factors which activate or inhibit coagulation, nor for fibrinolytic factors. On the contrary, the ratio between these factors deviates dangerously from their physiological equilibrium. Grave therapeutic consequences can be expected therefrom. In addition, most of the tested batches already contain heparin in quantities within the therapeutic range in spite of the fact that the manufacturer neglects to mention this detail. Finally, the method of preparation pushes the pH values far into the alkaline range. This fact alone could have fatal consequences if this product was administered to severely ill patients. In general, we can only express our surprise that the Bundesgesundheitsamt (German Health Board), as the official body responsible for approving this product, has agreed to its distribution on the basis of notification that changes would be made to an existing approved product. The following facts, in our opinion, should have determined the renewal of the licence for sale: As opposed to the approved product--a plasma derived from individual donors--the 'virus-free' plasma is a pooled plasma. As opposed to the approved product--a deep-frozen plasma derived from individual donors--the 'virus-free' plasma is a pooled lyophilized plasma. As opposed to the approved product which is not subjected to any chemical processing, the 'virus-free' product is treated with tri(n)butylphosphate and an unspecified detergent. These virucidal properties are assumed on the basis of consequential logic rather than proven by hard experimentally determined fact. Data confirming the efficiency of the process based on legitimate animal experiments has not be presented. In spite of all the efforts to improve safety in transfusion medicine we believe that if certain nonprofit-making blood banks are to retain their credibility, they should be subjected to the same stringent laws and regulations that are applied to the rest of the German pharmaceutical industry.(ABSTRACT TRUNCATED AT 400 WORDS) PMID: 1725667 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------