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At least 199 records · Page 11Linked to original sources

PubMed1995

Deformed expression in the Drosophila central nervous system is controlled by an autoactivated intronic enhancer.

Deformed (Dfd) is a Drosophila homeotic selector gene required for normal development of maxillary segment morphology in the larval and adult head. Consistent with this function, Dfd transcripts are restricted to epidermal, mesodermal and neural cells in the embryonic mandibular and maxillary primordia. Previous studies have identified a far upstream element in Dfd sequences which functions as an epidermal-specific autoregulatory enhancer. In a search through 35 kb of Dfd sequences for additional transcriptional control elements, we have identified a 3.2 kb DNA fragment containing an enhancer that mimics the expression of Dfd in the subesophageal ganglion of the embryonic central nervous system. This Neural autoregulatory enhancer (NAE) maps in the large Dfd intron just upstream of the homeobox exon and requires Dfd protein function for its full activity. A 608 bp NAE subfragment retains regulatory function that is principally localized in the subesophageal ganglion. This small region of the Drosophila melanogaster genome contains numerous blocks of sequence conservation with a comparable region from the Dfd locus of D.hydei. A pair of conserved blocks of NAE sequence match a Dfd protein binding site in the epidermal autoregulatory element, while another conserved sequence motif is repeated multiple times within the 608 bp subelement.

Animals↗

PubMed1997

Sequencing, expression pattern and RFLP mapping of a senescence-enhanced cDNA from Zea mays with high homology to oryzain gamma and aleurain.

Sequence analysis of a 1.4 kb clone from a cDNA library of senescing Zea mays leaves reveals an open reading frame for a 360 amino acid protein. Both the DNA and deduced amino acid sequences are highly homologous to the cysteine proteinases oryzain gamma and aleurain. Northern analysis demonstrates that the corresponding RNA level increases during natural leaf senescence, seedling germination and in chilling of tolerant maize lines, but decreases in a sensitive line. The mRNA level also decreases in regreening leaves, in dark-induced senescence and in nutrient or water stress. Southern and RFLP analysis provide evidence that the gene has two copies, on chromosomes 2 and 7.

Amino Acid Sequence↗

PubMed1992

A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically.

We have identified an estrogen-responsive enhancer element (DH3 ERE) in the estrogen-induced DNAase I-hypersensitive region III of the chicken ovalbumin gene, which is located approximately 3.3 kb upstream from the mRNA start site and does not contain palindromic ERE. Four TGACC half-palindromic motifs, separated from each other by more than 100 bp, are responsible for conferring estrogen inducibility either to the proximal ovalbumin gene promoter or to heterologous promoters. Thus, widely spaced half-palindromic ERE motifs can act synergistically. Each half-palindromic motif was shown to bind the estrogen receptor (ER) with a low efficiency in vitro. However, two widely spaced half-palindromic motifs bound the ER cooperatively, much more efficiently than expected from binding to isolated half-ERE motifs. The ovalbumin promoter half-palindromic ERE motif located close to the TATA box was required for the activity of the distal DH3 ERE, but could be replaced by the binding sites of other transactivators.

Animals↗

PubMed1995

Improved adenovirus vector provides herpes simplex virus ribonucleotide reductase R1 and R2 subunits very efficiently.

We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the activity of the ectopic major late promoter (MLP) in recombinant Ad. In 293 cells infected with helper-free Ad recombinants generated with the pAdBM5 transfer vector, both herpes simplex virus (HSV) ribonucleotide reductase R1 and R2 subunits represent the most abundant polypeptides, accounting for as much as 15-20% of total cellular proteins. Our data suggest that this level of expression is probably very close to the upper limit of the system. Furthermore, when compared to the widely utilized baculovirus (Bac)/Sf9 expression system, the improved Ad vector showed a better performance for the production and purification of active HSV-2 ribonucleotide reductase R1 and R2 subunits. The R2 subunit was about 5-fold more abundant in recombinant Ad-infected 293 cells than in Bac-infected Sf9 cells while the R1 subunit was produced at roughly similar levels with either system. However, the amount of active soluble R1 obtained from recombinant Ad-infected 293 cells was at least 5 times higher because most of the R1 produced in Sf9 cells was insoluble.

Adenoviruses, Human↗

PubMed1989

The enhancer-like sequence 3' to the A gamma gene is polymorphic in human populations.

Cloning and sequencing of the enhancer 3' of the A gamma globin gene of a particularly low G gamma and HbF sickle cell anemia (SCA) patient unexpectedly revealed three base changes (T----C, C----A, and A----G at sites +2285, +2460, and +2676) previously associated with the Seattle-type HPFH, thus leading the authors to suspect that the three mutations were polymorphic. The determination of the incidence of the mutations among various ethnic groups allowed the authors to conclude that this is a widely spread polymorphism, thus excluding any role of these base changes in the determination of the hereditary persistence of fetal hemoglobin (HPFH) phenotype. The origin of these three mutations is not clear because they appear linked, and the same bases (C, A, G) are found in homologous position in the 3' of the normal G gamma gene. As C, A, G at positions +2285, +2460, and +2676 are found with a 100% frequency in African SS patients and presumably among normal Africans (to explain the extremely high frequency among normal American blacks), it is likely that this was the sequence preceding the division of races. The presence of T, C, and A at the same positions apparently occurred after the divergence between blacks and the other races, that is, within the last 1 million years.

Base Sequence↗

PubMed1995

High resolution 2D-NMR studies indicating complete assignments and conformational characteristics of the NF-kappa B binding enhancer element of HIV-LTR.

The asymmetrical DNA duplex [5'd(AAGGGACTTTCC)].[5'-d(GGAAAGTCCCTT)] has been studied by one- and two-dimensional NMR techniques. The sequence is comprised of the actual 10 base-pair long binding site for the transcription factor NF-kappa B in the enhancer sequence of the long term repeat (LTR) region of HIV and SIV types of retroviruses associated with the AIDS syndrome. Two additional A.T base-pairs are also included on one end for an added interest in the 12-bp duplex sequence with a pseudo dyad-symmetric disposition of the oligopurine and oligopyrimidine segments, as it appears in the HIV-1 genome. Phase-sensitive two-dimensional spectra (NOESY, ROESY, COSY and TOCSY) were obtained at three different temperatures (5, 15 and 25 degrees C) for a complete assignment of the non-exchangeable protons by tracing through sequence specific intra- and internucleotide connectivities. 2D-NOESY spectra were also acquired in aqueous (90% H2O-D2O) solutions, with two different methods of water signal suppression, to assign the exchangeable protons from specific NOE correlations. Adenine H2 protons were assigned by the use of NOE correlations and from T1 relaxation time measurements. The general spectral features and semi-quantitative interproton distance estimates indicate a B-DNA type conformation. However, some distinctly unusual features associated with the nucleotides at and immediately adjacent to both the 5'-and 3'-ends of AAA/TTT and GGG/CCC segments were noted. The complete assignments, and the observed characteristics, will be of significant value in studying the complexes of this transcriptionally active DNA domain with the protein and other rationally designed DNA binding agents.

Base Sequence↗

PubMed1997

Phosphatidylcholine signaling in response to CSF-1.

The formation of cell membrane following CSF-1 stimulation of a macrophage cell line is coordinated with cell cycle progression. The majority of membrane phospholipid accumulates during the S phase and results from cell-cycle dependent oscillations in the rates of phosphatidylcholine biosynthesis and degradation. Both synthesis and degradation are enhanced during the G1 phase, resulting in a high rate of phosphatidylcholine turnover. Degradation of phosphatidylcholine after CSF-1 stimulation is mediated by a phospholipase C, and the release of diacylglycerol during G1 phase is biphasic. The degradation essentially stops during the S phase, thus allowing biosynthesis to supply the necessary membrane for cell division and doubling. The degradation of phosphatidylcholine during G1 signals the downstream activation of c-fos and junB transcription and can be mimicked by incubation of the macrophage cells with exogenous bacterial phospholipase C. In contrast, the expression of c-myc transcripts normally associated with CSF-1 stimulation is severely compromised in phospholipase C-treated cells, indicating that the diacylglycerol signals a pathway distinct from the pathway that governs c-myc activation. Constitutive expression of c-myc complements phospholipase C activity and permits the growth of cells in the presence of exogenous bacterial enzyme and the absence of CSF-1. Protein kinase C is not required to mediate the diacylglycerol signal that supports cell growth. GTP exchange on Ras is not enhanced, and MAP kinase activity is not stimulated in response to phosphatidylcholine degradation by exogenous phospholipase C. The 85 kDa cytoplasmic phospholipase A2 is activated, however, as well as a novel protein we have called p96. Rapid serine phosphorylation of p96 follows stimulation of cells with either CSF-1 or exogenous phospholipase C. Analysis of the murine cDNA encoding p96 reveals an amino-terminal domain with significant similarity to the amino-terminal domain of the Drosophila-disabled gene product and a carboxy-terminal domain containing proline-rich sequences characteristic of SH3 binding regions. The sequence of p96 suggests an interactive role for this unique protein in the CSF-1 signal transduction cascade.

Animals↗

PubMed1989

Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays.

We describe a novel approach to detect antibodies to synthetic peptide antigens in solid-phase radioimmunoassays, using a multiple antigen peptide (MAP) system. The MAPs consist of multiple copies of peptides that are synthesized as single units on a branching lysyl matrix using a solid-phase peptide synthesis method. The efficacy of the MAP approach in solid-phase immunoassays was compared with the conventional approach using a monomeric peptide of the immunodominant epitope of the circumsporozoite proteins of two species of malaria. Two monomeric peptides with 12 and 17 residues were found to bind poorly to plastic surfaces at a concentration up to 30 micrograms/ml, and showed no immunoreactivity to specific polyclonal or monoclonal antibodies, while the corresponding MAP-containing peptides showed excellent binding capacity and immunoreactivity at a concentration of 0.11 microgram/ml. The immunoreactivity of MAP-containing peptides was also superior to that of monomeric peptides conjugated to a protein carrier. The effects of various arrangements of lysyl branching of MAP on antigenicity were also studied, and the optimal number for lysyl branching of MAP was found to be octameric. Thus, the MAP, by enhancing the coating capacity and the avidity of synthetic peptides, provides increased sensitivity and reliability for the use of synthetic peptide to study antigen-antibody interactions on solid surfaces.

Antibodies↗

PubMed1996

The expression of a reporter protein, beta-galactosidase, is preserved during maturation and aging in some cells of the adult Drosophila melanogaster.

The effects of maturation and aging on cell stability and maintenance of protein expression have been examined in adult Drosophila melanogaster. Counting the number of cells present in the antenna of the adult fly revealed little loss in cell number with aging. Enhancer map-marked genes expressing beta-galactosidase (beta-gal) in the antenna and an Rh1 opsin reporter gene construct expressing beta-gal in the R1-6 photoreceptor cells of the compound eye revealed no alteration in spatial distribution or amount of beta-gal with aging. A heat shock-inducible promoter coupled to the expression of beta-gal, hsp70-lacZ, revealed that the rate and amount of induction of beta-gal after heat shock is preserved during aging but the rate of decay of beta-gal may be slightly delayed in older animals. These studies suggest that the ability to express a reporter protein, beta-galactosidase, is preserved in at least a subset of cells in the aging fly.

Animals↗

PubMed1999

Chronic administration of adenosine A3 receptor agonist and cerebral ischemia: neuronal and glial effects.

We have previously shown that chronic administration of the selective A3 receptor agonist N6-(3-iodobenzyl)-5'-N-methylcarboxoamidoadenosine (IB-MECA) leads to a significant improvement of postocclusive cerebral blood flow, and protects against neuronal damage and mortality induced by severe forebrain ischemia in gerbils. Using immunocytochemical methods we now show that chronic with IB-MECA results in a significant preservation of ischemia-sensitive microtubule associated protein 2 (MAP-2), enhancement of the expression of glial fibrillary acidic protein (GFAP), and a very intense depression of nitric oxide synthase in the brain of postischemic gerbils. These changes demonstrate that the cerebroprotective actions of chronically administered IB-MECA involve both neurons and glial cells, and indicate the possibility of distinct mechanisms that are affected in the course of chronic administration of the drug.

Adenosine↗

PubMed1998

Effects of endothelin-3 on water and sodium excretion during extracellular volume expansion.

The aim of the present study was to elucidate the role of an IV dose of endothelin-3 (ET-3) (5 ng Kg-1 min-1) on mean arterial pressure (MAP), on diuresis and natriuresis in control and in volume expanded anesthetized rats. A systemic infusion of ET-3 in normal rats (Group I) increased MAP and produced a trend of increasing diuresis, without changes in natriuresis. A 10% body weight expansion (Group II) increased diuresis and natriuresis without changes in MAP. The simultaneous infusion of ET-3 and expansion with saline (Group III) resulted in an increase in MAP, an enhanced diuretic response, and a natriuresis of similar magnitude to that observed in Group II. These results suggest that the diuresis produced by a low dose of exogenous ET-3 in control rats, is independent of sodium excretion. Furthermore, the enhanced diuresis caused by ET-3 during expansion is greater than the addition of ET-3 and expansion effects, suggesting that new mechanisms are triggered in order to maintain volume and salt homeostasis in this state.

Animals↗

PubMed1998

Cardiac MR imaging in congenital heart disease.

Magnetic resonance imaging is a unique and insightful tool for the assessment of structure and function in congenital heart disease. For anatomic assessment, the large field of view, lack of limitation by patient size, and ability to create three-dimensional surface displays from routine imaging acquisitions offer several advantages over other modalities. The ability of magnetic resonance imaging to assess the volume and mass of bizarre ventricular shapes accurately and myocardial tissue and blood tagging as well as phase encoded velocity mapping has enhanced research in pediatric cardiology. Newer techniques, such as oxygen-sensitive magnetic resonance imaging and echo-planar magnetic resonance imaging, promise even further advances in research and in clinical applications.

Heart Defects, Congenital↗

PubMed1998

Optical time-of-flight chemical detection: absorption-modulated fluorescence for spatially resolved analyte mapping in a bidirectional distributed fiber-optic sensor.

A continuous chemically sensitive optical fiber is used with optical time-of-flight chemical detection (OTOF-CD) for spatially resolved analyte mapping. To enhance signal levels and to improve their reproducibility, two novel principles for signal generation and processing are introduced. In the first, the fluorescene of an analyte-insensitive fluorophore is monitored as a function of the evanescent wave absorption of an analyte-sensitive indicator. The resulting signal levels are well above those encountered in optical time domain reflectometry methods that rely upon backscattering for spatially resolved detection. As a result, the method could significantly expand the range of species that can be detected with absorption reagents used in OTOF sensors. The second method raises signal-to-noise ratios by 3-4.5-fold for measurements made at the far ends of the sensing fiber. It functions by sending probe laser pulses into and monitoring their return sequentially from both ends of the sensing fiber. Because the two pulses provide complementary information, only the first half of each of the collected waveforms is used for analyte quantitation. The introduced concepts were experimentally verified with a distributed sensor constructed from a 40-m-long continuous chemically sensitive optical fiber. This sensing element was produced by immobilization of an ammonia-sensitive absorbing reagent (phenol red) and an analyte-insensitive fluorophore (rhodamine 640) into the original silicone cladding of the plastic-clad silica fiber.

Fiber Optic Technology↗

PubMed1994

Enhanced detection in brain activation maps using a multifiltering approach.

Current methods for detecting activation foci in positron emission tomography difference images include a low pass filtering step aimed at improving the signal-to-noise ratio. However, we show that detection sensitivity depends both on the activation signal and the filter sizes. Therefore, we propose to improve current detection methods by using a multifiltering strategy that is shown to be more sensitive when various kinds of signals are present in the brain activation images.

Brain↗

PubMed1996

Macrorestriction analysis of Caenorhabditis elegans genomic DNA.

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a "macrorestriction mapping" procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is < 220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located approximately 400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.

Animals↗

PubMed1992

Relapse of paranoid psychotic state in methamphetamine model of schizophrenia.

The study of the clinical course of methamphetamine (MAP) psychosis yields insights into the biological aspect of the relapse of the paranoid psychotic state with hallucination in schizophrenia. A series of MAP psychosis studies in Japan conducted over a period of more than four decades revealed three types of clinical courses of MAP psychosis after discontinuation of MAP: transient type, prolonged type, and persistent type. Identification of the latter two indicates a lasting change in the brain that produces and maintains a schizophrenia-like paranoid psychotic state without MAP. The characteristic course seen in the transient type is acute recurrence of the psychotic state after a long remission period, almost identical to the initial episode, due to reuse of MAP or to psychological stressors. Such lasting vulnerability of the brain to schizophrenia-like psychotic symptoms may be caused by a lasting sensitization of the brain to the psychotogenic action of MAP resulting from its chronic abuse. Experimental studies using animals sensitized to MAP-induced stereotypy suggest that lasting enhancement of MAP-induced dopamine release in the striatum and nucleus accumbens is related to the development and expression of brain vulnerability to schizophrenic symptoms.

Animals↗

PubMed2000

Effects of arsenic-, platinum-, and gold-containing drugs on the disposition of exogenous selenium in rats.

Having found that the electrophilic model compound sulfobromophthalein markedly altered the fate of exogenous selenium in the body by reacting in vivo with nucleophilic selenium metabolites, the effects of metal-containing drugs with expected selenium reactivity were tested on biliary, urinary, and pulmonary excretion. Tissue distribution of selenium in selenite-injected rats was also examined. Coadministration with [(75)Se]selenite (10 micromol/kg, iv) of the trypanosomicid arsenicals (100 micromol/kg, iv) trimelarsan (TMA) or melarsoprol (MAP), the antitumor cisplatin (25 micromol/kg, iv), or the antirheumatic gold sodium thiomalate (25 or 50 micromol/kg, iv) significantly altered the disposition of (75)Se, whereas carboplatin (100 micromol/kg, iv) did not produce such an effect. The most dramatic alterations included the approximately 20-fold increase in the biliary excretion rate of selenium in response to TMA and MAP, the almost complete cessation of the exhalation of selenium as dimethyl selenide after administration of the arsenic- and gold-containing drugs, and the manifold accumulation of selenium in the blood plasma following gold injection. Direct chemical reaction of the drugs with nucleophilic selenite metabolites in the body may underlie these alterations. The tight coordination in time and extent observed between the biliary excretion of arsenic and selenium in rats receiving either of the arsenicals and selenite supports this hypothesis. However, attempts to detect selenium-containing biliary metabolites of TMA and MAP have failed, possibly owing to their instability. In summary, the arsenic-, platinum- and gold-containing drugs significantly influence the fate of exogenous selenium, whereby they may adversely affect the availability of this essential element for synthesis of selenoenzymes. Furthermore, the capability of TMA and MAP to enhance the biliary and total excretion of selenium renders these drugs significant candidates for antidotes in selenium intoxication.

Animals↗

PubMedDate not supplied

Cerebral blood volume maps with dynamic contrast-enhanced T1-weighted FLASH imaging: normal values and preliminary clinical results.

PURPOSE: In this article we investigate the application of a method that uses the relaxation effect of bolus-like injected Gd-DTPA to quantify regional cerebral blood volume (rCBV). The aim of the study was to determine if the method provides correct rCBV values in healthy subjects as well as to obtain additional diagnostic information for patients with a glioma or stroke. METHOD: Twenty healthy subjects, 12 patients with brain infarctions, and 18 patients with gliomas were examined. A series of 64 sequential images of one slice was recorded during bolus transit with a FLASH sequence. The measured signal intensity-time curves were converted pixel-wise to concentration-time curves from which the rCBV images were calculated applying the indicator dilution method. RESULTS: An average value for gray and white matter of 4.4 +/- 1.6 vol% was obtained for the group of healthy subjects. The grading of the tumors could be classified according to the differences of their corresponding rCBV values. Fifty percent of the infarct patients had to be excluded from the analysis in the acute phase due to mispositioning of the slice and data degradation by gross motion artifact. Different rCBV values were found for areas that develop later into gliotic scars or cystic necrosis. CONCLUSION: The proposed method is easy to apply in clinical routine MR investigations and provides valuable information for noninvasive, preoperative assessment of tumor grading. It can also provide additional criteria for estimating the histological outcome and with it the degree of ischemia in stroke patients.

Adolescent↗