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At least 1,027 records · Page 57Linked to original sources
PubMed2006

Replication of an oxidized abasic site in Escherichia coli by a dNTP-stabilized misalignment mechanism that reads upstream and downstream nucleotides.

Abasic sites (AP) and oxidized abasic lesions are often referred to as noninstructive lesions because they cannot participate in Watson-Crick base pairing. The aptness of the term noninstructive for describing AP site replication has been called into question by recent investigations in E. coli using single-stranded shuttle vectors. These studies revealed that the replication of templates containing AP sites or the oxidized abasic lesions resulting from C1'- (L) and C4'-oxidation (C4-AP) are distinct from one another, suggesting that structural features other than Watson-Crick hydrogen bonds contribute to controlling replication. The first description of the replication of the abasic site resulting from formal C2'-oxidation (C2-AP) is presented here. Full-length and single-nucleotide deletion products are observed when templates containing C2-AP are replicated in E. coli. Single nucleotide deletion formation is largely dependent upon the concerted effort of pol II and pol IV, whereas pol V suppresses frameshift product formation. Pol V utilizes the A-rule when bypassing C2-AP. In contrast, pol II and pol IV utilize a dNTP-stabilized misalignment mechanism to read the upstream and downstream nucleotides when bypassing C2-AP. This is the first example in which the identity of the 3'-adjacent nucleotide is read during the replication of a DNA lesion. The results raise further questions as to whether abasic lesions are noninstructive lesions. We suggest that abasic site bypass is affected by the local biopolymer structure in addition to the structure of the lesion.

Base Pairing↗
PubMed1996

Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248-271 are dispensable for dimer formation.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization [Laughrea, M., & Jetté, L. (1994) Biochemistry 33, 13464-13474; Skripkin et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4945-4949] posits that the 248-270 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is the full or at least the core dimerization domain of HIV-1 RNA. Here, we show by nested deletion analysis that the 3' boundary of the HIV-1 dimerization domain is immediately downstream of hairpin 248-270 and that the isolated region 248-271 dimerizes at least as readily as longer RNAs. Among various HIV-1Lai RNA transcripts containing hairpin 248-270, all form two types of dimer, as is implicit in the kissing-loop model. The high-stability dimer resists semidenaturing conditions and the low-stability dimer cannot, which is consistent with the model. At physiological temperatures, low-stability dimers are usually formed, as if dimerization without nucleocapsid proteins corresponded to loop-loop interaction without switching from intra- to interstrand hydrogen bonding. Our results show that the 3' DLS (a sequence immediately 3' from the 5' splice junction and originally thought to be the dimerization domain of the HIV-1 genome) and adjacent nucleotides are not necessary for efficient dimerization of HIV-1Lai RNA at low and high ionic strength. Upstream of hairpin 248-270 exists another "DLS-like" sequence that we name 5' DLS: like the isolated 3' DLS, the isolated 5' DLS forms an apparently nonphysiological structure that can become substantially dimeric at high ionic strength.

Base Sequence↗
PubMed1998

Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to polyketide biosynthesis.

Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called "modules", each of which is responsible for one cycle of chain extension and processing. The modular nature of these enzymes suggests that the biosynthetic pathway might be rationally reprogrammed by manipulation of synthases at the domain level. Although, several examples of successful engineering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of "downstream" active sites to nonnatural substrates. Here, we report that the terminal modules of DEBS, which normally process highly functionalized intermediates, are competent to carry out their natural functions on smaller, more simple substrates. Expressed in the absence of other DEBS proteins, the DEBS3 protein, which normally carries out the final two extension cycles in the synthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed with a C3 carboxylic acid. This substrate is then extended through two condensation cycles to form a triketide. Tolerance of the "shortened" intermediates in the biosynthesis of this triketide, in combination with results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyketide synthases. Interestingly, priming of DEBS3 appears to proceed, not by acyltransfer from propionyl-CoA, but by decarboxylation of an enzyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry 36, 1846-1851] and suggests that, in the absence of an acceptable primer (or transferred intermediate), decarboxylative priming of ketosynthase domains may be a general property of modular polyketide synthases.

Amino Acid Sequence↗
PubMedDate not supplied

Effect of oxygen delignification operating parameters on downstream enzymatic hydrolysis of softwood substrates.

Enhanced oxygen delignification of softwood pulp samples (taken upstream and downstream of a commercial oxygen delignification unit) improved the initial rate of enzymatic saccharification and overall yield of monomeric sugars by 62-82% and 76-80%, respectively. Laboratory-scale experiments were used to examine the effect of a broad range of operating parameters (temperature, time, caustic concentration, and oxygen partial pressure) on the effectiveness of oxygen delignification. Using empirical models, kappa number (residual lignin content) was found to effectively predict final conversion to monomeric sugars. Application of oxygen delignification to sulfite mill knots resulted in smaller (20-25%) reduction in lignin content. However, using a combination of oxygen delignification and particle size reduction, up to 80% of the carbohydrate in the reject knots could be converted to fermentable sugars.

Carbohydrates↗
PubMedDate not supplied

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.

Animals↗
PubMedDate not supplied

Reactive extraction for downstream separation of 1,3-propanediol.

The downstream separation of 1,3-propanediol from dilute aqueous solution was studied. A process combining reversible reaction of 1, 3-propanediol with acetaldehyde to 2-methyl-1,3-dioxane and a simultaneous extraction of the product by organic solvent appears to be technically feasible and attractive. The dioxane yield was 91-92%, the overall conversion of 1,3-propanediol was ca. 98%, and recovery of dioxane into the organic extractant was 75%.

Acetaldehyde↗
PubMed2003

Opening of tartrate acetals using dialkylboron bromide: evidence for stereoselectivity downstream from ring fission.

Johnson-type acetals derived from dimethyl tartrate give, after opening with Me(2)BBr and cuprate displacement, secondary alcohols with high diastereoselectivity (>30:1). The mechanism proposed for the induction of diastereoselectivity is downstream from the ring fission. It implies a direct participation of the Lewis acid as a source of nucleophile and the stereospecific transformation of the resulting bromo acetal through an invertive and temperature-dependent process. The acetals are prepared by reaction of the desired aldehyde with dimethyl tartrate. Removal of the auxiliary is accomplished through SmI(2) reduction or by an addition-elimination protocol using methoxide.

Journal Article↗
PubMed2000

AtZFP1, encoding Arabidopsis thaliana C2H2 zinc-finger protein 1, is expressed downstream of photomorphogenic activation.

C2H2 zinc-finger proteins play important roles in plant development including floral organogenesis, leaf initiation, lateral shoot initiation, gametogenesis and seed development. The gene for one such protein from Arabidopsis, AtZFP1 (Arabidopsis thaliana zinc-finger protein 1), is expressed at high levels in the shoot apex, including the apical meristem, developing leaves and the developing vascular system. In light-grown seedlings, AtZFP1 expression is induced about three days after germination, before the expansion of the true leaves. Dark-grown plants, in which photomorphogenesis is repressed, have no detectable AtZFP1 expression in the shoot apex. Under conditions which induce or mimic photomorphogenic development including growth in the light, shifting dark-grown plants to continuous light or growth on cytokinin in the dark, high levels of AtZFP1 expression are detected. Furthermore, AtZFP1 expression does not depend on active photosynthesis as shown by analysis of plants grown on the carotenoid biosynthetic inhibitor norflurazon. These results are discussed in relation to a possible role for AtZFP1 in shoot development, downstream of photomorphogenic activation.

Arabidopsis↗
PubMed2000

Downstream molecular determinants of response to 5-fluorouracil and antifolate thymidylate synthase inhibitors.

Thymidylate synthase (TS) is an essential enzyme for the de novo synthesis of thymidylate and subsequently DNA synthesis. TS has been used as a target for cancer chemotherapy in the development of fluoropyrimidines such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine and of novel folate-based TS inhibitors such as ZD1694 (Tomudex, Raltitrexed), ZD9331, LY231514 (ALIMTA, Pemetrexed), AG337 (Thymitaq, Nolatrexed) and AG331. Although TS has been considered as a target for chemotherapy, the precise mechanism by which TS inhibition leads to cell death is still not completely resolved. TS inhibition results in depletion of dTTP, an essential precursor for DNA, and an increase in dUTP. This results in the so-called thymine-less death due to misincorporation of dUTP into DNA; its excision, catalysed by uracil-DNA glycosylase, results in DNA damage. Both this imbalance in dTTP/dUTP and DNA damage can result in induction of downstream events, leading to apoptosis. On the other hand a specific interaction exists between oncogenes and TS, by binding of TS protein to the p53 and c-myc RNA, while wt p53 can also inhibit TS promotor activity. TS inhibition by either 5-FU or antifolates can also result in a depression of TS protein mediated inhibition of TS mRNA translation leading to induction of more TS protein synthesis, and p53 protein may further deregulate this process. These complex indirect and direct interactions between oncogenes and TS may have as yet unclear clinical implications, since most data are based on in vitro or in vivo studies and some results are contradictive. In some preliminary clinical studies evidence was postulated for a combined prognostic role for TS and p53. This knowledge should be used to design clinical studies with the aim to deliver effective treatment to potentially sensitive patients both in the adjuvant setting and in advanced stage disease.

Antimetabolites, Antineoplastic↗
PubMed2001

Myc and mammary cancer: Myc is a downstream effector of the ErbB2 receptor tyrosine kinase.

The proto-oncogene c-myc encodes a transcription factor which plays a major role in the regulation of normal cellular proliferation and is aberrantly expressed in many breast tumors. In a normal cell Myc expression levels are tightly regulated being subject to many layers of control. Errantly expressed Myc collaborates with other oncogenes to promote transformation. In this review we will focus on the association between abnormal Myc expression and mammary cancer. In particular, we will discuss the role of Myc as a downstream effector of the ErbB2 receptor tyrosine kinase which is overexpressed and constitutively activate in many mammary tumors. The cooperation between Myc and ErbB2 in transformation will be discussed in relation to clinical studies on Myc in human cancer and with consideration of transgenic models of Myc-induced mammary cancer. Data from our laboratory will be presented showing that deregulated ErbB2 activity strongly stimulates cytoplasmic signaling pathways which in turn impinge on Myc at multiple levels causing its deregulated expression.

Animals↗
PubMed2002

Allele-specific underacetylation of histone H4 downstream from promoters is associated with X-inactivation in human cells.

We have used a novel approach to investigate the histone H4 acetylation status at X-inactivated genes compared with their active counterparts. Immunoprecipitation with a sheep antibody that preferentially binds multiply-acetylated H4 isoforms was used to select hyperacetylated chromatin from a human female lymphoblastoid cell line exhibiting non-random X-inactivation as a result of an X/autosome translocation. The distribution of active and inactive gene sequences between the immunoprecipitated and bulk chromatin was compared at four X-linked loci containing intragenic polymorphic microsatellite repeats to allow identification of individual alleles by polymerase chain reaction. We find that DNA sequences corresponding to transcriptionally silent alleles are consistently under-represented in the hyperacetylated fraction. As the microsatellite repeat sequences used to identify alleles range in distance from 6.5 kb to 25 kb downstream of promoters, we conclude that differential H4 acetylation of active and silent chromatin is not confined to regions involved in the initiation of transcription, contrary to previous reports.

Acetylation↗
PubMed2006

The influence of downstream arterial perfusion parameters on doppler pulsatility index and resistance index in an in-vitro perfusion model.

BACKGROUND: To investigate in vitro how downstream perfusion parameters influence pulsatility index (PI), resistance index (RI) and their constituting Doppler velocities. MATERIALS AND METHODS: We analyzed the influence of resistance, compliance, reflection coefficient and input impedance on PI and RI in an in-vitro model of arterial flow. RESULTS: The nominators of PI and RI were determined by resistance. The numerators were determined by compliance and by the reflection coefficient. There were close relationships of PI and RI with resistance under the condition of constant compliance, but not when compliance was variable. CONCLUSION: PI and RI consist of velocity terms which are independently influenced by different parameters of impedance: compliance, reflection coefficient and resistance. These findings explain the contradictory results reported for the relationship between the indices and peripheral resistance in studies where compliance and reflection effects were not considered.

Animals↗
PubMed1980

Sea urchin histone mRNA termini are located in gene regions downstream from putative regulatory sequences.

S1 nuclease mapping of the Psammechinus miliaris embryonic histone mRNAs locates the 5' termini in or adjacent to a short sequence homology (5'pyCATTCpu3') downstream from the putative RNA polymerase II regulatory sequence (5'TATAAATA3') or related sequences. The 3' termini map just after a sequence containing GC-rich hyphenated dyad symmetry, a feature of most known terminator sequences.

Animals↗
PubMed1992

Raf functions downstream of Ras1 in the Sevenless signal transduction pathway.

Specification of the R7 cell fate in the developing Drosophila eye requires activation of the Sevenless (Sev) receptor tyrosine kinase, located on the surface of the R7 precursor cell, by its interaction with the Boss protein, expressed on the surface of the neighbouring R8 cell. Four genes that participate in the intracellular transmission of this signal have so far been identified and molecularly characterized: Ras1, Sos, Gap1 and sina (refs 4-8). The Drosophila homologue of the mammalian Raf-1 serine/threonine kinase, which has been implicated in signal transduction pathways activated by many receptor tyrosine kinases (reviewed in refs 9 and 10), is encoded by the raf locus (also known as l(1)polehole, Draf-1 or Draf). Here we show that the Drosophila Raf serine/threonine kinase also plays a crucial role in the R7 pathway: the response to Sev activity is dependent on raf function, and a constitutively activated Raf protein can induce R7 cell development in the absence of sev function. We also present genetic evidence suggesting that Raf acts downstream of Ras1 and upstream of Sina in this signal transduction cascade.

Animals↗
PubMed1993

Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of Notch.

During neurogenesis in Drosophila, groups of equipotential, neurally competent cells choose between epidermal and neural fates. Notch, a phylogenetically conserved transmembrane protein, may act as a receptor in a lateral signalling pathway in which a single neural precursor is chosen from each group and the neural fate of the other cells is inhibited, causing them to differentiate into epidermis. Possible intracellular transduction events mediating signals from Notch are, however, unknown. shaggy is also required for the lateral signal and encodes serine/threonine protein kinases with homology to the glycogen synthase kinase-3 (GSK-3) enzymes that act in signal transduction pathways in vertebrates. We report here that, in transgenic flies, GSK-3 beta can substitute for shaggy, and we also present a study of epistatic relationships between shaggy and gain and loss of function alleles of Notch. The results indicate that shaggy/GSK-3 is part of a signalling pathway downstream of Notch.

Animals↗
PubMed1996

A gene complex acting downstream of dpp in Drosophila wing morphogenesis.

Localized expression of the transforming growth factor-beta (TGF-beta) homologue decapentaplegic (dpp) is crucial for Drosophila wing development. Here we show that spalt and spalt-related (sal and salr), two closely related genes that encode transcription factors, are expressed in response to dpp in a central territory of the wing imaginal disc, where they are required for the patterning of the wing. They are among the first identified elements that act downstream of dpp in wing development. The phenotypic consequences of misexpression of sal and salr suggest that an important outcome of dpp activity is the subdivision of the wing disc into territories smaller than lineage compartments, through the regulation of transcription-factor-encoding genes such as sal and salr.

Animals↗
PubMed1996

c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption.

The primary defect in mice lacking the c-src gene is osteopetrosis, a deficiency in bone resorption by osteoclasts. Osteoclasts express high levels of the c-Src protein and the defect responsible for the osteopetrotic phenotype of the c-src-deficient (src-) mouse is cell-autonomous and occurs in mature osteoclasts. However, the specific signalling pathways that require c-Src expression for normal osteoclast activity have not been elucidated. We report here that the proto-oncogene product c-Cbl is tyrosine-phosphorylated in a Src-dependent manner in osteoclasts, where the two proteins colocalize on some vesicular structures. In vitro bone resorption by osteoclast-like cells (OCLs) is inhibited by both c-src and c-cbl antisense oligonucleotides. Furthermore, tyrosine phosphorylation of c-Cbl and the localization of c-Cbl-containing structures to the peripheral cytoskeleton are impaired in resorption-deficient c-src- OCLs, as well as in wild-type OCLs that have been treated with c-src antisense oligonucleotides. These results indicate that c-Cbl may act downstream of c-Src in a signalling pathway that is required for bone resorption.

3T3 Cells↗
PubMed2005

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Animals↗