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PubMed2006

Aquaporin subfamily with unusual NPA boxes.

Aquaporins have been identified based on highly conserved two asparagine-proline-alanine (NPA) boxes that are important for the formation of a water-permeating pore. Some aquaporin-like sequences, however, have less conserved NPA boxes. Although they have lower homology with conventional aquaporins, they should be included in aquaporin family based on their conserved six transmembrane domains and hydrophobic NPA box-like repeats. They are widely distributed in multicellular organisms. Only SIPs from plants and AQP11/12 from mammals were examined previously and found to be localized inside the cell. Intracellular localization will be a common feature of these aquaporin-like proteins since most of them have positively charged amino acid clusters at the carboxy-termini similar to di-lysine motif (-KKXX) for an endoplasmic reticulum retention signal. Accordingly, they are tentatively named subcellular-aquaporins in this review. Currently, studies on their functions and biological roles are limited. SIPs were shown to function as water channels and the disruption of AQP11 produced neonatally fatal polycystic kidneys. Further works on subcellular-aquaporins will reveal new insights into the roles of aquaporins.

Alanine↗

PubMed2006

Assembly into snoRNP controls 5'-end maturation of a box C/D snoRNA in Saccharomyces cerevisiae.

The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5'-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5'-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3'-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Our data suggest a functional coupling between snoRNP assembly and 5'-end maturation of independently transcribed box C/D snoRNAs.

3' Flanking Region↗

PubMed2007

Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast.

Ribose methylation of eukaryotic rRNA is directed by box C/D small nucleolar RNAs (snoRNAs), which pinpoint the nucleotide to be methylated in specific position within the rRNA sequence. Here, we report the identification of a novel double-guide C/D box snoRNA termed snR88 that directs methylation of two previously undetermined sites in 25S rRNA from the fission yeast. Knockout of the predicted TATA box of the snR88 gene resulted in the complete blocking of its expression, showing that snR88 is an independently transcribed gene and dispensable for yeast viability. The depletion of snR88 abolished 25S rRNA methylation at U2304 and U2497 simultaneously. Interestingly, an unusual pause of reverse transcription at U2495 was observed, which implies an unknown structure of 25S rRNA related to ribose methylation at U2497 in the fission yeast.

Base Sequence↗

PubMed2004

Genetically-based behavioral traits influence the effects of Shuttle Box avoidance overtraining and extinction upon intertrial responding: a study with the Roman rat strains.

In the present study, we evaluated the effects of extended exposure procedures to contextual fear cues as a way of reducing intertrial responses (ITRs), a characteristic stereotyped behavior displayed in 2-way Shuttle Box avoidance overtraining. We used rat strains psychogenetically selected for efficient (Roman high-avoidance; RHA) versus poor 2-way Shuttle Box performance (Roman low-avoidance; RLA), the former being fearless/impulsive relative to the latter. Additionally, Sprague-Dawley (SD) rats were compared with RHAs to have a measure of the ITRs exhibited by another, non-selected group of animals. The main findings were that after extended exposure RHAs diminished their ITRs without affecting avoidance performance, although they emitted a pattern of ITRs somewhat excessive as compared with SDs. We concluded that three factors could influence the expression of ITRs in RHA rats: (1) the repetitive and stereotyped responding induced by 2-way Shuttle Box overtraining; (2) contextual fear conditioning; and (3) a genetic background associated with high impulsiveness.

Animals↗

PubMed2004

Guide RNAs with 5' caps and novel box C/D snoRNA-like domains for modification of snRNAs in metazoa.

BACKGROUND: Spliceosomal snRNAs and ribosomal RNAs in metazoans contain numerous modified residues that are functionally important. The most common modifications are site-specific 2'-O-methylation and pseudouridylation, both directed by small ribonucleoprotein particles. Each particle is composed of a short guide RNA and a set of several proteins. All previously characterized modification guide RNAs in metazoa are encoded in and processed from introns. RESULTS: We have identified and characterized three novel guide RNAs for conserved 2'-O-methylation of U2, U4, and U12 snRNAs. Two guides, termed mgU2-25/61 and mgU12-22/U4-8, appear to be independently transcribed as judged by the presence of methylated guanosine caps at their 5' ends and upstream promoters similar to those of telomerase RNA. These guide RNAs are each composed of a canonical box C/D snoRNA and a novel box C/D snoRNA-like domain, where the C'/D' motif, rather than C/D, can be folded into a conserved kink-turn structure. The snoRNA-like domains are predicted to direct 2'-O-methylation of invariant G residues that occupy analogous positions in the U2 and U12 snRNA secondary structures. A third guide, mgU2-19/30 RNA, is composed of two canonical box C/D snoRNA domains encoded within a single intron. CONCLUSIONS: This is the first description in metazoan cells of 5'-capped modification guide RNAs that appear to be independently transcribed. Since plant, yeast, and protozoan guide RNAs are mostly independently transcribed, the identification of such RNAs argues that ancestral metazoans possessed independently transcribed guide RNAs and only later, during the evolution of metazoan organisms, did the guide RNA genes shift to introns.

Animals↗

PubMed2005

The APC subunit Doc1 promotes recognition of the substrate destruction box.

BACKGROUND: Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. RESULTS: To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. CONCLUSIONS: Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2006

"Black box" 101: How the Food and Drug Administration evaluates, communicates, and manages drug benefit/risk.

A prominently displayed boxed warning, the so-called "black box," is added to the labeling of drugs or drug products by the Food and Drug Administration when serious adverse reactions or special problems occur, particularly those that may lead to death or serious injury. Healthcare providers are often not knowledgeable about the origin, meaning, and implications of these "black box" warnings. In this review, our goal is to provide insight into how the Food and Drug Administration evaluates, communicates, and manages drug benefit/risk. We discuss drug labeling, the emphasis on safety throughout the drug approval process, legislative initiatives for safe use of drugs in children, and postmarketing safety surveillance. In addition, we encourage health care providers to report drug reactions to the Food and Drug Administration's MedWatch program. A discussion of new Food and Drug Administration initiatives to improve drug safety processes and methods to serve the public better are highlighted.

Drug Labeling↗

PubMed2006

SRY-directed DNA bending and human sex reversal: reassessment of a clinical mutation uncovers a global coupling between the HMG box and its tail.

Sex-reversal mutations in human SRY cluster within its high-mobility group box, a conserved motif of DNA bending. A classical substitution at the crux of this angular domain (M64I) has been reported to impair DNA bending but not DNA binding, implying that sharp bending is required for transcriptional activation and testis determination. Surprisingly, we report that this defect was an inadvertent consequence of protein truncation: in the intact protein, sharp DNA bending is restored by the basic tail of the high-mobility group box. Structural coupling between box and tail is tuned to the native DNA bend angle, damping conformational fluctuations and enabling bidirectional induced fit within the bent complex. M64I-associated sex reversal is instead caused by the impaired function of a flanking non-classical nuclear localization signal (NLS). Similar impairment is caused by M64A, suggesting that mislocalization is due to loss of an M64-specific function and not gain of a non-native I64-specific function. Transcriptional activity, attenuated by mislocalization, is rescued by fusion of a heterologous NLS. In a male embryonic gonadal cell line, M64I and M64A SRY-NLS fusion proteins exhibit native transcriptional activation of Sox9, a key step in testicular differentiation. Our results suggest that male development is robust to subtle alterations in SRY-DNA architecture but depends critically on nuclear localization. The previously unsuspected role of M64 within a non-classical NLS may contribute to its invariance among SOX-related and LEF-1-related transcription factors.

Active Transport, Cell Nucleus↗

PubMed2006

Training of A3-A3 side-to-side anastomosis in a deep corridor using a box with 6.5-cm depth: technical note.

BACKGROUND: Cerebral revascularization in the deep surgical field is technically challenging. Especially, side-to-side anastomosis like A3-A3 could be technically more difficult compared with end-to-side anastomosis. To improve surgeon's dexterity and maneuverability in the deep surgical field, the authors developed an easily accessible and well-simulating training system using prosthetic tubes and a box. METHODS: Two prosthetic tubes (silicon tube, 1.2 mm in diameter) are mounted in parallel on the bottom of 6.5-cm-deep emptied 'tissue paper box.' The orifice of the box is restricted to 2 x 2 cm to simulate a deep and narrow surgical corridor. Using bayonet-shaped micro needle holder and forceps, the side-to-side anastomosis of the tubes is performed with 10-0 nylon under operative microscope. RESULTS: Prosthetic tubes well simulated real A3-A3 anastomosis. From the standpoint of technical difficulty, this training system needed slightly higher level of dexterity compared with real A3-A3 anastomosis because of narrower and deeper surgical corridor, and the wall of prosthetic tube was slightly thicker and more inflexible. After this training, the surgical technique in real A3-A3 anastomosis was improved. CONCLUSIONS: This training system worked well to ease the transition from anastomosis in shallow surgical field to deep and narrow surgical field. The prosthetic tube we used approximates real A3 relatively well, and the ease in setting up this system enabled repeated practice, which resulted in steep learning curve of the technique.

Aged↗

PubMed2007

Prevalence of RHD 1227A and hybrid Rhesus box in the general Chinese population.

The Rhesus (Rh) blood group is the most polymorphic blood group system. Serological analyses revealed that only 0.1% to 0.4% of the Chinese population exhibited a D-negative phenotype. In contrast to D-negative Caucasian persons, who are mostly associated with a deletion of RHD between the upstream and downstream Rhesus boxes, approximately 30% of apparently D-negative Chinese were actually DEL, potentially harboring a grossly intact RHD gene. The authors' previous detection of the RHD 1227A allele in all DEL samples prompted the conclusion that the RHD 1227A gene is highly prevalent in DEL populations of Chinese ethnicity, which indicates that it may be a marker for the rapid detection of DEL. Currently, random surveys for RHD 1227 A allele and for hybrid Rhesus box were conducted among 399 Han Chinese in Taiwan. The estimated frequencies of RHD 1227G/G, RHD 1227A/G, RHD A/A, RHD 1227G/-, RHD 1227A/-, and RHD-/- were 0.89054, 0.0118, 0.00016, 0.0414, 0.00055, and 0.00192, respectively. These results were consistent with the prevalence of D-negative and DEL in persons of Chinese ethnicity. The authors conclude that detection of RHD 1227A and hybrid Rhesus box alleles is predictive of Rh phenotypes in the Chinese population.

Adolescent↗

PubMed2002

Murine H-rev107 gene encoding a class II tumor suppressor: gene organization and identification of an Sp1/Sp3-binding GC-box required for its transcription.

H-rev107, which belongs to class II tumor suppressor genes, is ubiquitously expressed in normal cells, but is downregulated in many carcinomas and tumor cell lines. Sequence analysis showed that the murine H-rev107 gene is composed of five exons and four introns. Transfections revealed that 7.6 kb of the H-rev107 promoter directed a high level expression of the reporter gene. There were no significant differences in promoter activity when various 5'-deletion promoter constructs from -7.6 kb to -113 bp were employed. By further deletion and mutation analysis, we found that the region between -83 and -75 containing a GC-box was essential for promoter activity in NIH3T3 or REF52 fibroblasts expressing H-rev107 at moderate to high levels. Gelshifts demonstrated in vitro binding of Sp1 and Sp3 to this GC-box. Cotransfection of Sp1 and Sp3 functionally stimulated promoter activity in SL2 cells. By chromatin immunoprecipitation assays, we observed in vivo binding of Sp1 and Sp3 to the proximal promoter region in NIH3T3 cells and liver, concluding that the transcription of the H-rev107 gene is dependent on Sp1/Sp3-binding to the -83/-75 GC-box.

3T3 Cells↗

PubMed2003

Molecular cloning and characterization of the human ASB-8 gene encoding a novel member of ankyrin repeat and SOCS box containing protein family.

We have cloned a new member of human ankyrin repeat and SOCS box containing protein family (ASB), designed as hASB-8, from a human placental cDNA library and further extended by 5(') and 3(')-RACE. The full-length cDNA was 2545bp in length, with a predicted open reading frame encoding a protein of 288 amino acids, which was 96% identical to mouse ASB-8 protein. Computer analysis revealed that the deduced amino acid sequence of the human ASB-8 contained four Ankyrin repeats and one SOCS box. The gene had four exons separated by three introns and was mapped to human chromosome 12q13. Human ASB-8 mRNA was expressed at the highest level of expression in skeletal muscle and at a varied level of expression in heart, brain, placenta, liver, kidney, and pancreas. The transcript of hASB-8 was not detected in adult normal lung tissue, but found in lung carcinoma cell lines SPC-A1, A549, and NCI-H446. Subcellular localization analysis showed that the EGFP-tagged hASB-8 protein was localized at cytoplasm in human hepatocellular carcinoma cell line BEL-7402. We also provided evidence that hASB-8 could interact with Elongin B-C complex in vitro. Furthermore, transfection with the truncated mutant of hASB-8 cDNA lacking SOCS box could suppress cell growth of lung adenocarcinoma SPC-A1 cells in vitro, which suggests that this gene might be related to the development of lung cancer.

Adult↗

PubMed1990

CC Ar GG boxes, cis-acting elements with a dual specificity. Muscle-specific transcriptional activation and serum responsiveness.

The influence of different CC Ar GG boxes derived from either muscle-specific or serum-responsive genes, on the specificity of different promoters has been investigated. Inserted upstream from an 85 base-pair long minimal promoter of the human cardiac alpha-actin gene, a single copy of both the cognate CC Ar GG element (HCA1) and the c-fos gene serum response element (SRE) stimulate transcription four- to fivefold more efficiently in C2 myogenic cells than in L fibroblastic cells, SRE being two- to threefold more active than HCA1. Inserted upstream from the ubiquitous Herpes simplex thymidine kinase (HSV-tk) promoter, multimerized CC Ar GG boxes behave as strong muscle-specific activating elements, about 20-fold more active in myogenic C2 cells than in L fibroblasts and hepatoma HepG2 cells. They also confer serum responsiveness on the HSV-tk promoter. Efficiency of HCA1 and SRE tetramers in conferring both muscle specificity and serum responsiveness is roughly similar. It appears, therefore, that regardless of their origin (either muscle-specific or serum-responsive genes) CC Ar GG boxes behave by themselves as both muscle-specific activating and serum-responsive elements.

Actins↗

PubMed2001

Transcriptional regulation of galectin-10 (eosinophil Charcot-Leyden crystal protein): a GC box (-44 to -50) controls butyric acid induction of gene expression.

Galectin-10 (gal-10, also known as Charcot-Leyden crystal protein) is a member of the galectin family of beta-galactoside binding proteins that is expressed uniquely in eosinophilic and basophilic leukocytes. To gain a better understanding of galectin gene expression, we present an analysis of the transcriptional regulation of the gene encoding gal-10. Analysis of the minimal promoter revealed nine consensus-binding sites for transcription factors, including several that are also found in the minimal promoters of galectins -1, -2, and -3. The decrease in gal-10 promoter activity after disruption of either the GC box (-44 to -50) or the Oct site (-255 to -261) suggests that these sites, along with the previously characterized GATA and EoTF sites, are necessary for full promoter activity. By supershift analysis, we demonstrate binding of the transcription factors Sp1 and Oct1 to the consensus GC box and the Oct site, respectively. Similar to gal-1, gal-10 expression is induced by butyric acid, an effect that is lost upon ablation of the GC box. Additionally, we demonstrate AML3 binding to the consensus AML site and YY1 binding to the Inr sequence, both elements functioning as silencers in the gal-10 promoter.

Base Sequence↗

PubMed1999

Lipid grafts of egg-box complex: a new supramolecular biovector for 5-fluorouracil delivery.

An attempt was made to improve the pharmacokinetic behaviour of 5-fluorouracil (5-FU) by incorporating it into lipoprotein imitating synthetic carrier 'supramolecular biovector (SMBV)' which is an important prerequisite for achieving its better therapeutic performance against cancer. The polysaccharide core of SMBVs was prepared by ionotropic gelation technique by cross-linking polyguluronate units in the alginate molecules with calcium ions to form so called 'egg-box structure'. The formulation and process variables were optimized to obtain particles of nanometer size range. Hydrophobization was carried out by fatty-acylation on the surface followed by phospholipid coating. Palmitoyl polyethylene glycol (p-PEG) was anchored to impart stealth behaviour. The scanning electron microscopy showed discrete spheres of average diameter 748 nm. Polydispersity was estimated to be 0.37. Overall zeta potential was -21.3 mV. The drug loading capacity and encapsulation efficiency was found to be 10.0% and 97.9%, respectively. The release from drug solution (AP) followed zero-order kinetics. Higuchi release pattern was obtained for egg-box complex cores (AP1) while first-order pattern was followed for fatty acylated (AP2) and lipid coated cores before (AP3) and after p-PEG anchoring (AP4). The amount of drug liberated in 24 h was in the order AP > AP1 > AP2 > AP4 > AP3. The release pattern obtained was a combined effect of drug diffusion through egg-box matrix as well as partitioning in hydrophobic layer and p-PEG layer around the SMBV. The stability study showed negligible leakage and no appreciable change in particle size upon storage at different temperatures which is an indication of good stability of SMBV formulation. The plasma clearance data revealed increase in circulation half-life of drug and bioavailability. Tissue distribution data obtained was a result of competitive uptake of formulations from tissue macrophages and lymphatics depending upon its surface characteristics and residence period in vascular system. The enhanced delivery of drug to lymphatics and improvement in its half-life render SMBVs useful for control of metastasis and tumour growth.

Alginates↗

PubMed2004

Fluorescence-based assays for RGS box function.

Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Galphabetagamma) to catalyze GTP loading and, consequently, activation of Galpha subunits and the liberation of Gbetagamma. Galpha.GTP and Gbetagamma are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Galpha subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Galpha and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Galpha subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Galpha subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Galpha proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Galpha activation by aluminum tetrafluoride.

Amino Acid Substitution↗

PubMed2002

eIF4A: the godfather of the DEAD box helicases.

eIF4A has long been considered the "gold standard" for DEAD box helicases. In large measure, this reflected two items: first, the role of eIF4A in protein synthesis initiation was relatively well established. Second, a wide variety of biochemical studies had established the ability of eIF4A to bind nucleic acids in an ATP-dependent manner, to hydrolyze ATP in an RNA-dependent manner, and to unwind RNA duplexes in an ATP-dependent manner. In this article, these basic observations are reviewed for biochemical consistency and also interpreted in light of the available crystal structures for DEAD box proteins. The role of non-processive vs. processive helicase activity in protein synthesis is discussed. Also examined is the influence of ancillary protein factors (eIF4B, eIF4G, and eIF4H) on this activity. Finally, the "real" role(s) for eIF4A helicase activity in protein synthesis is discussed and related to other circumstances that likely also involve the use of non-processive or slightly processive DEAD box helicases (ribosome biosynthesis, RNA splicing).

Amino Acid Motifs↗

PubMed1999

Effects of SCH23390 and raclopride on anxiety-like behavior in rats tested in the black-white box.

Dopamine (DA) systems are activated by stress, and this response has as a corollary the induction of stress-related behaviors such as anxiety. In mice, D2 receptor blockade produces an apparent anxiogenic effect, although locomotor impairments might have been present. We investigated the effects of D1 and D2 antagonists on a variety of anxiety-like behaviors induced by the black-white box in rats and carefully screened for any locomotor deficits. Adult male Lister hooded rats were injected with either the D1 antagonist SCH23390 (0. 0.1. or 0.25 mg/kg i.p.) or the D2 antagonist raclopride (0, 0.05, or 0.10 mg/kg i.p.) 20 min prior to being placed into the white chamber of the black-white box (n = 8-10/group). Rats were videotaped and the tapes were scored for latency to exit the white chamber, latency to reenter the white chamber, time spent in the white chamber, intercompartmental crossing, and locomotor activity. ANOVA revealed no effect of the D1 antagonist SCH23390 on any behavioral measure. However, the raclopride-treated rats left the white area sooner than control rats (p < 0.01). Raclopride-treated rats also exhibited delayed reentry times to the white chamber compared to control rats (p < 0.01) and spent significantly less time in the white chamber (p < 0.05). Neither SCH23390 nor raclopride affected locomotor activity in a manner that confounded these behaviors. These results confirm that D2 receptor blockade enhances anxiety in rats tested in the black-white box.

Animals↗