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PubMedDate not supplied

Association of platelet-derived growth factor-induced protein with nuclear material.

Platelet-derived growth factor (PDGF) has been proposed to initiate the cell-cycle traverse of density-arrested BALB/c-3T3 cells by rendering quiescent cells 'competent' to respond to 'progression' factors contained in platelet-poor plasma (PPP). PDGF-treated cells remain competent for many hours following PDGF removal; subsequent addition of PPP triggers G0-G1 traversal and entry into S phase. Numerous observations suggest that the competent state reflects the existence of a stable PDGF-induced 'second signal' as opposed to a persistent association of PDGF with cells. Several unique proteins have been shown to be synthesized in response to PDGF; of these, the dose-dependent production of 'pI' (molecular weight 29,000) was found to correlate closely with the induction of competence. We report here the further characterization of pI in terms of its time-dependent synthesis, intracellular location and stability. Electrophoretic analysis of nuclear and non-nuclear extracts of PDGF-treated BALB/c-3T3 cells demonstrated that pI is synthesized during the first 6 h of G0-G1, is associated with nuclear material and is stable for 9-12 h. These findings are consistent with the proposed role of pI as the cellular mediator of PDGF.

Animals↗
PubMed1998

Solid-phase reactors as high stability reagent sources in flow analysis: selective flow injection spectrophotometric determination of cysteine in pharmaceutical formulations.

The flow injection spectrophotometric determination of cysteine was carried out by reaction with cobalt(II) ions entrapped in a polymeric material and filling a packed-bed reactor; the released cobalt(II) complexed with the amino acid was monitored at 360 nm. The method worked with a high repeatability, even with independent reactors, days and solutions. Selectivity of the procedure was tested with twenty different foreign compounds found in pharmaceutical formulations containing cysteine, parent amino acids included; no serious interferences were observed. The calibration graph for cysteine was linear over the range 1-90 micrograms ml-1 with a relative standard deviation of 0.8% at 60 micrograms ml-1 (n = 158). The calculated sample throughput was 90 h-1. The method was applied to determine the content of cysteine in pharmaceutical formulations.

Cobalt↗
PubMed2005

IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities.

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711-->Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1(S711E) displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1(S711E) retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1(S711E) completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1(S711E) is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

Aconitate Hydratase↗
PubMed2001

Probing opioid receptor-ligand interactions by employment of indolizidin-9-one amino acid as a constrained Gly(2)-Gly(3) surrogate in a leucine-enkephalin mimic.

The relationship between the conformation and biological activity of Leu-enkephalin was studied using (2S,6R,8S)-9-oxo-8-N-(Boc)amino-1-azabicyclo[4.3.0]nonane-2-carboxylic acid [(2S,6R,8S)-1, I(9)AA] as a constrained Gly(2)-Gly(3) dipeptide surrogate. [I(9)AA](2,3)-Leu-enkephalin 12 was assembled using solid-phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I(9)AA](2,3)-Leu-enkephalin 12 exhibited affinities for the mu- and delta-opioid receptors that were three orders of magnitude lower than that of Leu-enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and 3J(NH-CalphaH) coupling constants for 12 could not support a hydrogen-bonded beta-turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu-enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.

Amino Acids↗
PubMed1995

Fluoxetine in patients with major depressive disorder--a responder analysis.

Fluoxetine is a selective serotonin re-uptake inhibitor and thus being a specific antidepressant has its specific responder. In order to attempt a differential description of fluoxetine responders we collected data of 219 patients with major depressive disorder (MDD; DSM-III R) in an open multicenter (phase IV) study, which were further subclassified. According to the treatment plan, these patients were to be treated with 20 mg fluoxetine daily for a period of five weeks. CGI, HAMD, SDS, undesired side effects, laboratory and physiological variables were recorded at weekly intervals. Predictors for response were searched for by means of discriminatory analysis. The stability of the relations found was verified in two randomized halves of the data. It was found that those patients are probably good responders who at base-line have a high Covi anxiety score, but who according to Raskin score, self-assessment of depression (SDS), and CGI are not particularly severely depressed and show a relatively favorable therapeutic response in the first week of treatment.

Adolescent↗
PubMed2003

Dissipative discrete breathers: periodic, quasiperiodic, chaotic, and mobile.

The properties of discrete breathers in dissipative one-dimensional lattices of nonlinear oscillators subject to periodic driving forces are reviewed. We focus on oscillobreathers in the Frenkel-Kontorova chain and rotobreathers in a ladder of Josephson junctions. Both types of exponentially localized solutions are easily obtained numerically using adiabatic continuation from the anticontinuous limit. Linear stability (Floquet) analysis allows the characterization of different types of bifurcations experienced by periodic discrete breathers. Some of these bifurcations produce nonperiodic localized solutions, namely, quasiperiodic and chaotic discrete breathers, which are generally impossible as exact solutions in Hamiltonian systems. Within a certain range of parameters, propagating breathers occur as attractors of the dissipative dynamics. General features of these excitations are discussed and the Peierls-Nabarro barrier is addressed. Numerical scattering experiments with mobile breathers reveal the existence of two-breather bound states and allow a first glimpse at the intricate phenomenology of these special multibreather configurations.

Journal Article↗
PubMed2001

Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Adult↗
PubMed2003

Down-regulation of the mitochondrial translation system during terminal differentiation of HL-60 cells by 12-O-tetradecanoyl-1-phorbol-13-acetate: comparison with the cytoplasmic translation system.

Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly down-regulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPA-induced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation (in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.

Blotting, Northern↗
PubMed2006

Polyamine analogues down-regulate estrogen receptor alpha expression in human breast cancer cells.

The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor alpha (ERalpha) and ERalpha target genes in ER-positive human breast cancer cell lines, whereas neither ERbeta nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERalpha in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERalpha transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERalpha minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine-inhibited ERalpha expression, suggesting that AP-1 is a positive regulator of ERalpha expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERalpha induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells.

Breast Neoplasms↗
PubMed2003

K+ and Na+-induced self-assembly of telomeric oligonucleotide d(TTAGGG)n.

The telomeric DNA oligomers, d(TTAGGG)(n), where n=1, 2, 4, could self-associate into the multi-stranded structures in appropriate condition, exhibited different CD spectra. The presense of Na(+) was more advantage to facilitate the formation of anti-parallel conformation, but the presense of K(+) enhanced their thermal stability. Spectroscopic analysis of 3, 3'-diethyloxadicarbocyanine (DODC) showed the formation of hairpin quadruplex structures for d(TTAGGG)(2) and d(TTAGGG)(4), but d(TTAGGG) could not. The four-stranded tetraplexes and branched nanowire formed in the presense of K(+) or Na(+) alone were observed by atomic force microscopy (AFM). The ability of d(TTAGGG)(n) to self-assemble into four-stranded tetraplexes and nanowires depends strongly on the number of repeating units and ionic environment. A model to explain how these structures formed is proposed.

Cations, Monovalent↗
PubMed1983

Field validation study of NO2 personal passive samplers in a "diesel" haulage underground mine.

Three passive samplers are now commercially available for NO2. This field validation, conducted in an underground mine, attempted to address both the precision and accuracy of the three now commercially available. The probable sources of NO2 were identified as diesel engines and blasting operations. Comparative sampling was conducted with the passive samplers versus the standard "baseline" impingement method. The three NO2 samplers were as follows: 1) PRO-TEK (DuPont); 2) Palmes (MDA); 3) VaporGard (MSA). Three sets of data consisting of impingers and passive sampler results were taken on top of a moving diesel vehicle over a three-day period. An expanded metal screen was welded in a "free standing" plane above the vehicle to serve as a sampling platform. The evaluation of concentration data suggested that correlations of accuracy and precision versus the impinger method were best for the Palmes and VaporGard samplers. The PRO-TEK sampler does not seem to produce accurate data, but it is somewhat precise. Factors of sensitivity, accuracy, precision, cost, ease of analysis, and stability must be weighed.

Air Pollutants, Occupational↗
PubMed1989

Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells.

During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP proteins whose behavior is also rapidly modified by a heat shock at 45 degrees C, whereas no effect is observed at 42 degrees C. Using a monoclonal antibody, we show that in situ the antigens are available only when the cells are heat shocked at 45 degrees C. Subcellular fractionation shows that in normal cells the antigens are associated with the bulk of hnRNP (50-200S). During heat shock, whereas the overall characteristics of the bulk of preexisting hnRNP are unchanged, these antigens rapidly switch to a subpopulation of hnRNP with larger average size (50 to less than 300S) and increased stability. Structural analysis of the associated hnRNP in normal and stressed cells shows that in both cases the antigens are associated with the nuclear matrix subcomplex of hnRNP, which in situ is part of the internal nuclear matrix. Such hnRNP antigens, which are rapidly redistributed during a heat shock at the upper temperature range of the stress response, might well be involved in splicing and/or transport control.

Antibodies, Monoclonal↗
PubMed2005

Low intake of calcium, folate, nicotinic acid, vitamin E, retinol, beta-carotene and high intake of pantothenic acid, biotin and riboflavin are significantly associated with increased genome instability--results from a dietary intake and micronucleus index survey in South Australia.

The aim of this study was to determine the association between dietary intake, determined using a food frequency questionnaire, and genome damage in lymphocytes measured using the micronucleus (MN) assay. The study, performed on 190 healthy individuals (mean age 47.8 years, 46% males), also examined whether a supplementation with beta-carotene, vitamins C and E along with zinc (ACEZn), in a randomized trial for 6 months, improves genome stability. Multivariate analysis of baseline data showed that (1) the highest tertile of intake of vitamin E, retinol, folic acid, nicotinic acid (preformed) and calcium is associated with significant reductions in MN frequency, i.e. -28, -31, -33, -46 and -49%, respectively (P < 0.005) relative to the lowest tertile of intake and (2) the highest tertile of intake of riboflavin, pantothenic acid and biotin was associated with significant increases in MN frequency, i.e. +36% (P = 0.054), +51% (P = 0.021), and +65% (P = 0.001), respectively, relative to the lowest tertile of intake. Mid-tertile beta-carotene intake was associated with an 18% reduction in MN frequency (P = 0.038); however, the highest tertile of intake (>6400 microg/day) resulted in an 18% increment in MN frequency. Supplementation with ACEZn significantly reduced the MN index by 13% (P = 0.038). The study also showed interactive additive effects such as the protective effect of increased calcium intake (-46%) and the exacerbating effect of riboflavin (+42%) on increased genome damage caused by low folate intake. The results from this study illustrate the strong impact of a wide variety of micronutrients and their interactions on genome health, depending on the level of intake.

Adult↗
PubMed2003

Sumoylation is involved in beta-catenin-dependent activation of Tcf-4.

Sumoylation is involved in mediating protein-protein interactions, subcellular compartmentalization and protein stability. Our analysis of various Wnt signaling molecules revealed that one of them, Tcf-4, is sumoylated at the endogenous level. At least one sumoylation site, Lys297, of Tcf-4 was identified. The sumoylation of Tcf-4 was enhanced by PIASy, a SUMO E3 enzyme, and inhibited by Axam, a desumoylation enzyme. Although PIASy did not affect the interaction of Tcf-4 with beta-catenin or DNA, Tcf-4, SUMO-1 and PIASy were co-localized in the nucleus and present in a complex in the PML body. PIASy enhanced beta-catenin-dependent transcriptional activity of Tcf-4, whereas Axam inhibited it. Reduction of the protein level of Axam by RNA interference led to an increase in sumoylation of Tcf-4 and activation of Tcf-4. Furthermore, beta-catenin and PIASy activated Tcf-4(K297R), in which Lys297 was changed to arginine, less than wild-type Tcf-4. These results suggest that sumoylation of Tcf-4 is involved in beta-catenin-dependent and Tcf-4-mediated gene expression in the Wnt signaling pathway.

Animals↗
PubMed1998

Novel mutations in the RAD3 and SSL1 genes perturb genome stability by stimulating recombination between short repeats in Saccharomyces cerevisiae.

Maintaining genome stability requires that recombination between repetitive sequences be avoided. Because short, repetitive sequences are the most abundant, recombination between sequences that are below a certain length are selectively restricted. Novel alleles of the RAD3 and SSL1 genes, which code for components of a basal transcription and UV-damage-repair complex in Saccharomyces cerevisiae, have been found to stimulate recombination between short, repeated sequences. In double mutants, these effects are suppressed, indicating that the RAD3 and SSL1 gene products work together in influencing genome stability. Genetic analysis indicates that this function is independent of UV-damage repair and mutation avoidance, supporting the notion that RAD3 and SSL1 together play a novel role in the maintenance of genome integrity.

Adenosine Triphosphatases↗
PubMed2002

Ligand-specific conformations of an ionotropic glutamate receptor.

A simple in silico procedure is proposed with a view to predict the agonist or antagonist character of new, AMPA-type Glu receptor channel ligands. Based on the experimental binding domain structures, the orientation of a single Lys residue close to the ligand binding core was found to be diagnostic of ligand-induced conformational changes. Acting as a switch, the position of the Lys residue indicates the agonist or antagonist character of AMPA receptor ligands, known to bind to the receptor. Stability centre analysis substantiated the key role this switch might play in ligand-induced conformational changes.

Binding Sites↗
PubMedDate not supplied

Psychometric testing of fatigue instruments for use with cancer patients.

BACKGROUND: Cancer treatment-related fatigue (CRF) is a common side effect of cancer treatment. A problem identified in most reviews of CRF is lack of sound approaches to measurement that are congruent with the conceptualization of CRF as a self-perceived state. The diversity of instruments available to measure fatigue and the lack of comprehensive testing of several promising instruments with cancer patients undergoing treatment provided the rationale for this study. The purpose of this article is to report the results of psychometric testing of several fatigue instruments in patients undergoing cancer treatment. OBJECTIVES: The aims of this study were to determine the reliability, validity, and responsiveness of each instrument and to determine the ability of each instrument to capture CRF. METHODS: Existing fatigue instruments with published psychometric information that indicated suitability for further testing were selected and included the Profile of Mood States Short Form fatigue subscale (F_POMS-sf), Multidimensional Assessment of Fatigue (MAF), Lee Fatigue Scale (LFS), and the Multidimensional Fatigue Inventory (MFI). Data were collected at a university-based clinical cancer center and a freestanding comprehensive cancer center. Subjects completed all study instruments, which were presented in random order, at a time when CRF was expected to be high and again when it was expected to be low. A subset of subjects completed the instruments within 48 hours of one of the data collection points when CRF was expected to be relatively unchanged to provide stability data. RESULTS: Reliability estimates using Cronbach's alpha indicated that all instruments examined had good internal consistency. Test-retest correlations showed good stability for total scores on all the instruments, but some subscales of the LFS and MFI had marginal stability. Factor analysis of all instruments indicated that only the LFS and the F_POMS-sf fully supported their construct validity. All of the instruments showed responsiveness to changes in CRF related to treatment. CONCLUSIONS: The results of the study provide researchers and clinicians with detailed comparisons of the performance of established fatigue measures in cancer patients undergoing treatment to use when selecting measures of CRF.

Adult↗