Biomedical subjects

M Buck

Publications and source records attributed to M Buck.

At least 73 records · Page 4Linked to original sources

PubMed1999

Cisplatin and gemcitabine treatment for malignant mesothelioma: a phase II study.

PURPOSE: We performed a phase II study of combined cisplatin 100 mg/m2, given intravenously on day 1, and gemcitabine 1,000 mg/m2, given intravenously on days 1, 8, and 15 of a 28-day cycle for six cycles among patients with advanced measurable pleural mesothelioma. PATIENTS AND METHODS: Pleural tumor was measured at three levels on computed tomographic scans at study entry and before the second, fourth, and sixth cycles and every 2 months thereafter to disease progression. Of the 21 patients treated, 19 were male; the median age was 62 years (range, 46 to 74 years); 62% had epithelial tumors; and 18 were classified as tumor-node-metastasis system stage III or IV. Ninety-four cycles were given (median, six; mean, 4.5 per patient), with a mean relative dose intensity of cisplatin 96.7% and gemcitabine 82.5%. RESULTS: Best objective responses achieved were as follows: complete response, no patients; partial response, 10 patients (complete response + partial response, 47.6% [95% confidence interval, 26.2% to 69.0%]); no change, nine patients; and progressive disease, two patients. Median response duration was 25 weeks, progression-free survival was 25 weeks, and overall survival was 41 weeks. Nine of the 10 responders (90%) and three of nine patients with no change had significant symptom improvement. Serial measurements of vital capacity were performed on three of the responders; all showed a significant increase during the time of remission. Toxicity was mainly gastroenterologic and hematologic. Grade 3 nausea and vomiting occurred in 33% of patients, grade 3 leukopenia in 38%, grade 3 thrombocytopenia in 14%, and grade 4 thrombocytopenia in 19%. CONCLUSION: Combined cisplatin and gemcitabine is an active combination in malignant mesothelioma and produces symptomatic benefit in responding patients.

Aged↗

PubMed1999

Limited proteolysis of the IGF binding protein-2 (IGFBP-2) by a specific serine protease activity in early breast milk.

IGF in milk possibly promote maturation of the gastrointestinal tract in newborns. We studied the composition of milk samples derived from 99 healthy women at regular intervals during a period beginning 3 d and ending 6 mo after birth. The concentrations measured by RIA on d 3 were 10.7+/-0.4 ng/mL for IGF-II, 1.9+/-0.1 ng/mL for IGF-I, 100+/-5 ng/mL for IGF binding protein-3 (IGFBP-3), and 2163+/-108 ng/mL for IGFBP-2. All factor concentrations decreased by up to 70% in the course of the 6 mo. The most striking finding was an IGFBP-2-specific protease activity. Protease assays performed by incubation of 125I-IGFBP-2 with milk yielded fragments of 14, 16, 23, and 25 kD. 125I-IGFBP-3 was not cleaved. Proteolysis occurred only in milk from mothers until 4 wk postpartum and could be visualized by immunoblots. Since the affinity of the fragments to 125I-IGF-II was low, they were not demonstrable by ligand blot. Inhibitor studies and pH optimizing classified the IGFBP-2 protease as an Me2(+)-dependent serine protease with a pH optimum of 7 to 8. The proteolytic activity of further milk proteins, known as IGFBP proteases, was analyzed. Epidermal growth factor receptor peptide and prostate-specific antigen did not cleave IGFBP-2, although the protease activity correlated (r = 0.84, p < 0.00003) with the prostate-specific antigen concentration in milk. The y-nerve growth factor cleaved 125I-IGFBP-2, but in a completely different manner than the milk protease. In conclusion, the IGFBP-2 protease in milk is most probably a kallikrein. The specific proteolysis of IGF/IGFBP-2 complexes may increase the biologic availability of IGF in early milk. This mechanism may promote growth of the maternal breast epithelium and maturation of the gastrointestinal tract of newborns.

Female↗

PubMed1999

Stability of mutations in a Sphingomonas strain.

Sphingomonas sp. strain RW1 is able to mineralise dibenzofuran and dibenzo-p-dioxin. Three mutants were constructed that could not use dibenzofuran or dibenzo-p-dioxin as a carbon source but were able to grow with the succeeding metabolites of the pathway. Two different mutagenic agents were applied, a chemical treatment with 1-methyl-3-nitro-1-nitrosoguanidine, resulting in mutants RW1-N6 and RW1-N7, and a biological insertion mutagenesis with the mini-Tn5 transposon pBSL118, resulting in mutant RW1-M3. Southern blot analysis and PCR experiments confirmed a single insertion of the mini-Tn5 into one of the genes coding for the oxygenase component of the dibenzofuran 4,4a-dioxygenase system. The genetic stability of these mutants was examined after growth with complex medium under nonselective conditions. All three mutants failed to revert to wild-type metabolic functions.

Blotting, Southern↗

PubMed1998

Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP.

Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both.

B-Lymphocytes↗

PubMed1998

Structure of d(TGCGCA)2 and a comparison to other DNA hexamers.

The X-ray crystal structure of d(TGCGCA)2 has been determined at 120 K to a resolution of 1.3 A. Hexamer duplexes, in the Z-DNA conformation, pack in an arrangement similar to the 'pure spermine form' [Egli et al. (1991). Biochemistry, 30, 11388-11402] but with significantly different cell dimensions. The phosphate backbone exists in two equally populated discrete conformations at one nucleotide step, around phosphate 11. The structure contains two ordered cobalt hexammine molecules which have roles in stabilization of both the Z-DNA conformation of the duplex and in crystal packing. A comparison of d(TGCGCA)2 with other Z-DNA hexamer structures available in the Nucleic Acid Database illustrates the elusive nature of crystal packing. A review of the interactions with the metal cations Na+, Mg2+ and Co3+ reveals a relatively small proportion of phosphate binding and that close contacts between metal ions are common. A prediction of the water structure is compared with the observed pattern in the reported structure.

Binding Sites↗

PubMed1998

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Bacterial Proteins↗

PubMed1998

Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins.

Alcohol based cosolvents, such as trifluoroethanol (TFE) have been used for many decades to denature proteins and to stabilize structures in peptides. Nuclear magnetic resonance spectroscopy and site directed mutagenesis have recently made it possible to characterize the effects of TFE and of other alcohols on polypeptide structure and dynamics at high resolution. This review examines such studies, particularly of hen lysozyme and beta-lactoglobulin. It presents an overview of what has been learnt about conformational preferences of the polypeptide chain, the interactions that stabilize structures and the nature of the denatured states. The effect of TFE on transition states and on the pathways of protein folding and unfolding are also reviewed. Despite considerable progress there is as yet no single mechanism that accounts for all of the effects TFE and related cosolvents have on polypeptide conformation. However, a number of critical questions are beginning to be answered. Studies with alcohols such as TFE, and 'cosolvent engineering' in general, have become valuable tools for probing biomolecular structure, function and dynamics.

Magnetic Resonance Spectroscopy↗

PubMed1998

Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression.

The Epstein-Barr virus (EBV)-encoded BARF0 open reading frame gene products are consistently expressed in EBV-positive Burkitt's lymphoma (BL) cell lines, nasopharyngeal carcinoma cell lines, and lymphoblastoid cell lines (LCLs). Here we show that the BARF0 sequence includes an HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope. By using theoretically predicted HLA A2 binding motifs and peptide-loaded antigen presentation-deficient T2 cells, polyclonal BARF0-specific CD8(+) CTLs were isolated from four different healthy EBV-seropositive donors but not from two seronegative donors. These CTL lines recognized the peptide epitope LLWAARPRL, which was found to be conserved in 33 of 34 virus strains originating from Caucasian, African, and Asian individuals. The BARF0-specific CTL lines could lyse EBV-negative BL cells stably transfected with the BARF0 gene but did not kill HLA A2-matched EBV-positive BL cells and LCLs in a standard 51Cr release assay. Reverse transcriptase PCR analysis demonstrated that these EBV-positive cell lines expressed significantly lower levels of BARF0 mRNA than transfected cells. This data indicated that the BARF0 epitope could be endogenously processed; however, antigen levels in the target cell were a limiting factor for the effective interaction between BARF0-expressing cells and CTLs. The limited expression of BARF0 antigen in EBV-infected BL cells and LCLs might contribute to the escape of immune recognition from virus-specific CTLs present in the host.

Amino Acid Sequence↗

PubMed1998

TNF-alpha inhibits liver collagen-alpha 1(I) gene expression through a tissue-specific regulatory region.

Although tumor necrosis factor-alpha (TNF-alpha) inhibits collagen-alpha 1(I) gene expression in cultured hepatic stellate cells, assessment of its effects on hepatic collagen expression is complicated by the confounding variables of tissue necrosis and inflammation. Therefore, we analyzed whether chronically elevated serum TNF-alpha affects constitutive hepatic collagen metabolism in vivo by inoculating nude mice with Chinese hamster ovary (CHO) cells secreting TNF-alpha (TNF-alpha mice) or with control CHO cells (control mice). Before the onset of weight loss, collagen synthesis and collagen gene expression were inhibited in the liver of TNF-alpha mice. In transgenic mice, after 8 h, TNF-alpha (500 ng at 0 and 5 h) inhibited the liver expression of the collagen-alpha 1(I)-human growth hormone (hGH) transgene containing the first intron and -440 bp of the 5' region. Similarly, in cultured hepatic stellate cells isolated from these transgenic animals, the -440 bp collagen-alpha 1(I)-hGH transgene was responsive to TNF-alpha treatment independent of the activation of these cells. Transfection studies in stellate cells allowed further characterization of this TNF-alpha-responsive segment to -220 bp of the 5' region. Because in the skin the inhibitory effect of TNF-alpha involves a regulatory region of the collagen-alpha 1(I) gene beyond -440 bp, we herein identify a novel tissue-specific regulation of collagen-alpha 1(I) gene by TNF-alpha.

Animals↗

PubMed1998

Long- and short-term D-alpha-tocopherol supplementation inhibits liver collagen alpha1(I) gene expression.

We analyzed the role of oxidative stress on liver collagen gene expression in vivo. Long- and short-term supplementation with the lipophilic antioxidant D-alpha-tocopherol (40 IU/day for 8 wk or 450 IU for 48 h) to normal C57BL/6 mice selectively decreased liver collagen mRNA by approximately 70 and approximately 60%, respectively. In transgenic mice, the -0.44 kb of the promoter and the first intron of the human collagen alpha1(I) gene were sufficient to confer responsiveness to D-alpha-tocopherol. Inhibition of collagen alpha1(I) transactivation in primary cultures of quiescent stellate cells from these transgenic animals by D-alpha-tocopherol required only -0.44 kb of the 5' regulatory region. This regulation resembled that of the intact animal following D-alpha-tocopherol treatment and indicates that D-alpha-tocopherol may act directly on stellate cells. Transfection of stellate cells with collagen-LUC chimeric genes allowed localization of an "antioxidant"-responsive element to the -0.22 kb of the 5' region excluding the first intron. These findings suggest that oxidative stress, independently of confounding variables such as tissue necrosis, inflammation, cell activation, or cell proliferation, modulates in vivo collagen gene expression.

Animals↗

PubMed1998

The ups and downs of a kidney transplant.

While a kidney transplant is not touted by the renal community as a cure, 38,000 people are currently waiting for a chance to have one. There are pros and cons of being on dialysis versus having a kidney transplant: Dialysis means a 12-hour commitment each week, needle sticks, delicate care of your graft or fistula, and a careful watch on fluid intake. For transplant patients, flu and infections are a constant threat, as is weight gain, bone disease, and the lifetime cost and dependency of immunosuppressive medications. But most patients who have a transplant--and those who have had one and lost the graft--say they will take the risks of dealing with a suppressed immune system over dialysis. Marilyn Buck, a recipient of a kidney from her 24-year-old son in 1993, agrees. But it hasn't always been easy. Here is her story.

Adaptation, Psychological↗

PubMed1997

Probing the assembly of transcription initiation complexes through changes in sigmaN protease sensitivity.

The alternative bacterial sigmaN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of sigmaN, the assembly of end-labeled Klebsiella pneumoniae sigmaN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free sigmaN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that sigmaN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.

Bacterial Proteins↗

PubMed1997

Nucleoprotein complex formation by the enhancer binding protein nifA.

The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.

Azotobacter vinelandii↗

PubMed1997

Structural and dynamical properties of a denatured protein. Heteronuclear 3D NMR experiments and theoretical simulations of lysozyme in 8 M urea.

Oxidized and reduced hen lysozyme denatured in 8 M urea at low pH have been studied in detail by NMR methods. 15N correlated NOESY and TOCSY experiments have provided near complete sequential assignment for both 1H and 15N resonances. Over 900 NOEs, including 130 (i, i + 2) and 23 (i, i + 3) NOEs, could be identified by analysis of the NOESY spectra of the denatured states, and 3J(HN, Halpha) coupling constants and 15N relaxation rates have been measured. The coupling constant and NOE data were analyzed by comparisons with theoretical predictions from a random coil polypeptide model based on amino acid specific phi,psi distributions extracted from the protein data bank. There is significant agreement between predicted and experimental NMR parameters suggesting that local conformations of the denatured states are largely determined by short-range interactions within the polypeptide chain. This result is supported by the observation that the chemical shift, coupling constant, and NOE data are little affected by whether or not the four disulfide bridge cross-links are formed in the denatured protein. The relaxation data, however, show significant differences between the oxidized and reduced protein. Analysis of the relaxation data in terms of simple dynamics models provides evidence for weak clustering of hydrophobic groups near tryptophan residues and increased barriers to motion in the more compact conformers formed when the polypeptide chain is cross-linked by the disulfide bridges. Using this information, a structural description of these denatured states is given in terms of an ensemble of conformers, which have a complex relationship between their local and global characteristics.

Amino Acid Sequence↗

PubMed1997

Two domains within sigmaN (sigma54) cooperate for DNA binding.

The sigma-N (sigmaN) subunit of the bacterial RNA polymerase is a sequence specific DNA-binding protein. The RNA polymerase holoenzyme formed with sigmaN binds to promoters in an inactive form and only initiates transcription when activated by enhancer-binding positive control proteins. We now provide evidence to show that the DNA-binding activity of sigmaN involves two distinct domains: a C-terminal DNA-binding domain that directly contacts DNA and an adjacent domain that enhances DNA-binding activity. The sequences required for the enhancement of DNA binding can be separated from the sequences required for core RNA polymerase binding. These results provide strong evidence for communication between domains within a transcription factor, likely to be important for the function of sigmaN in enhancer-dependent transcription.

Amino Acid Sequence↗

PubMed1997

Acceleration of the folding of hen lysozyme by trifluoroethanol.

The refolding of a partially structured state of hen lysozyme formed in 60% (v/v) 2,2,2-trifluoroethanol (TFE) has been studied using hydrogen exchange pulse labelling monitored by 2D 1H NMR, and by stopped flow fluorescence and CD measurements. The results are compared with similar studies of the refolding of the protein denatured in 6 M guanidine hydrochloride (GuHCl). Two conclusions have emerged from these studies. First, provided that the refolding conditions are identical, the two denatured states fold with very similar kinetics, despite the fact the extensive secondary structure is present in the TFE-denatured state but not in the protein denatured in 6 M GuHCl. This arises because of the rapid equilibration of structure in the species formed in the initial stage of folding. Second, whilst addition of GuHCl to the refolding buffer decreases the rate of folding, low concentrations of TFE increase the rate of folding. The result is consistent with slow steps in the refolding of lysozyme being associated primarily with the reorganisation of hydrophobic interactions rather than of hydrogen bonded structure.

Animals↗

PubMed1997

Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism.

Enhancer-dependent transcription in bacteria requires the alternative transcription factor sigma N (sigma 54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of sigma N by circular dichroism (CD) analysis. The sigma N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of sigma N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36 degrees C. The secondary structure displays a two-state melting curve with a second Tm of 85 degrees C. The amino-terminal Region I activation domain together with the acidic Region II does not contribute to the two-state melting. In marked contrast, the integrity of the C-terminal DNA-binding domain is required for the two-state melting. Measurements of pKb also demonstrated that a C-terminal part of sigma N, but not regions I or I + II, is required for the structural integrity of sigma N at high pH. Measurements of pKa suggested that alpha-helical structures are important in sigma N for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of sigma N do not require regions I or I + II but were strongly diminished by C-terminal truncation of sigma N. Promoter DNA binding resulted in a conformational change in sigma N, permitting the determination of a binding constant. A typical B-DNA conformation was adopted by the promoter DNA. Implications for the modular domain organization of sigma N, the function of C-terminal sequences, and domain communication and its role in activation of transcription are discussed.

Base Sequence↗

PubMed1997

DNA-binding domain mutants of sigma-N (sigmaN, sigma54) defective between closed and stable open promoter complex formation.

The sigmaN RNA polymerase binds promoters in a transcriptionally inactive form. Activation by enhancer binding positive control proteins results in the formation of an open promoter complex. In the closed complex, DNA sequences melted upon activation are close contacted by the sigmaN C-terminal DNA-binding domain. Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigmaN function. Mutants defective in supporting sigmaN-dependent growth and in vivo promoter activation were obtained. The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro. However, they were defective in response to activator in vitro. They failed in the formation of heparin-stable promoter complexes characteristic of open promoter complexes. The sigmaN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition. The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in sigmaN are discussed.

Alanine↗