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1.
Transl Psychiatry ; 7(1): e1008, 2017 01 24.
Article in English | MEDLINE | ID: mdl-28117844

ABSTRACT

Several previous functional magnetic resonance imaging (fMRI) studies have demonstrated the predictive value of brain activity during emotion processing for antidepressant response, with a focus on clinical outcome after 6-8 weeks. However, longitudinal studies emphasize the paramount importance of early symptom improvement for the course of disease in major depressive disorder (MDD). We therefore aimed to assess whether neural activity during the emotion discrimination task (EDT) predicts early antidepressant effects, and how these predictive measures relate to more sustained response. Twenty-three MDD patients were investigated once with ultrahigh-field 7T fMRI and the EDT. Following fMRI, patients received Escitalopram in a flexible dose schema and were assessed with the Hamilton Depression Rating Scale (HAMD) before, and after 2 and 4 weeks of treatment. Deactivation of the precuneus and posterior cingulate cortex (PCC) during the EDT predicted change in HAMD scores after 2 weeks of treatment. Baseline EDT activity was not predictive of HAMD change after 4 weeks of treatment. The precuneus and PCC are integral components of the default mode network (DMN). We show that patients who exhibit stronger DMN suppression during emotion processing are more likely to show antidepressant response after 2 weeks. This is, to our knowledge, the first study to show that DMN activity predicts early antidepressant effects. However, DMN deactivation did not predict response at 4 weeks, suggesting that our finding is representative of early, likely treatment-related, yet unspecific symptom improvement. Regardless, early effects may be harnessed for optimization of treatment regimens and patient care.


Subject(s)
Antidepressive Agents/therapeutic use , Brain/physiopathology , Citalopram/therapeutic use , Depressive Disorder, Major/drug therapy , Adolescent , Adult , Depressive Disorder, Major/physiopathology , Emotions , Female , Functional Neuroimaging , Gyrus Cinguli/physiopathology , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Neural Pathways/physiopathology , Parietal Lobe/physiopathology , Treatment Outcome , Young Adult
2.
Protein Expr Purif ; 120: 42-50, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26687898

ABSTRACT

N(pro) fusion technology, a highly efficient system for overexpression of proteins and peptides in Escherichia coli, was further developed by splitting the autoprotease N(pro) into two fragments to generate a functional complementation system. The size of the expression tag is thus reduced from 168 to 58 amino acids, so by 66%. Upon complementation of the fragments auto-proteolytic activity is restored. This process has been shown for three model proteins of different size, a short 16 aa-peptide, MCP-1, and lysozyme. Moreover, the complementation was still functional after immobilization of the N-terminal fragment to a solid support which enables recycling of the immobilized fragment. This strategy enhances overall productivity of N(pro) Fusion Technology and thus allows more efficient production of recombinant proteins with reduced costs and in higher yields. Overall, the N(pro) complementation system has, depending on the size of the target molecule, potential to increase the productivity up to 4 fold for batch refolding and even more for on-column refolding strategies by the proven possibility of regeneration of the immobilized fragment.


Subject(s)
Escherichia coli/genetics , Peptide Hydrolases , Recombinant Fusion Proteins/isolation & purification , Up-Regulation , Amino Acid Sequence , Chemokine CCL2/isolation & purification , Chemokine CCL2/metabolism , Chromatography, Affinity , Escherichia coli/metabolism , Molecular Sequence Data , Muramidase/isolation & purification , Muramidase/metabolism , Peptides/isolation & purification , Peptides/metabolism , Protein Refolding , Recombinant Fusion Proteins/metabolism
3.
J Chem Phys ; 132(22): 224503, 2010 Jun 14.
Article in English | MEDLINE | ID: mdl-20550404

ABSTRACT

We introduce a sparse-matrix algorithm that allows for the simulation of two-dimensional infrared (2DIR) spectra in systems with many coupled chromophores. We apply the method to bulk water, and our results are based on the recently developed ab initio maps for the vibrational Hamiltonian. Qualitative agreement between theory and experiment is found for the 2DIR spectra without the use of any fitting or scaling parameters in the Hamiltonian. The calculated spectra for bulk water are not so different from those for HOD in D(2)O, which we can understand by considering the spectral diffusion time-correlation functions in both cases. We also calculate the ultrafast anisotropy decay, which is dominated by population transfer, finding very good agreement with experiment. Finally, we determine the vibrational excitation diffusion rate, which is more than two orders of magnitude faster than the diffusion of the water molecules themselves.

4.
J Chem Phys ; 131(14): 144511, 2009 Oct 14.
Article in English | MEDLINE | ID: mdl-19831456

ABSTRACT

We study theoretically the steady-state and ultrafast vibrational spectroscopy, in the OD-stretch region, of dilute HOD in aqueous solutions of sodium bromide. Based on electronic-structure calculations on clusters containing salt ions and water, we develop new spectroscopic maps that enable us to undertake this study. We calculate OD-stretch absorption line shapes as a function of salt concentration, finding good agreement with experiment. We provide molecular-level understandings of the monotonic (as a function of concentration) blueshift, and nonmonotonic line width. We also calculate the frequency time-correlation function, as measured by spectral diffusion experiments. Here again we obtain good agreement with experiment, finding that at the highest salt concentration spectral diffusion slows down by a factor of 3 or 4 (compared to pure water). For longer times than can be accessed experimentally, we find that spectral diffusion is very complicated, with processes occurring on multiple time scales. We argue that from 6 to 40 ps, relaxation involves anionic solvation shell rearrangements. Finally, we consider our findings within the general context of the Hofmeister series, concluding that this series must reflect only local ordering of water molecules.


Subject(s)
Bromides/chemistry , Sodium Compounds/chemistry , Vibration , Water/chemistry , Deuterium Oxide/chemistry , Diffusion , Electrons , Hydroxides/chemistry , Molecular Dynamics Simulation , Salts/chemistry , Solutions , Spectrophotometry, Infrared
5.
J Phys Chem B ; 113(13): 4125-30, 2009 Apr 02.
Article in English | MEDLINE | ID: mdl-19425249

ABSTRACT

We present theoretical calculations of the vibrational sum-frequency susceptibility for the water liquid/vapor interface. Our approach builds on previous calculations by us and others, using the time-averaging approximation within the mixed quantum/classical formulation for coupled vibrational chromophores, and electric-field maps for transition frequencies, dipoles, polarizabilities, and intramolecular vibrational couplings. We compare our results for the imaginary part of the susceptibility to those from recent experiments, and comment about the effects of intermolecular vibrational coupling and the assignment of features in the spectrum.

6.
J Chem Phys ; 129(21): 214705, 2008 Dec 07.
Article in English | MEDLINE | ID: mdl-19063573

ABSTRACT

An electronic structure/molecular dynamics approach, originally developed to describe the vibrational spectroscopy of the OH stretch of dilute HOD in liquid D(2)O, is applied to the vibrational sum-frequency spectroscopy of the liquid/vapor interface of this system. In both cases the OH stretch is effectively decoupled from the OD stretches, allowing it to act as a local probe of structure and dynamics. A mixed quantum/classical expression for the vibrational sum-frequency response that includes the effect of motional narrowing is used to calculate the resonant susceptibility. Despite being developed for the bulk liquid, our method works well for the surface in that the real and imaginary parts of the resonant susceptibility are in good agreement with experiment. We explore the nature of hydrogen bonding at the interface as well as its impact on the sum-frequency spectrum. It is found that the spectrum is dominated by single-donor molecules with a total of two or three hydrogen bonds.

7.
J Chem Phys ; 128(22): 224511, 2008 Jun 14.
Article in English | MEDLINE | ID: mdl-18554033

ABSTRACT

IR and Raman (parallel- and perpendicular-polarized) spectra in the OH stretch region for liquid water were measured some years ago, but their interpretation is still controversial. In part, this is because theoretical calculation of such spectra for a neat liquid presents a formidable challenge due to the coupling between vibrational chromophores and the effects of motional narrowing. Recently we proposed an electronic structure/molecular dynamics method for calculating spectra of dilute HOD in liquid D(2)O, which relied on ab initio calculations on clusters to provide a map from nuclear coordinates of the molecules in the liquid to OH stretch frequencies, transition dipoles, and polarizabilities. Here we extend this approach to the calculation of couplings between chromophores. From the trajectories of the fluctuating local-mode frequencies, transition moments, and couplings, we use our recently developed time-averaging approximation to calculate the line shapes. Our results are in good agreement with experiment for the IR and Raman line shapes, and capture the significant differences among them. Our analysis shows that while the coupling between chromophores is relatively modest, it nevertheless produces delocalization of the vibrational eigenstates over up to 12 chromophores, which has a profound effect on the spectroscopy. In particular, our results demonstrate that the peak in the parallel-polarized Raman spectrum at about 3250 wavenumbers is collective in nature.

8.
J Chem Phys ; 127(10): 104105, 2007 Sep 14.
Article in English | MEDLINE | ID: mdl-17867735

ABSTRACT

For an isolated resonance of an isolated chromophore in a condensed phase, the absorption line shape is often more sharply peaked than the distribution of transition frequencies as a result of motional narrowing. The latter arises from the time-dependent fluctuations of the transition frequencies. It is well known that one can incorporate these dynamical effects into line shape calculations within a semiclassical approach. For a system of coupled chromophores, both the transition frequencies and the interchromophore couplings fluctuate in time. In principle one can again solve this more complicated problem with a related semiclassical approach, but in practice, for large numbers of chromophores, the computational demands are prohibitive. This has led to the development of a number of approximate theoretical approaches to this problem. In this paper we develop another such approach, using a time-averaging approximation. The idea is that, for a single chromophore, a motionally narrowed line shape can be thought of as a distribution of time-averaged frequencies. This idea is developed and tested on both stochastic and more realistic models of isolated chromophores, and also on realistic models of coupled chromophores, and it is found that in all cases this approximation is quite satisfactory, without undue computational demands. This approach should find application for the vibrational spectroscopy of neat liquids, and also for proteins and other complicated multichromophore systems.

9.
Proc Natl Acad Sci U S A ; 104(36): 14215-20, 2007 Sep 04.
Article in English | MEDLINE | ID: mdl-17576923

ABSTRACT

We present improvements on our previous approaches for calculating vibrational spectroscopy observables for the OH stretch region of dilute HOD in liquid D2O. These revised approaches are implemented to calculate IR and isotropic Raman spectra, using the SPC/E simulation model, and the results are in good agreement with experiment. We also calculate observables associated with three-pulse IR echoes: the peak shift and 2D-IR spectrum. The agreement with experiment for the former is improved over our previous calculations, but discrepancies between theory and experiment still exist. Using our proposed definition for hydrogen bonding in liquid water, we decompose the distribution of frequencies in the OH stretch region in terms of subensembles of HOD molecules with different local hydrogen-bonding environments. Such a decomposition allows us to make the connection with experiments and calculations on water clusters and more generally to understand the extent of the relationship between transition frequency and local structure in the liquid.


Subject(s)
Deuterium/chemistry , Spectrophotometry, Infrared/methods , Spectrum Analysis, Raman/methods , Water/chemistry , Electrons , Gases/chemistry , Hydrogen/chemistry , Hydrogen Bonding , Indicator Dilution Techniques , Oxygen/chemistry , Temperature
10.
Zoonoses Public Health ; 54(1): 16-22, 2007.
Article in English | MEDLINE | ID: mdl-17359442

ABSTRACT

Qualitative and quantitative contamination of ready-to-eat food-stuffs with the pathogen Listeria monocytogenes was studied in 1586 samples collected from 103 supermarkets (n = 946) and 61 households (n = 640) in Vienna, Austria. Seventeen groups of ready-to-eat foods were classified into three risk categories for contamination (CP1-CP3). Three to four samples were randomly collected at the retail level from each CP. Regarding the households, the sampling procedure was started with food items of CP1, and if not available, was continued with sampling of food items of CP2 and finally of CP3. Additionally, 184 environmental samples (swabs from the kitchen area, dust samples from the vacuum cleaner) and faecal samples (household members and pet animals) were included. One-hundred and twenty-four (13.1%) and 45 (4.8%) samples out of 946 food samples collected from food retailers tested positive for Listeria spp. and L. monocytogenes, respectively, with five smoked fish samples exceeding the tolerated limit of 100 CFU/g food. Food-stuffs associated with the highest risk of contamination were twice as frequently contaminated with L. monocytogenes as food-stuffs associated with a medium risk of contamination. Products showing the highest contamination rate were fish and seafood (19.4%), followed by raw meat sausages (6.3%), soft cheese (5.5%) and cooked meat products/patés (4.5%). The overall contamination rate of foods collected at the household level was more than two times lower. Only 5.6% and 1.7% of 640 food-stuffs analysed tested positive for Listeria spp. and L. monocytogenes, respectively. However, CP1 foods were rarely collected. Pulsed-field gel electrophoresis (PFGE) typing of the collected L. monocytogenes isolates revealed a high degree of diversity between the isolates, with some exceptions. PFGE typing of isolates harvested from green-veined cheese revealed a match among strains, although the manufacturer seemed to be distinguishable. Typing of household strains revealed an epidemiological link within one family. In this case, food-stuffs and the kitchen environment were contaminated by an indistinguishable isolate. In addition, the same isolate was collected from a pooled faecal sample of the household members suggesting that consumption of even low contaminated food items (<100 CFU/g) results in Listeria shedding after the passage through the gut.


Subject(s)
Food Contamination/analysis , Food Microbiology , Listeria/isolation & purification , Listeriosis/transmission , Meat Products/microbiology , Animals , Austria , Consumer Product Safety , Humans , Listeria monocytogenes/isolation & purification , Listeriosis/veterinary , Risk Factors , Zoonoses
11.
J Immunol ; 164(6): 3169-76, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10706707

ABSTRACT

The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3' end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology.


Subject(s)
Adjuvants, Immunologic/physiology , Alternative Splicing/immunology , Blood Proteins/genetics , Adjuvants, Immunologic/biosynthesis , Adjuvants, Immunologic/chemistry , Amino Acid Sequence , Base Sequence , Blood Proteins/biosynthesis , Blood Proteins/chemistry , Blood Proteins/physiology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Immunosuppressive Agents/pharmacology , Intracellular Signaling Peptides and Proteins , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Membrane Proteins/biosynthesis , Molecular Sequence Data , Protein Isoforms/chemistry , Protein Isoforms/genetics , Sequence Analysis, Protein , Transcription, Genetic/immunology , U937 Cells
12.
Mol Cell Biochem ; 193(1-2): 5-11, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10331631

ABSTRACT

Recent evidence obtained with transgenic knockout mice suggests that the enzyme poly(ADP-ribose)polymerase (PARP) does not play a direct role in DNA break processing. Nevertheless, inactivation of the catalytic or the DNA nick-binding functions of PARP affects cellular responses to genotoxins at the level of cell survival, sister chromatid exchanges and apoptosis. In the present report, we conceptualize the idea that PARP is part of a DNA break signal mechanism. In vitro screening studies revealed the existence of a protein family containing a polymer-binding motif of about 22 amino acids. This motif is present in p53 protein as well as in MARCKS, a protein involved in the regulation of the actin cytoskeleton. Biochemical analyses showed that these sequences are directly targeted by PARP-associated polymers in vitro, and this alters several molecular functions of p53- and MARCKS protein. PARP-deficient knockout mice from transgenic mice were found to exhibit several phenotypic features compatible with altered DNA damage signaling, such as downregulation and lack of responsiveness of p53 protein to genotoxins, and morphological changes compatible with MARCKS-related cytoskeletal dysfunction. The knockout phenotype could be rescued by stable expression of the PARP gene. We propose that PARP-associated polymers may recruit signal proteins to sites of DNA breakage and reprogram their functions.


Subject(s)
Adenosine Diphosphate Ribose/metabolism , DNA Damage , Poly(ADP-ribose) Polymerases/physiology , Animals , DNA-Binding Proteins/metabolism , Humans , Mice , Mice, Knockout , Mice, Transgenic , Models, Genetic , Tumor Suppressor Protein p53/metabolism
13.
FEBS Lett ; 449(2-3): 259-63, 1999 Apr 23.
Article in English | MEDLINE | ID: mdl-10338144

ABSTRACT

Until recently, poly(ADP-ribosyl)ation was supposed to be confined only to polymerizing(ADP-ribosyl)transferase/(ADP-ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP-ribosyl)transferase homologues from mouse and man that lack all of the N-terminal DNA binding and BRCA1 C-terminus domains and will be designated polymerizing(ADP-ribosyl)transferase-2 as distinguished from the classical polymerizing(ADP-ribosyl)transferase (polymerizing(ADP-ribosyl)transferase-1). The murine polymerizing(ADP-ribosyl)transferase-2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue ('APP', GenBank database AF069298). Expression of the murine polymerizing(ADP-ribosyl)transferase-2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP-ribosyl)ation activity might account for most of the residual poly(ADP-ribosyl)ation observed in polymerizing(ADP-ribosyl)transferase-1(-/-) mice.


Subject(s)
Arabidopsis/enzymology , Caenorhabditis elegans/enzymology , Poly(ADP-ribose) Polymerases/genetics , Amino Acid Sequence , Animals , Arabidopsis/genetics , Base Sequence , Caenorhabditis elegans/genetics , Conserved Sequence , DNA, Complementary , Humans , Introns , Mammals , Mice , Mice, Knockout , Molecular Sequence Data , Multigene Family , Sequence Homology, Amino Acid , Tissue Distribution , Transcription, Genetic
14.
Oncogene ; 18(3): 713-9, 1999 Jan 21.
Article in English | MEDLINE | ID: mdl-9989821

ABSTRACT

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.


Subject(s)
Apoptosis , Glucocorticoids/pharmacology , Growth Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Division , Enzyme Activation , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Leukemia-Lymphoma, Adult T-Cell , Peptide Hydrolases/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger , Tumor Cells, Cultured
15.
Eur J Clin Invest ; 28(9): 695-700, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9767367

ABSTRACT

BACKGROUND: Long-term or high-dosage consumption of vitamin C may play a role in calcium oxalate kidney stone formation. The present study was undertaken to determine the biochemical and physicochemical risk factors in a male subject who developed haematuria and calcium oxalate crystalluria after ingestion of large doses of ascorbic acid for 8 consecutive days. METHODS: Twenty-four-hour urine samples were collected before and during the ascorbic acid ingestion period as well as after the detection of haematuria. A special procedure was implemented for urine collections to allow for oxalate, ascorbate and other urinalysis. Oxalate was determined in the presence of EDTA to prevent in vitro conversion to ascorbic acid, whereas ascorbate itself was determined by manual titration in a redox method using the dye dichlorophenolindophenol. Urinalysis data were used to compute calcium oxalate relative supersaturations and Tiselius risk indices, whereas scanning electron microscopy was used to examine urinary deposits. RESULTS: Oxalate excretion increased by about 350% during ascorbate ingestion before haematuria. Ascorbate concentrations also increased dramatically but appeared to reach a plateau maximum. Increasing calcium excretion was accompanied by decreasing potassium and phosphate values. The calcium oxalate relative supersaturation and Tiselius risk index increased during vitamin C ingestion and large aggregates of calcium oxalate dihydrate crystals were observed by scanning electron microscopy immediately after the detection of haematuria. CONCLUSION: High percentage metabolic conversion of ascorbate to oxalate in this subject caused relative hyperoxaluria and crystalluria, the latter manifesting itself as haematuria. Clinicians need to be alerted to the potential dangers of large dose ingestion of vitamin C in some individuals.


Subject(s)
Ascorbic Acid/administration & dosage , Ascorbic Acid/adverse effects , Calcium Oxalate/urine , Hematuria/chemically induced , Hyperoxaluria/chemically induced , Adult , Ascorbic Acid/pharmacokinetics , Calcium Oxalate/chemistry , Crystallization , Humans , Kidney Calculi/chemically induced , Male , Microscopy, Electron, Scanning , Risk Factors , Time Factors
16.
Biochemistry ; 37(40): 14181-8, 1998 Oct 06.
Article in English | MEDLINE | ID: mdl-9760255

ABSTRACT

The human leukemia U937 cells differentiate into monocyte/macrophage-like cells when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). We observed that during this process, both protein and mRNA levels for PARS markedly decreased in U937 cells. Through deletion analysis of the PARS regulatory gene, we found that the sequence within the first intron region was responsible for the TPA-dependent repression. Electrophoretic mobility shift assays (EMSAs) and Southwestern blot analysis indicate that this element bound specifically to a nuclear protein. TPA treatment abolished the binding of the protein in U937 cells but not in HeLa cells. DNase I footprinting data show that the cis regulatory element is located between residues 328 and 383. We further examined the function of this cis element (BS207) in a basal promoter regulatory reporter construct and found that this cis element (BS207) functions as an enhancer via the binding of an unknown trans-acting factor. TPA treatment diminished the binding activity of the factor in U937 cells, resulting in a decrease in the enhanced activity to the basal level. These results suggest that abolishment of the binding of a special nuclear protein to the first intron of the PARS gene is related to the TPA-responsive downregulation of PARS in U937 cells.


Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Poly(ADP-ribose) Polymerases/genetics , Regulatory Sequences, Nucleic Acid/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Base Sequence , Cell Differentiation/drug effects , DNA, Neoplasm/metabolism , Down-Regulation/drug effects , Humans , Introns , Molecular Sequence Data , Monocytes/drug effects , Monocytes/enzymology , Monocytes/pathology , Mutagenesis, Site-Directed , Neoplasm Proteins/genetics , Poly(ADP-ribose) Polymerases/biosynthesis , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding/genetics , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Repressor Proteins/genetics , Sequence Deletion , U937 Cells
17.
Biol Chem ; 379(4-5): 621-3, 1998.
Article in English | MEDLINE | ID: mdl-9628368

ABSTRACT

Infection of Escherichia coli cells with bacteriophage T1 induces synthesis of a bacteriophage-specific DNA methyltransferase (M.EcoT1, EC No: 2.1.1.72) with a specificity for adenine residues in the sequence 5'-GATC-3'. Purification of M.EcoT1 allowed the determination of the coding sequence of the gene (Schneider-Scherzer et al., 1990). The peptide of the entire coding sequence was over-expressed as a histidine-hexapeptide tagged protein in E. coli. Affinity purification using a Ni2+ chelating (Ni-NTA) resin yielded a recombinant enzyme with almost the same enzymatic properties as the protein purified from T1 infected E. coli cells. Interestingly, in both purification procedures, a protein with a molecular weight of 50000 was found to copurify with M.EcoT1. The N-terminal amino acid sequence identified these proteins in both cases as E. coli enolase (EC No: 4.2.1.11).


Subject(s)
Escherichia coli/enzymology , Phosphopyruvate Hydratase/metabolism , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , T-Phages/enzymology , DNA/metabolism , Escherichia coli/virology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S-Adenosylmethionine/metabolism , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , T-Phages/genetics
18.
Clin Chem Lab Med ; 36(3): 143-7, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9589801

ABSTRACT

The present study was undertaken to determine the effect of ingestion of large doses of vitamin C on urinary oxalate excretion and on a number of other biochemical and physicochemical risk factors associated with calcium oxalate urolithiasis. A further objective was to determine urinary ascorbate excretion and to relate it qualitatively to ingested levels of the vitamin and oxalate excretion. Ten healthy males participated in a protocol in which 4 g ascorbic acid was ingested for 5 days. Urines (24 h) were collected prior to, during and after the protocol. The urine collection procedure was designed to allow for the analysis of oxalate in the presence and absence of an EDTA preservative and for the analysis of ascorbic acid by manual titration using 2,6 dichlorophenolindophenol. Physicochemical risk factors such as the calcium oxalate relative supersaturation and Tiselius risk index were calculated from urine composition. The results showed that erroneously high analytical oxalate levels occur in the asence of preservative. In the preserved samples there was no significant increase in oxalate excretion at any stage of the protocol. Ascorbate excretion increased when vitamin C ingestion commenced but levelled out after 24 hours suggesting that saturation of the metabolic pool is reached within 24 hours after which ingested ascorbic acid is excreted unmetabolized in the urine. While transient statistically significant changes occurred in some of the biochemical risk factors, they were not regarded as being clinically significant. There were no changes in either the calcium oxalate relative supersaturation or Tiselius risk index. It is concluded that ingestion of large doses of ascorbic acid does not affect the principal risk factors associated with calcium oxalate kidney stone formation.


Subject(s)
Ascorbic Acid/adverse effects , Calcium Oxalate/urine , Kidney Calculi/chemically induced , 2,6-Dichloroindophenol , Adult , Ascorbic Acid/administration & dosage , Ascorbic Acid/urine , Humans , Kidney Calculi/urine , Male , Risk Factors , South Africa
19.
Oncogene ; 15(20): 2429-37, 1997 Nov 13.
Article in English | MEDLINE | ID: mdl-9395239

ABSTRACT

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.


Subject(s)
Apoptosis/physiology , Caspases , Cysteine Endopeptidases/physiology , Gene Expression Regulation, Leukemic , Leukemia-Lymphoma, Adult T-Cell/pathology , Neoplasm Proteins/physiology , Tumor Suppressor Protein p53/physiology , Alleles , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Caspase 1 , Caspase 8 , Caspase 9 , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Dactinomycin/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Genes, p53 , Heterozygote , Humans , Neoplasm Proteins/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Phenotype , Poly(ADP-ribose) Polymerases/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Fusion Proteins/physiology , Temperature , Transfection , Tubulin/metabolism , Tumor Cells, Cultured/drug effects , Vincristine/pharmacology
20.
FEBS Lett ; 402(1): 36-40, 1997 Jan 27.
Article in English | MEDLINE | ID: mdl-9013854

ABSTRACT

Glucocorticoids (GC) induce programmed cell death (apoptosis) in immature lymphocytes and are an essential component in the therapy of acute lymphatic leukemia. The mechanism underlying GC-induced apoptosis particularly in leukemia cells is, however, not well understood. Most forms of apoptosis seem to employ a common final effector pathway characterized by specific proteolytic events mediated by interleukin 1beta-converting enzyme (ICE) and/or other ICE-like cysteine proteases. These events may result in the morphologic changes characteristic of apoptosis. To determine whether a similar proteolytic pathway is activated during GC-induced leukemia cell apoptosis, we investigated poly(ADP-ribose) polymerase (PARP), a typical target of ICE-like proteases, during GC-induced apoptosis of the human acute T-cell leukemic cell line CEM-C7H2. Our studies showed proteolytic PARP cleavage suggestive of activation of ICE-like proteases that preceeded morphologic signs of apoptosis. We further established stably transfected CEM-C7H2 sublines expressing the cowpox virus protein CrmA that inhibits some, but not all, ICE-like proteases. GC-induced PARP cleavage and apoptosis were neither inhibited nor delayed in crmA-expressing cell lines. In contrast, crmA expression rendered the same lines resistant to Apo1/Fas-induced PARP cleavage and apoptosis. Thus, different proteases might be activated during the effector phases of GC-and Apo1/Fas-induced apoptosis in human leukemia cells.


Subject(s)
Apoptosis , Cysteine Endopeptidases/metabolism , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Leukemia-Lymphoma, Adult T-Cell/pathology , Poly Adenosine Diphosphate Ribose/metabolism , Serpins/physiology , Viral Proteins , Caspase 1 , Humans , Leukemia-Lymphoma, Adult T-Cell/enzymology , Serpins/genetics , Transfection , Tumor Cells, Cultured , fas Receptor/immunology , fas Receptor/physiology
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