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Biomedical subjects

M Buck

Publications and source records attributed to M Buck.

At least 19 recordsLinked to original sources
PubMed2006

Phase II trial of weekly docetaxel for patients with relapsed ovarian cancer who have previously received paclitaxel--ANZGOG 02-01.

OBJECTIVE: To determine the response rate of weekly docetaxel in women with relapsed epithelial ovarian cancer previously treated with paclitaxel and at least one line of platinum-based chemotherapy. METHODS: In this multi-center phase II trial, 37 patients with relapsed disease were enrolled and treated with weekly docetaxel at 35 mg/m for 5 out of 6 consecutive weeks. Two patient cohorts were considered, those who progressed or relapsed within 4 months (N=7) or at greater than 4 months (N=30) from the time of completing their last course of paclitaxel. RESULTS: Patients in both cohorts received a median of 2 cycles of treatment (range; 1-4). In evaluable patients, the combined overall response rate, using both CA125 and RECIST response criteria was 18.9% (7/37; 95% CI; 10-34%). The combined overall progression-free survival was 3.1 months (95% CI; 2.5-3.8), and the combined overall survival was 12.3 months (95% CI; 8.2-16.4). Treatment was generally well tolerated with the only grade 4 toxicity being skin toxicity (3%). The most common grade 3 toxicities were fatigue (14%) and watery eyes (8%) with grade 3 neutropenia observed in only 5% of patients. CONCLUSION: Weekly docetaxel is well tolerated and has activity in patients with relapsed ovarian cancer previously treated with platinum and paclitaxel.

Antineoplastic Agents↗
PubMed2006

A second paradigm for gene activation in bacteria.

Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA(+) (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the sigma(54) factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The sigma(54) factor then controls the conformational changes in the RNAP required to form the open promoter complex.

Bacteria↗
PubMed2005

Self-assembled monolayers of semifluorinated alkaneselenolates on noble metal substrates.

Self-assembled monolayers (SAMs) formed from semifluorinated dialkyldiselenol (CF(3)(CF(2))(5)(CH(2))(2)Se-)(2) (F6H2SeSeH2F6) on polycrystalline Au(111) and Ag(111) were characterized by high-resolution X-ray photoelectron spectroscopy, infrared reflection absorption spectroscopy, near edge X-ray absorption fine structure spectroscopy, scanning tunneling microscopy, and contact angle measurements. The Se-Se linkage of F6H2SeSeH2F6 was found to be cleaved upon the adsorption, followed by the formation of selenolate-metal bond. The resulting F6H2Se SAMs are well-ordered, densely packed, and contamination-free. The packing density of these films is governed by the bulky fluorocarbon part, which exhibits the expected helical conformation. A noncommensurate hexagonal arrangement of the F6H2Se molecules with an average nearest-neighbor spacing of about 5.8 +/- 0.2 A, close to the van der Waals diameter the fluorocarbon chain, was observed on Au(111). The orientation of the fluorocarbon chains in the F6H2Se SAMs does not depend on the substrate-the average tilt angle of these moieties was estimated to be about 21-22 degrees on both Au and Ag.

Journal Article↗
PubMed2005

Self-assembled monolayers of aromatic selenolates on noble metal substrates.

Self-assembled monolayers (SAMs) formed from bis(biphenyl-4-yl) diselenide (BBPDSe) on Au(111) and Ag(111) substrates have been characterized by high-resolution X-ray photoelectron spectroscopy, near-edge X-ray absorption fine structure spectroscopy, infrared reflection absorption spectroscopy, water contact angle measurements, and scanning tunneling microscopy (STM). BBPDSe was found to form contamination-free, densely packed, and well-ordered biphenyl selenolate (BPSe) SAMs on both Au and Ag. Spectroscopic data suggest very similar packing density, orientational order, and molecular inclination in BPSe/Au and BPSe/Ag. STM data give a similar intermolecular spacing of 5.3 +/- 0.4 A on both Au and Ag but exhibit differences in the exact arrangement of the BPSe molecules on these two substrates, with the (2 square root[3] x square root[3])R30 degrees and (square root[3] x square root[3])R30 degrees unit cells on Au and Ag, respectively. There is strong evidence for adsorbate-mediated substrate restructuring in the case of Au, whereas no clear statement on this issue can be made in the case of Ag. The film quality of the BPSe SAMs is superior to their thiol analogues, which is presumably related to a better ability of the selenolates to adjust the surface lattice of the substrate to the most favorable 2D arrangement of the adsorbate molecules. This suggests that aromatic selenolates represent an attractive alternative to the respective thiols.

Absorption↗
PubMed2005

Cisplatin and gemcitabine induction chemotherapy followed by concurrent chemoradiotherapy or surgery for locally advanced non-small cell lung cancer.

BACKGROUND: We report a study of induction chemotherapy followed by concurrent chemoradiotherapy for stage IIIA/IIIB non-small cell lung cancer. METHODS: Patients received two cycles of induction chemotherapy with cisplatin 100 mg/m(2) on day 1 and gemcitabine 1000 mg/m(2) on days 1, 8, and 15 of a 28-day cycle. If the disease was resectable [corrected] surgery was followed with two further cycles. If unresectable, patients received cisplatin 100 mg/m(2) day 1, 29 with 5-fluorouracil 1000 mg/m(2) per 24 h continuous infusion for 96 h on days 2-5 and days 30-33 of the radiotherapy administration. Radiation therapy consisted of 63 Gy, 35 fractions, 7 weeks. RESULTS: Of 48 patients, 40% had a partial response to induction chemotherapy. Four of eleven patients with stage IIIA tumours had resectable disease. The remaining seven patients plus 37 with stage IIIB disease had chemoradiotherapy. Response at the completion of all therapy was 62% (IIIA 73%, IIIB 59%). For all patients the median survival was 15.3 months: 1 year and 3 years, 58% and 25%, respectively. Those with IIIB disease responding to induction chemotherapy had significantly superior survival to those that did not respond (37 months vs 11 months; P = 0.005). This remained significant from a landmark at 8 weeks after the start of treatment (P = 0.01). CONCLUSION: These results are equivalent to other studies using induction chemotherapy prior to concurrent chemoradiotherapy. Response to induction chemotherapy may have major prognostic significance.

Adult↗
PubMed2004

Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA+ transcriptional activator protein.

Enhancer-dependent transcriptional activators that act upon the sigma54 bacterial RNA polymerase holoenzyme belong to the extensive AAA+ superfamily of mechanochemical ATPases. Formation and collapse of the transition state for ATP hydrolysis engenders direct interactions between AAA+ activators and the sigma54 factor, required for RNA polymerase isomerization. A DNA fork junction structure present within closed complexes serves as a nucleation point for the DNA melting seen in open promoter complexes and restricts spontaneous activator-independent RNA polymerase isomerization. We now provide physical evidence showing that the ADP.AlF(x) bound form of the AAA+ domain of the transcriptional activator protein PspF changes interactions between sigma54-RNA polymerase and a DNA fork junction structure present in the closed promoter complex. The results suggest that one functional state of the nucleotide-bound activator serves to alter DNA binding by sigma54 and sigma54-RNA polymerase and appears to drive events that precede DNA opening. Clear evidence for a DNA-interacting activity in the AAA+ domain of PspF was obtained, suggesting that PspF may make a direct contact to the DNA component of a basal promoter complex to promote changes in sigma54-RNA polymerase-DNA interactions that favour open complex formation. We also provide evidence for two distinct closed promoter complexes with differing stabilities.

Adenosine Diphosphate↗
PubMed2002

Mechanochemical ATPases and transcriptional activation.

Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone-like binding activities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase-activating proteins.

Adenosine Triphosphatases↗
PubMed2001

Nuclear export of phosphorylated C/EBPbeta mediates the inhibition of albumin expression by TNF-alpha.

Decreased albumin expression is a frequent feature of cachexia patients afflicted with chronic diseases, including cancer, and a major contributor to their morbidity. Here we show that tumor necrosis-alpha (TNF-alpha) treatment of primary mouse hepatocytes or TNF-alpha overexpression in a mouse model of cachexia induces oxidative stress, nitric oxide synthase (NOS) expression and phosphorylation of C/EBPbeta on Ser239, within the nuclear localization signal, thus inducing its nuclear export, which inhibits transcription from the albumin gene. SIN-1, a NO donor, duplicated the TNF-alpha effects on hepatocytes. We found similar molecular abnormalities in the liver of patients with cancer-cachexia. The cytoplasmic localization and association of C/EBPbeta-PSer239 with CRM1 (exportin-1) in TNF-alpha-treated hepatocytes was inhibited by leptomycin B, a blocker of CRM1 activity. Hepatic cells expressing the non-phosphorylatable C/EBPbeta alanine mutant were refractory to the inhibitory effects of TNF-alpha on albumin transcription since the mutant remained localized to the nucleus. Treatment of TNF-alpha mice with antioxidants or NOS inhibitors prevented phosphorylation of C/EBPbeta on Ser239 and its nuclear export, and rescued the abnormal albumin gene expression.

Active Transport, Cell Nucleus↗
PubMed2001

Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange.

The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules.

Actins↗
PubMed2001

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action.

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Adenosine Diphosphate↗
PubMed2001

Regulatory sequences in sigma 54 localise near the start of DNA melting.

Transcription initiation by the enhancer-dependent sigma(54) RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of sigma(54) were used to map sigma(54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma(54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in sigma(54)-DNA contact and sigma(54) conformation. The centre is close to the active site of the RNA polymerase and includes sigma(54) regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.

Ascorbic Acid↗
PubMed2001

In vitro roles of invariant helix-turn-helix motif residue R383 in sigma(54) (sigma(N)).

In vitro DNA-binding and transcription properties of sigma(54) proteins with the invariant Arg383 in the putative helix-turn-helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme-promoter complexes in which limited DNA opening downstream of the -12 GC element has occurred. Unlike wild-type sigma(54), holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the -1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the sigma(54) holoenzyme, which includes the -12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of sigma(54).

Arginine↗
PubMed2001

DNA melting within a binary sigma(54)-promoter DNA complex.

The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.

Bacterial Proteins↗
PubMed2001

C/EBPbeta phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival.

Upon activation by liver injury, hepatic stellate cells produce excessive fibrous tissue leading to cirrhosis. The hepatotoxin CCl(4) induced activation of RSK, phosphorylation of C/EBPbeta on Thr(217), and proliferation of stellate cells in normal mice, but caused apoptosis of these cells in C/EBPbeta-/- or C/EBPbeta-Ala(217) (a dominant-negative nonphosphorylatable mutant) transgenic mice. Both C/EBPbeta-PThr(217) and the phosphorylation mimic C/EBPbeta-Glu(217), but not C/EBPbeta-Ala(217), were associated with procaspases 1 and 8 in vivo and in vitro and inhibited their activation. Our data suggest that C/EBPbeta phosphorylation on Thr(217) creates a functional XEXD caspase substrate/inhibitor box (K-Phospho-T(217)VD) that is mimicked by C/EBPbeta-Glu(217) (KE(217)VD). C/EBPbeta-/- and C/EBPbeta-Ala(217) stellate cells were rescued from apoptosis by the cell permeant KE(217)VD tetrapeptide or C/EBPbeta-Glu(217).

Acetylcysteine↗