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At least 217 records · Page 12Linked to original sources

PubMed2003

Luminescent CdSe/CdS core/shell nanocrystals in dendron boxes: superior chemical, photochemical and thermal stability.

The surface ligands, generation-3 (G3) dendrons, on each semiconductor nanocrystal were globally cross-linked through ring-closing metathesis (RCM). The global cross-linking of the dendron ligands sealed each nanocrystal in a dendron box, which yielded box-nanocrystals. Although the dendron ligands coated CdSe nanocrystals (CdSe dendron-nanocrystals) were already quite stable, the stability of CdSe box-nanocrystals against chemical, photochemical, and thermal treatments were dramatically improved in comparison to that of the original dendron-nanocrystals. Furthermore, the box structure of the ligands monolayer coupled with the stable inorganic CdSe/CdS core/shell nanocrystals resulted in a class of extremely stable nanocrystal/ligands complexes. The band edge photoluminescence of the core/shell dendron-nanocrystals and box-nanocrystals were partially remained, and could be further brightened through controlled chemical oxidation or photooxidation. Practically, the stability of the box-nanocrystals is sufficient for most fundamental studies and technical applications. The box-nanocrystals may represent a general solution for the commonly encountered instability for many types of colloidal nanocrystals. The size distribution of the empty dendron boxes formed by the dissolution of the inorganic nanocrystals in concentrated HCl was very narrow. The empty boxes as new types of polymer capsules are soluble in solution, mesoporous, and with a very thin but stable peripheral. Those nanometer-sized cavities should be of interest for many purposes in the field of solution host-guest chemistry.

Journal Article↗

PubMed1999

Interaction between composite elements in the napA promoter: both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression.

During seed maturation, the transcriptional activity of napin genes is regulated by developmental signals involving the transcriptional activator ABI3 and abscisic acid (ABA). To localize cis elements involved in the seed-specific activity of the napin napA promoter, a systematic analysis was performed focusing on two major element complexes, the B-box and RY/G. Substitution mutation analysis using promoter-reporter gene fusions in stable transgenic tobacco showed synergistic interactions between elements within these complexes. The distal part of the B-box shows similarities to abscisic acid response elements and the proximal portion contains a CA-rich element. In vitro studies involving Exonuclease III protection and electrophoretic mobility shift assays revealed binding by nuclear proteins to elements within the B-box. The distal and proximal parts of the B-box were found to bind distinct nuclear protein complexes. By gain-of-function analysis with a tetramer of the B-box fused to a truncated (-46) cauliflower mosaic virus (CaMV) 35S minimal promoter, it was demonstrated that the B-box mediates strong activity in seeds. Further, it was shown that the elements in the B-box constitute an ABA-responsive complex, since the B-box tetramer mediates ABA-responsiveness in vegetative tissues to a construct containing the CaMV virus 35S enhancer (-343 to -90). Thus, the seed-specific activity of the napA promoter relies on the combinatorial interaction between the RY/G complex and the B-box ABA-responsive complex during the ABA response in seed development.

2S Albumins, Plant↗

PubMedDate not supplied

Comparative chromosome mapping of a conserved homoeo box region in mouse and human.

Specific genes are assumed to regulate pattern formation in the mammalian embryo, but as yet none has been identified unequivocally. It is possible that such genes in mammals may be identified by virtue of a conserved coding sequence, because many of the Drosophila melanogaster homoeotic and segmentation genes, which have crucial roles in the regulation of segmental pattern formation during embryonic development, contain a 180-base pair (bp) DNA sequence, the homoeo box, and that sequences homologous to the Drosophila homoeo box are also present in 6-10 copies in higher animals, including mammals. Although the assumption that the homoeo box identifies genes responsible for pattern formation in mammals remains to be validated, it is a particularly attractive hypothesis given the strong conservation of homoeo boxes over vast evolutionary distances. Here we report the localization of a human homoeo box region, previously cloned and shown to contain two homoeo boxes within a sequence of 5-kilobases (kb), to the long arm of chromosome 17. We show that two single-copy homoeo box-flanking probes derived from this region strongly hybridize to single-copy restriction fragments in mouse genomic DNA and that these conserved homoeo box-flanking sequences map to mouse chromosome 11. This may be significant as several genes that map to chromosome 17 in human also map to chromosome 11 in the mouse, implying that a segment of mouse chromosome 11 is homologous to a region of human chromosome 17. Taken together, these data suggest that the homoeo box region detected with our probes is highly conserved in human and mouse.

Animals↗

PubMed2003

Structural insights into the U-box, a domain associated with multi-ubiquitination.

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val-->Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.

Amino Acid Sequence↗

PubMed2006

Bilirubin oxidation products (BOXes) and their role in cerebral vasospasm after subarachnoid hemorrhage.

Many factors have been postulated to cause delayed subarachnoid hemorrhage (SAH)-induced vasospasm, including hemoglobin, nitric oxide, endothelin, and free radicals. We propose that free radicals (because of the high levels that are produced in the blood clots surrounding blood vessels after SAH) act on bilirubin, biliverdin, and possibly heme to produce BOXes (Bilirubin OXidized Products). Bilirubin oxidation products act on vascular smooth muscle cells to produce chronic vasoconstriction and vasospasm combined with a vasculopathy because of smooth muscle cell injury. This review summarizes recent evidence that BOXes play a role in SAH-induced vasospasm. The data supporting a role for BOXes includes (1) identification of molecules in cerebrospinal fluid (CSF) of patients with vasospasm after SAH that have structures consistent with BOXes; (2) BOXes are vasoactive in vitro and mimic the biochemical actions of CSF of patients with vasospasm; (3) BOXes are vasoactive in vivo, constricting rat cerebral vessels; and (4) there is a correlation between clinical occurrence of vasospasm and BOXes concentration in our preliminary study of patients with SAH. Since oxidation of bilirubin, biliverdin, and perhaps heme is proposed to produce BOXes that contribute to vasospasm, either blocking bilirubin formation, inactivating bilirubin or BOXes, or removing all of the blood clot before vasospasm are potential treatment targets.

Animals↗

PubMed2004

Box length search algorithm for molecular simulation of systems containing periodic structures.

We have developed a box length search algorithm to efficiently find the appropriate box dimensions for constant-volume molecular simulation of periodic structures. The algorithm works by finding the box lengths that equalize the pressure in each direction while maintaining constant total volume. Maintaining the volume at a fixed value ensures that quantitative comparisons can be made between simulation and experimental, theoretical or other simulation results for systems that are incompressible or nearly incompressible. We test the algorithm on a system of phase-separated block copolymers that has a preferred box length in one dimension. We also describe and test a Monte Carlo algorithm that allows the box lengths to change while maintaining constant volume. We find that the box length search algorithm converges at least two orders of magnitude more quickly than the variable box length Monte Carlo method. Although the box length search algorithm is not ergodic, it successfully finds the box length that minimizes the free energy of the system. We verify this by examining the free energy as determined by the Monte Carlo simulation.

Journal Article↗

PubMed2006

Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase.

Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions.

Amino Acid Sequence↗

PubMed1997

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Amino Acid Sequence↗

PubMed1998

An additional role for the F-box motif: gene regulation within the Neurospora crassa sulfur control network.

The F-box represents a protein motif originally identified as a conserved amino-terminal domain within the Neurospora crassa negative regulator sulfur controller-2. Recently, F-boxes have been found within a number of cell cycle regulatory proteins, where they mediate ubiquitin-driven proteolytic events required for major cell cycle transitions. F-box function, however, is not restricted solely to cell cycle pathways. Here we present evidence expanding F-box function to encompass gene regulatory processes independent of the cell cycle through in vivo analysis of an F-box acting within the N. crassa sulfur regulatory network. The Neurospora sulfur circuit features a set of regulatory genes acting to modulate gene expression based on environmental sulfur conditions. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, as well as the negative regulatory gene scon-2+. Through site-directed mutagenesis of the SCON2 F-box, we have generated a sulfur auxotrophic phenotype previously unobserved in any scon-2 mutant. Using Northern analysis, we have traced this auxotrophy to a complete shutdown of cys-3+ gene expression. We have further analyzed F-box function by constructing a series of chimeric SCON2 proteins containing swapped F-box domains from the yeast transcriptional inhibitor Met30p and the Candida albicans cell cycle regulator Cdc4p. The ability of these chimeric proteins to restore partial wild-type sulfur regulation in vivo emphasizes the universal nature of this motif and confirms the functional importance of the F-box within noncell cycle regulatory pathways.

Amino Acid Sequence↗

PubMed1998

NF-Y organizes the gamma-globin CCAAT boxes region.

The CCAAT-binding activator NF-Y is formed by three evolutionary conserved subunits, two of which contain putative histone-like domains. We investigated NF-Y binding to all CCAAT boxes of globin promoters in direct binding, competition, and supershift electrophoretic mobility shift assay; we found that the alpha, zeta, and proximal gamma CCAAT boxes of human and the prosimian Galago bind avidly, and distal gamma CCAAT boxes have intermediate affinity, whereas the epsilon and beta sequences bind NF-Y very poorly. We developed an efficient in vitro transcription system from erythroid K562 cells and established that both the distal and the proximal CCAAT boxes are important for optimal gamma-globin promoter activity. Surprisingly, NF-Y binding to a mutated distal CCAAT box (a C to T at position -114) is remarkably increased upon occupancy of the high affinity proximal element, located 27 base pairs away. Shortening the distance between the two CCAAT boxes progressively prevents simultaneous CCAAT binding, indicating that NF-Y interacts in a mutually exclusive way with CCAAT boxes closer than 24 base pairs apart. A combination of circular permutation and phasing analysis proved that (i) NF-Y-induced angles of the two gamma-globin CCAAT boxes have similar amplitudes; (ii) occupancy of the two CCAAT boxes leads to compensatory distortions; (iii) the two NF-Y bends are spatially oriented with combined twisting angles of about 100 degrees. Interestingly, such distortions are reminiscent of core histone-DNA interactions. We conclude that NF-Y binding imposes a high level of functionally important coordinate organization to the gamma-globin promoter.

Base Sequence↗

PubMed2002

The hU3-55K protein requires 15.5K binding to the box B/C motif as well as flanking RNA elements for its association with the U3 small nucleolar RNA in Vitro.

The 15.5K protein directly binds to the 5' stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of 15.5K to the box B/C motif. The association of hU3-55K with the U3BC RNA was found to be also dependent on a conserved RNA structure that flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our data support a new structural model of the box B/C region of the U3 snoRNA in which the box B/C motif is base-paired to form a structure highly similar to that of both the U4 5' stem-loop and the box C/D motif.

Amino Acid Sequence↗

PubMed2006

VMD2 promoter requires two proximal E-box sites for its activity in vivo and is regulated by the MITF-TFE family.

The retinal pigment epithelium (RPE) is crucial for the function and survival of retinal photoreceptors. VMD2 encodes bestrophin, an oligomeric chloride channel that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Previously, we defined the VMD2 upstream region from -253 to +38 bp as being sufficient to direct RPE-specific expression in the eye, and we suggested microphthalmia-associated transcription factor (MITF) as a possible positive regulator. Here we show that in transgenic mice the -154 to +38 bp region is sufficient for RPE expression, and mutation of two E-boxes, 1 and 2, within this region leads to loss of promoter activity. A yeast one-hybrid screen using bait containing E-box 1 identified clones encoding MITF, TFE3, and TFEB, and chromatin immunoprecipitation with antibodies against these proteins enriched the VMD2 proximal promoter. Analysis using in vivo electroporation with constructs containing mutation of each E-box indicated that expression in native RPE requires both E-boxes, yet in vitro DNA binding studies suggested that MITF binds well to E-box 1 but only minimally to E-box 2. MITF knockdown by small interfering RNA (siRNA) in cell culture revealed a strong correlation between MITF and VMD2 mRNA levels. Sequential transfection of a luciferase construct with expression vectors following MITF siRNA revealed that TFE3 and TFEB can also transactivate the VMD2 promoter. Taken together, we suggest that VMD2 is regulated by the MITF-TFE family through two E-boxes, with E-box 1 required for a direct interaction of MITF-TFE factors and E-box 2 for binding of the as yet unidentified factor(s).

Animals↗

PubMed1999

Effects of box features on spine loading during warehouse order selecting.

Low back disorders in distribution centres or warehouses have been identified as an area of elevated risk in many industries. The task of an order selector requires workers manually to lift boxes from storage bins to a mobile pallet. This study explored the effect of box features and box location when lifting from a pallet in a storage bin upon spine loading. Ten experienced warehouse workers were asked to lift boxes from a pallet while the size, weight, handle features and location of the box on a pallet were changed. An EMG-assisted model was employed to assess spine compression, lateral shear and anterior-posterior shear during the lifts. The position from which the worker lifted a box on a pallet had the most profound effect on spine loading while the lower level of the pallet represented the greatest loadings on the spine. Box weight did not appear to be a feasible means of controlling spine loading unless its position on the pallet could also be controlled. The inclusion of handles had an effect similar to reducing the box weight by 4.5 kg, whereas box size did not effectively affect spine loading. The mechanisms by which these factors affect spine loading are discussed.

Adult↗

PubMed1997

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.

Bacterial Proteins↗

PubMed1999

Nucleolar localization signals of box H/ACA small nucleolar RNAs.

The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3' hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5' or 3' hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.

Animals↗

PubMed2002

Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP.

Methylation of the ribose 2'-hydroxyl, the most widespread modification of ribosomal and splicesomal RNAs, is guided by the box C/D class of small nucleolar RNAs (snoRNAs). Box C/D small nucleolar ribonucleoproteins (snoRNPs) contain four core proteins: fibrillarin, Nop56, Nop58 and 15.5 kDa. We constructed U25 snoRNAs containing a single photoactivatable 4-thiouridine at each U position within the conserved box C/D and C'/D' motifs. Proteins assembled on the snoRNA after injection into Xenopus oocyte nuclei were identified by cross-linking, and reconstituted particles characterized by functional rescue and mutational analyses. Our data argue that box C/D snoRNPs are asymmetric, with the C' box contacting Nop56 and fibrillarin, the C box interacting with Nop58, and the D and D' boxes contacting fibrillarin. No cross-link to 15.5 kDa was detected; its binding is disrupted by 4-thiouridine substitution in position 1 of the C box. Repositioning the guide sequence of U25 upstream of box D instead of D' revealed that both C/D motifs have the potential to function as guide centers, but, surprisingly, there was no alteration in protein cross-linking.

Animals↗

PubMed2000

The box C/D motif directs snoRNA 5'-cap hypermethylation.

The 5'-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2, 7-trimethylguanosine structure. 5'-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5'-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C' in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C' in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C' in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs.

Animals↗

PubMed2006

The role of high mobility group box-1 protein in severe sepsis.

PURPOSE OF REVIEW: Despite medical advances, mortality in severe sepsis remains high. As our understanding of the innate immune system has expanded, clinical trials have focused on inhibiting cytokines present early in the infectious process such as interleukin-1 and tumor necrosis factor-alpha, although with disappointing results. There is evidence that the nuclear protein high mobility group box-1 protein, when released extracellularly, acts as a persistent mediator of sepsis and is therefore a promising candidate for therapeutic intervention. This review summarizes current knowledge of the protein and highlights recent relevant findings. RECENT FINDINGS: High mobility group box-1 protein may be released into the circulation either due to necrosis of cells or by active release from macrophages and endothelial cells. Models of experimental sepsis in mice have shown a strong association between extracellular high mobility group box-1 protein and lethality. Treatments against the biological activities of high mobility group box-1 protein reduce lethality in these models. Other studies have shown high mobility group box-1 protein as a key regulator in acute and chronic inflammation. Recent findings confirm that high mobility group box-1 protein is persistently elevated in human patients with severe sepsis. SUMMARY: Despite all efforts, mortality in severe sepsis remains high. A massive amount of evidence indicates high mobility group box-1 protein as a delayed and important propagator of inflammation. Recent studies confirm persisting high levels of high mobility group box-1 protein in serum up to 1 week after hospitalization. Reducing levels of the protein by anti-high mobility group box-1 protein treatment may be one way to moderate uncontrolled inflammation seen in sepsis.

Animals↗