bluuebunny/crossref_metadata_2025 huggingface dataset:
A plasma lipoprotein containing only apolipoprotein Eand with gamma mobility on electrophoresis releases cholesterol fromcells.
Authors: Huang Y , von Eckardstein A , Wu S , Maeda N , Assmann G
Published: May 2006

Previous studies have identified lipid-poor high
density lipoproteins with electrophoretic pre-beta mobility as the initial
acceptors of cell-derived cholesterol in human plasma. These lipoproteins
contain apolipoprotein A-I (apo A-I) as their sole apolipoprotein. In the
present study, incubation of human plasma with [3H]cholesterol-laden skin
fibroblasts has led to the identification of another lipoprotein that serves as
a potent initial acceptor of cell-derived cholesterol. This lipoprotein, which
we term gamma-LpE, exhibits gamma mobility on agarose gel electrophoresis. As
determined by nondenaturing PAGE and by electron microscopy, the size of the
spherical particle ranges between 12 and 16 nm. SDS/PAGE and subsequent
immunoblotting identified apoE as its sole apolipoprotein. Plasma from normal
and apoA-I-deficient mice, but not from apoE-deficient mice, released
[3H]cholesterol from fibroblasts into a gamma-migrating lipoprotein. Cell
culture media from hepatoma cells or mouse peritoneal macrophages, both of which
contain apoE of cellular origin, also promoted efflux of [3H]cholesterol from
fibroblasts into a gamma-migrating fraction. This was not observed with cell
culture medium from fibroblasts alone. In conclusion, our results strongly
indicate the presence in human plasma of a lipoprotein containing only apoE,
gamma-LpE, which is secreted by peripheral cells and is a potent acceptor of
cell-derived cholesterol.
Structure, function, and chromosome mapping of thegrowth-suppressing human homologue of the murine gas1 gene.
Authors: Del Sal G , Collavin L , Ruaro M E , Edomi P , Saccone S , Valle G D , Schneider C
Published: May 2006

We describe the isolation, growth-suppressing
activity, and chromosomal localization of the human homologue of the murine
growth-arrest-specific gene gas1. Overexpression of h-gas1 is able to block cell
proliferation in the A549 lung carcinoma and the T24 bladder carcinoma cell
lines. No effect was observed when h-gas1 was introduced into the osteosarcoma
cell line SAOS-2 and into the adenovirus-type-5 transformed cell line 293. This
finding is related to our previous evidence that simian virus 40-transformed NIH
3T3 cells are also refractory to murine gas1 overexpression, suggesting that the
retinoblastoma and/or p53 gene products have an active role in mediating the
growth-suppressing effect of gas1. We also show that h-gas1 is on chromosome
9q21.3-22.1, in a region considered to be a fragile site. Altogether, the
results raise the possibility that h-gas1 may be a target for genetic
alterations leading to its inactivation in tumor cells.
Efficient detection of point mutations on color-codedstrands of target DNA.
Authors: Verpy E , Biasotto M , Meo T , Tosi M
Published: May 2006

Presently available methods for screening large
genetic regions for unknown point mutations are neither flawless nor
particularly efficient. We describe an approach, especially well suited to
identifying mutations present in the heterozygous state, that combines several
improvements in a protocol called fluorescence-assisted mismatch analysis
(FAMA). Appropriate gene regions of the wild-type and the putative mutant allele
are simultaneously amplified from genomic DNA by using the polymerase chain
reaction, and large DNA fragments, so far up to 800 bp, are end labeled with
strand-specific fluorophores. Aliquots are denatured and reannealed to form
heteroduplexes and subjected to conventional cytosine- and thymine-specific
modifications. Cleavages occurring on opposite strands are detected by
denaturing gel electrophoresis using an automated DNA sequencer. Since the DNA
fragments derived from the mutant allele are also end labeled, the number of
informative mispaired bases is doubled compared to conventional searches using
wild-type probes. The sensitivity of detection is also increased, because
differential fluorescent end labelling allows the identification and measurement
of strand-specific background cleavages at matched cytosine or thymine residues.
Automatic superimposition of tracings from different subjects allows mismatch
detection at sites that, because of the nature of the bases involved and of the
neighboring sequence, are known to be less susceptible to cleavage. The effects
of the latter parameters have been studied quantitatively on a series of point
mutations found in the human C1-inhibitor gene in patients affected by
hereditary angioedema. Dilution experiments have demonstrated that most
mutations are detected even when the mutant chromosome is diluted 10-fold or
more compared with the normal one.
Mutations in PRG1, a yeast proteasome-related gene, cause defects in nuclear division and are suppressed by deletion of a mitotic cyclin gene.
Authors: Friedman H , Snyder M
Published: May 2006

Proteasomes are ubiquitous complexes exhibiting proteolytic activity in vitro. The function(s) of these enzymes in vivo is not known. To investigate the in vivo role of proteasomes, four temperature-sensitive alleles of the Saccharomyces cerevisiae proteasome-related gene, PRG1, were constructed and analyzed. At both the permissive and restrictive temperatures, many prg1 cells have a large bud, contain replicated DNA, and have their nucleus positioned at the neck with a short spindle. These different phenotypes indicate a defect in nuclear division. Consistent with a nuclear division defect, prg1 mutant strains lose a dispensable chromosome at a higher frequency than wild-type cells. Importantly, deletion of CLB2, a gene encoding a mitotic cyclin, suppresses the temperature-sensitive growth phenotype of prg1 mutant strains. Our results indicate that proteasomes are important for nuclear division and suggest that they participate in degradation of the Clb2 protein (Clb2p).
Ribozyme-mediated attenuation of pancreatic beta-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion.
Authors: Efrat S , Leiser M , Wu Y J , Fusco-DeMane D , Emran O A , Surana M , Jetton T L , Magnuson M A , Weir G , Fleischer N
Published: May 2006

Phosphorylation of glucose to glucose 6-phosphate by glucokinase (GK; EC 2.7.1.2) serves as a glucose-sensing mechanism for regulating insulin secretion in beta cells. Recent findings of heterozygous GK gene mutations in patients with maturity-onset diabetes of the young (MODY), a form of type II (non-insulin-dependent) diabetes characterized by autosomal dominant inheritance, have raised the possibility that a decrease in beta-cell GK activity may impair the insulin secretory response of these cells to glucose. To generate an animal model for MODY we have expressed in transgenic mice a GK antisense RNA with a ribozyme element under control of the insulin promoter. Mice in two independent lineages had about 30% of the normal islet GK activity. Insulin release in response to glucose from in situ-perfused pancreas was impaired; however, the plasma glucose and insulin levels of the mice remained normal. These mice are likely to be predisposed to type II diabetes and may manifest increased susceptibility to genetic and environmental diabetogenic factors. They provide an animal model for studying the interaction of such factors with the reduced islet GK activity.
Convection-enhanced delivery of macromolecules in the brain.
Authors: Bobo R H , Laske D W , Akbasak A , Morrison P F , Dedrick R L , Oldfield E H
Published: May 2006

For many compounds (neurotrophic factors, antibodies, growth factors, genetic vectors, enzymes) slow diffusion in the brain severely limits drug distribution and effect after direct drug administration into brain parenchyma. We investigated convection as a means to enhance the distribution of the large and small molecules 111In-labeled transferrin (111In-Tf; M(r), 80,000) and [14C]sucrose (M(r), 359) over centimeter distances by maintaining a pressure gradient during interstitial infusion into white matter to generate bulk flow through the brain interstitium. The volume of distribution (Vd) containing > or = 1% concentration of infusion solution increased linearly with the infusion volume (Vi) for 111In-Tf(Vd/Vi, 6:1) and [14C]sucrose (Vd/Vi, 13:1). Twenty-four hours after infusion, the distribution of 111In-Tf was increased and more homogeneous, and penetration into gray matter had occurred. By using convection to supplement simple diffusion, enhanced distribution of large and small molecules can be obtained in the brain while achieving drug concentrations orders of magnitude greater than systemic levels.
Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon".
Authors: Dao V , Guenther R , Malkiewicz A , Nawrot B , Sochacka E , Kraszewski A , Jankowska J , Everett K , Agris P F
Published: May 2006

The efficiency of translation depends on correct tRNA-ribosome interactions. The ability of chemically synthesized yeast tRNA(Phe) anticodon domains to effectively inhibit the binding of native yeast tRNA(Phe) to poly(U)-programmed Escherichia coli 30S ribosomal subunits was dependent on a Mg(2+)-stabilized stem and an open anticodon loop, both facilitated by base modifications. Analysis of tRNA sequences has revealed that base modifications which negate canonical hydrogen bonding are found in 95% of those tRNA anticodon loop sequences with the potential to form two Watson-Crick base pairs across the loop. Therefore, we postulated that a stable anticodon stem and an open loop are prerequisites for ribosome binding. To test this hypothesis, DNA analogs of the yeast tRNA(Phe) anticodon domain were designed to have modification-induced, Mg(2+)-stabilized stems and open loops. The unmodified DNA analog neither bound to poly(U)-programmed 30S ribosomal subunits nor inhibited the binding of native tRNA(Phe). However, specifically modified DNA analogs did bind to ribosomal subunits and effectively inhibited tRNA(Phe) from binding. Thus, modification-dependent Mg(2+)-stabilized anticodon domain structures with open loops have evolved as the preferred anticodon conformations for ribosome binding.
Endosomal aspartic proteinases are required for invariant-chain processing.
Authors: Marić M A , Taylor M D , Blum J S
Published: May 2006

Immunogenic peptides are displayed in the context of class II histocompatibility proteins on the surface of antigen-presenting cells. Class II alpha and beta subunits bind the invariant chain (I-chain), a transmembrane glycoprotein which must dissociate prior to peptide presentation. Proteolytic release of I-chain in an acidic compartment is followed by class II alpha beta surface expression. Two distinct proteinases sequentially catalyze I-chain dissociation in B-lymphoblastoid cell lines. An aspartic proteinase initiates processing whereas a cysteine proteinase catalyzes the final stages of I-chain release. Inactivation of these enzymes prevents class II alpha beta maturation, demonstrating that acidic proteinases are essential for the generation of functional class II complexes. I-chain processing was localized to a dense endosomal compartment, suggesting this is the first site where class II alpha beta become accessible to peptides. I-chain fragments complexed with class II alpha beta accumulate in dense endosomes of B-lymphoblastoid cells treated with cysteine proteinase inhibitors. A signal for endosomal retention/targeting present in the cytoplasmic tail of these fragments may sequester class II alpha beta in this compartment until I-chain processing is complete.
Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2.
Authors: Buscail L , Delesque N , Estève J P , Saint-Laurent N , Prats H , Clerc P , Robberecht P , Bell G I , Liebow C , Schally A V
Published: May 2006

The effects of somatostatin analogues RC-160 and SMS-201-995 on tyrosine phosphatase and cell proliferation were investigated in COS-7 and NIH 3T3 cells expressing human somatostatin receptor subtype 1 or 2 (SSTR1 or SSTR2). Binding experiments were performed on membranes from COS-7 cells expressing human SSTR1 or SSTR2 using 125I-labeled [Tyr11]S-14 or [Tyr3]SMS-201-995, respectively. The somatostatin analogues RC-160 and SMS-201-995 exhibited low affinity for SSTR1 (IC50 of 0.43 and 1.5 microM, respectively) and high affinity for SSTR2 (IC50 of 0.27 and 0.19 nM). Addition of these analogues to cells expressing either SSTR1 or SSTR2 did not result in an inhibition of adenylate cyclase activity. In SSTR2-expressing cells, both analogues induced a rapid stimulation of a tyrosine phosphatase activity (EC50: RC-160, 2 pM; SMS-201-995, 6 pM) and an inhibition of serum-stimulated proliferation (EC50: RC-160, 6.3 pM; SMS-201-995, 12 pM). In SSTR1-expressing cells, only RC-160 induced stimulation of a tyrosine phosphatase activity. Both analogues caused an inhibition of cell proliferation at a concentration higher than 10 nM in accordance with their affinities for the SSTR1 receptor subtype. A good correlation between the affinities of RC-160 and SMS-201-995 for each receptor subtype and their potencies to inhibit cell proliferation suggests the involvement of these receptors in cell growth regulation. Tyrosine phosphatase was stimulated by both these analogues in SSTR2 and by RC-160 in SSTR1 at affinities similar to their ability to inhibit growth and bind to receptors, implicating tyrosine phosphatase as a transducer of the growth inhibition signal. We also found that mRNAs of receptor subtypes were variably expressed in different pancreatic and colon cancer cell lines, indicating the necessity of a precise analysis of receptor subtypes in target tissues before therapy with analogues.
Correction of accelerated autoimmune disease by early replacement of the mutated lpr gene with the normal Fas apoptosis gene in the T cells of transgenic MRL-lpr/lpr mice.
Authors: Wu J , Zhou T , Zhang J , He J , Gause W C , Mountz J D
Published: May 2006

MRL-lpr/lpr mice develop a generalized autoimmune disease which includes increased autoantibody production, glomerulonephritis, and development of lymphadenopathy. The lpr genetic defect has been identified as a mutation in the Fas apoptosis gene that results in low expression of Fas mRNA. To determine the significance of the lpr mutation and T cells in the development of the autoimmune disease, we constructed transgenic MRL-lpr/lpr mice using a full-length murine Fas cDNA under the regulation of the T-cell-specific CD2 promoter and enhancer. Here we show that the early correction of the lpr gene defect in T cells eliminates glomerulonephritis and development of lymphadenopathy and decreases the levels of autoantibodies. In this model, early correction of the lpr defect in T cells is sufficient to eliminate the acceleration of autoimmune disease even in the presence of B cells and other cells that express the mutant lpr gene.
Molecular evidence for the origin of birds.
Authors: Hedges S B
Published: May 2006

The major groups of amniote vertebrates appeared during a relatively short time span at the end of the Paleozoic Era, a fact that has caused difficulty in estimating their relationships. The fossil record suggests that crocodilians are the closest living relatives of birds. However, morphological characters and molecular sequence data from living amniotes have repeatedly challenged this hypothesis by indicating a bird-mammal relationship. DNA sequences from four slow-evolving genes (mitochondrial 12S and 16S rRNA, tRNAVal, and nuclear alpha-enolase) now provide strong statistical support for a bird-crocodilian relationship.
A form of immunologic tolerance through impairment of germinal center development.
Authors: Pulendran B , Karvelas M , Nossal G J
Published: May 2006

Primary immunization with the T-cell-dependent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to human serum albumin results in the development of two pathways of B-cell development, the extrafollicular pathway and the germinal center pathway. Soluble, deaggregated NP-human serum albumin injected before immunization results in a marked diminution of clonable higher-affinity antibody-forming cell precursors--i.e., a form of immunologic tolerance within the secondary B-cell repertoire. We describe here the cellular changes in the spleen that underlie this tolerance. Using multiparameter flow cytometry, we show that tolerant mice develop far fewer NP-binding, peanut agglutinin-positive, or germinal center cells than the control immunized mice; 14 days after challenge control spleens have approximately 2 x 10(5) such cell per spleen, whereas the tolerant mice have approximately 1 x 10(4) cells. Furthermore, we demonstrate by immunohistology a reduction in the number of germinal centers containing lambda-bearing cells, characteristic of the response of C57BL/6 mice to NP. Taken together, these data suggest an impairment of germinal center development in the tolerant mice.
A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated.
Authors: Pei G , Samama P , Lohse M , Wang M , Codina J , Lefkowitz R J
Published: May 2006

The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.
Protein S binds to and inhibits factor Xa.
Authors: Heeb M J , Rosing J , Bakker H M , Fernandez J A , Tans G , Griffin J H
Published: May 2006

Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investigated possible interaction of protein S with human factor Xa. Factor Xa, diisopropylphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of approximately 19 nM. In fluid phase, protein S bound to factor Xa with a Kd of approximately 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition of prothrombin conversion to thrombin by factor Xa was phospholipid-independent and was 1.6 times stimulated by Ca2+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at approximately 8 nM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mechanisms of anticoagulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.
Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide.
Authors: Predescu D , Horvat R , Predescu S , Palade G E
Published: May 2006

In a murine heart perfusion system, we were able to "turn off" the transport of derivatized albumin [dinitrophenylated albumin (DNP-albumin)] from the perfusate to the tissue, by preperfusing the system with 1 mM N-ethylmaleimide (NEM) for 5 min at 37 degrees C, followed by a 5-min perfusion of DNP-albumin in the presence of NEM. Using a postembedding immunocytochemical procedure, we showed that (i) a 30-sec to 1-min treatment of heart vasculature with 1 mM NEM reduces the transendothelial transport of DNP-albumin and nearly stops it after 5 min, and (ii) DNP-albumin is detected exclusively in plasmalemmal vesicles (PVs) while in transit across endothelial cells. Perfusion with 10 mM dithiothreitol for 1 min before NEM prevents the inhibition of vesicular transport. To quantitate the NEM effect on vesicular transport inhibition, we developed an ELISA and a dot-blot assay for measuring DNP-albumin in supernatants of perfused whole-heart homogenates. The results obtained indicate that the treatment of the heart vasculature with 1 mM NEM decreases the vesicular transport of DNP-albumin by 78-80%. Since NEM is known to inhibit the fusion of different types of vesicular carriers with their target membranes in other cell types and in in vitro reconstituted cellular systems, by alkylating a NEM-sensitive factor, we assume that the same mechanism applies in our in situ system. The decrease of vesicular transport can be explained by NEM preventing the fusion of recycling vesicles with their targets--i.e., the abluminal and luminal domains of the plasmalemma. The results open to question previous interpretations from other laboratories according to which plasmalemmal vesicles are sessile, immobile structures.
Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4.
Authors: Ford E , Ares M
Published: May 2006

Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.
Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 cells.
Authors: Rouillé Y , Westermark G , Martin S K , Steiner D F
Published: May 2006

Proglucagon is processed differentially in the pancreatic alpha cells and the intestinal L cells to yield either glucagon or glucagon-like peptide 1, respectively, structurally related hormones with opposing metabolic actions. Here, we have studied the processing of proglucagon in alpha TC1-6 cells, an islet-cell line transformed by simian virus 40 large tumor (T) antigen, a model of the pancreatic alpha cell. We found that these cells process proglucagon at certain dibasic cleavage sites to release glucagon and only small amounts of glucagon-like peptide 1, as demonstrated by both continuous and pulse-chase labeling experiments. Both normal islet alpha cells and alpha TC1-6 cells were shown to express the prohormone convertase PC2 at high levels, but not the related protease PC3. Expression of PC2 antisense RNA in alpha TC1-6 cells inhibited both PC2 production and proglucagon processing concomitantly. We conclude that PC2 is the key endoprotease responsible for proglucagon processing in cells with the alpha-cell phenotype.
A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease.
Authors: Hensley K , Carney J M , Mattson M P , Aksenova M , Harris M , Wu J F , Floyd R A , Butterfield D A
Published: May 2006

beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.
Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.
Authors: Meldal M , Svendsen I , Breddam K , Auzanneau F I
Published: May 2006

A solid-phase assay for the complete subsite mapping of the active site of endoproteases has been developed. A library of resin-bound protease substrates was synthesized both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer type of resin that allows proteases to diffuse into the interior and perform their catalytic activity. Anthranilic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet mixing of the beads and parallel washing procedures. After treatment with subtilisin Carlsberg, fluorescing beads were collected and subjected to peptide sequencing, affording the preferred sequences, their cleavage bond, and a semiquantitative estimation of the turnover. A statistical distribution of preferred amino acids was obtained for each subsite. The result was compared with data from kinetic studies in solution.
Purification and initial characterization of a potential plant vacuolar targeting receptor.
Authors: Kirsch T , Paris N , Butler J M , Beevers L , Rogers J C
Published: May 2006

Clathrin-coated vesicles are known to be involved in the transport of proteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of approximately 80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum sativum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the approximately 80 kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the approximately-80 kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.
SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function.
Authors: Goldblum S E , Ding X , Funk S E , Sage E H
Published: May 2006

SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of injury and induces rounding in adherent endothelial cells in vitro. To determine whether SPARC might influence endothelial permeability, we studied the effect of exogenous SPARC on the movement of 14C-labeled bovine serum albumin across postconfluent bovine pulmonary artery endothelial cells. SPARC increased (P < 0.02) transendothelial albumin flux in a dose-dependent manner at concentrations > or = 0.5 microgram/ml. At a fixed dose (15 micrograms/ml), exposure times > or = 1 h augmented (P < 0.005) albumin flux by 1.3- to 3.6-fold; this increase was blocked by anti-SPARC antibodies but not by inhibition of protein synthesis. Barrier dysfunction was not associated with loss of cell viability. Monolayers exposed to SPARC exhibited a rounded morphology and intercellular gaps. Prior stabilization of F-actin with phallicidin protected against the changes in barrier function (P = 0.0001) that were otherwise induced by SPARC. Bovine aortic and retinal microvascular endothelia also responded to SPARC. We propose that SPARC regulates endothelial barrier function through F-actin-dependent changes in cell shape, coincident with the appearance of intercellular gaps, that provide a paracellular pathway for extravasation of macromolecules.
A conserved sequence motif within the exceptionally diverse telomeric sequences of budding yeasts.
Authors: McEachern M J , Blackburn E H
Published: May 2006

Telomeric DNA sequences have generally been found to be remarkably conserved in evolution, typically consisting of repeated, very short sequence units containing clusters of G residues. Recently however the telomeric DNA of the asexual yeast Candida albicans was shown to consist of much longer repeat units. Here we report the identification of seven additional telomeric sequences from sexual and asexual budding yeast species. The telomeric repeat units from this group of relatively closely related species show more phylogenetic diversity in length (8-25 bp), sequence, and composition than has been seen previously throughout a wide phylogenetic range of other eukaryotes. We also show that certain strains of the asexual diploid species Candida tropicalis have two forms of telomeric repeats, which appear to differ by a single base pair. Despite their great diversity, the telomeric repeat units of C. albicans, Saccharomyces cerevisiae, and all of the species we have examined in this report share a conserved approximately 6-bp motif of T and G residues resembling more typical telomeric sequences.
The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes.
Authors: Toczyski D P , Matera A G , Ward D C , Steitz J A
Published: May 2006

Epstein-Barr virus (EBV), an oncogenic herpesvirus, encodes two small RNAs (EBERs) that are expressed at high levels during latent transformation of human B lymphocytes. Here we report that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22. Approximately half of the L22 in EBV-positive cells is contained within the EBER1 ribonucleoprotein (RNP) particle, whereas the other half residues in monoribosomes and polysomes. Immunofluorescence with anti-L22 antibodies demonstrates that L22 is localized in the cytoplasm and the nucleoli of uninfected human cells, as expected, whereas EBV-positive lymphocytes also show strong nucleoplasmic staining. In situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to EBER1 in vivo. Since incubation of uninfected cell extracts with excess EBER1 RNA does not remove L22 from preexisting ribosomes, in vivo binding of L22 by EBER1 may precede ribosome assembly. The gene encoding L22 has recently been identified as the target of a chromosomal translocation in certain patients with leukemia, suggesting that L22 levels may be a determinant in cell transformation.
Inactivation of gene expression in plants as a consequence of specific sequence duplication.
Authors: Flavell R B
Published: May 2006

Numerous examples now exist in plants where the insertion of multiple copies of a transgene leads to loss of expression of some or all copies of the transgene. Where the transgene contains sequences homologous to an endogenous gene, expression of both transgene and endogenous gene is sometimes found to be impaired. Several examples of these phenomena displaying different features are reviewed. Possible explanations for the observed phenomena are outlined, drawing on known cellular processes in Drosophila, fungi, and mammals as well as plants. It is hypothesized that duplicated sequences can, under certain circumstances, become involved in cycles of hybrid chromatin formation or other processes that generate the potential for modification of inherited chromatin structure and cytosine methylation patterns. These epigenetic changes could lead to altered transcription rates or altered efficiencies of mRNA maturation and export from the nucleus. Where the loss of gene expression is posttranscriptional, antisense RNA could be formed on accumulated, inefficiently processed RNAs by an RNA-dependent RNA polymerase or from a chromosomal promoter and cause the observed loss of homologous mRNAs and possibly the modification of homologous genes. It is suggested that the mechanisms evolved to help silence the many copies of transposable elements in plants. Multicopy genes that are part of the normal gene catalog of a plant species must have evolved to avoid these silencing mechanisms or their consequences.
An Escherichia coli homologue of eukaryotic potassium channel proteins.
Authors: Milkman R
Published: May 2006

A DNA sequence in Escherichia coli K-12 contains an evident gene, kch, which predicts a protein 417 residues long with extensive similarity to a group of eukaryotic potassium channel proteins in amino acid sequence, in the presence of six apparent transmembrane (S) regions, and in the potassium-specific P (or H5) "pore" region found between S5 and S6. Most of the kch gene, including all of these regions and the 5' flanking region, have been sequenced in 38 wild reference (ECOR) strains as well; variation is conservative, indicating the protein's importance to the species, possibly as a defense against osmotic shock. Since the major family of eukaryotic potassium channel proteins is thought to have evolved from a common ancestor, the evolutionary position of this evident bacterial homologue is of interest, particularly since its function may have changed less than those of eukaryotic channels in the last billion years. While cases of probable importation of eukaryotic genes into bacteria are known, there is no evidence that kch has been imported. The relevant properties of the Kch protein and further ways to investigate its evolutionary position are discussed.
Bromophenacyl bromide binding to the actin-bundling protein l-plastin inhibits inositol trisphosphate-independent increase in Ca2+ in human neutrophils.
Authors: Rosales C , Jones S L , McCourt D , Brown E J
Published: May 2006

Ligation of IgG Fc receptors on polymorphonuclear leukocytes causes an increase in the concentration of free intracytoplasmic Ca2+ ([Ca2+]i) which arises from release of intracellular stores but is independent of inositol 1,4,5-trisphosphate. We found that bromophenacyl bromide (BPB), an alkylating agent which inhibits leukocyte degranulation, adherence, and phagocytosis, inhibited IgG-stimulated increases in [Ca2+]i with an IC50 of 0.2 microM. In contrast, BPB had no effect on inositol 1,4,5-trisphosphate-dependent [Ca2+]i increases induced by fMet-Leu-Phe, complement fragment C5a, ATP, or platelet-activating factor. Using a monoclonal antibody specific for BPB, we identified in polymorphonuclear leukocytes a single cytosolic protein of 66 kDa and isoelectric point pH 5.6 which bound BPB when intact cells were treated with the alkylating agent. This BPB-binding protein was identified as l-plastin, a Ca(2+)-regulated actin-bundling protein. l-Plastin was found associated with the Triton X-100-insoluble cytoskeleton in polymorphonuclear leukocytes adherent to immune complexes, suggesting that BPB affects Fc receptor-mediated signal transduction by altering the actin cytoskeleton. Consistent with this hypothesis, both cytochalasin B and cytochalasin D inhibited the IgG-dependent increase in [Ca2+]i, without any effect on fMet-Leu-Phe-induced Ca2+ release. These data suggest that the actin cytoskeleton is essential for signal transduction from plasma membrane Fc receptors and that l-plastin has a critical role in activation of this pathway.
Dominant dystrophic epidermolysis bullosa: identification of a Gly-->Ser substitution in the triple-helical domain of type VII collagen.
Authors: Christiano A M , Ryynänen M , Uitto J
Published: May 2006

Epidermolysis bullosa (EB) represents a group of genodermatoses characterized by fragility and easy blistering of the skin. In the dystrophic forms of EB, blisters occur below the basement membrane of the skin, at the level of the anchoring fibrils. We have recently demonstrated tight genetic linkage between the type VII collagen gene (COL7A1) and both the dominant and recessive forms of dystrophic EB. We searched for mutations in dominant dystrophic EB by PCR amplification of genomic segments of COL7A1, followed by heteroduplex analysis. Examination of the PCR fragment corresponding to exon 73 of COL7A1 revealed a marked shift in the electrophoretic pattern in patients from a large Finnish dominant dystrophic EB family with genetic linkage to the COL7A1 locus (Z = 5.37, theta = 0). Sequence analysis revealed a G-->A transition at nucleotide 6118 in the triple helical domain of COL7A1, which converted a glycine residue to a serine (GGT-->AGT). This mutation occurs between interruptions 11 and 12 of the triple helix, in the seventh of a series of 24 uninterrupted Gly-Xaa-Yaa repeats. Pathogenetic glycine substitutions that disrupt the triple helix have been shown to exert a deleterious effect on the protein in several other disorders involving collagen genes. The clinical phenotype in this family probably arises due to a dominant negative mutation in type VII collagen, resulting in the formation of structurally abnormal anchoring fibrils.
Inhibition of cytochromes P4501A by nitric oxide.
Authors: Stadler J , Trockfeld J , Schmalix W A , Brill T , Siewert J R , Greim H , Doehmer J
Published: May 2006

Inflammatory stimulation of the liver leads to the induction of nitric oxide (NO) biosynthesis. Because NO binds to the catalytic heme moiety of cytochromes P450 (CYPs), we investigated whether NO interferes with specific CYP-dependent metabolic pathways. In a first experimental approach V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP1A1 and -1A2 were used. Incubation with the NO donors sodium nitroprusside and S-nitrosylacetylpenicillamine led to a concentration-dependent inhibition of all four CYP enzymes. CYP1A1 was more sensitive to the inhibitory effect of NO than CYP1A2. In the second part of the study, endogenous NO synthesis was induced in rat hepatocytes by incubation with a mixture of cytokines and endotoxin. Concurrently, as NO production in hepatocytes increased within 24 hr, a decrease in CYP1A1-dependent benzo[a]pyrene turnover was observed to almost undetectable levels. The competitive inhibitor of NO synthesis, NG-monomethyl-L-arginine, was able to significantly restore CYP1A1 activity in the presence of cytokines and endotoxin. Inhibition of hepatocellular CYP activity by NO was predominantly due to a direct effect on the enzymes. However, NO-dependent inhibition of CYP expression at a transcriptional level was also demonstrated. Our results indicate that inhibition of NO biosynthesis in patients suffering from systemic inflammatory response syndromes may help to restore biotransformation capacity of the liver.
Tax protein of human T-cell leukemia virus type I binds to the ankyrin motifs of inhibitory factor kappa B and induces nuclear translocation of transcription factor NF-kappa B proteins for transcriptional activation.
Authors: Hirai H , Suzuki T , Fujisawa J , Inoue J , Yoshida M
Published: May 2006

Human T-cell leukemia virus type I causes adult T-cell leukemia and tropical spastic paraparesis, and its regulator protein Tax has been implicated in the pathogenic activity of human T-cell leukemia virus type I. Tax activates transcription of viral and cellular genes through specific enhancers: the 21-bp enhancer of human T-cell leukemia virus type I, the nuclear factor kappa B (NF-kappa B)-binding site of the interleukin 2 receptor alpha gene, and the serum-responsive element of c-fos. Tax binds to enhancer-binding proteins including cAMP-responsive element-binding protein, cAMP-responsive element modulator, transcription factor NF-kappa B p50 and p67SRF, and associates with each enhancer DNA indirectly. In addition to this mechanism, we report here that Tax binds to inhibitory factor kappa B gamma (I-kappa B) gamma, which forms a complex with NF-kappa B protein heterodimer p50-p65 or homodimer p50-p50 and retains them in the cytoplasm. Tax binding to I-kappa B gamma induces nuclear translocation of NF-kappa B p65. In association with this nuclear translocation of p65, transcription directed by the kappa B enhancer is strongly activated. Tax binds to the ankyrin motifs of I-kappa B gamma, suggesting its possible interaction with many other proteins carrying ankyrin motifs contributing to various regulatory processes. This is a different mechanism of transcriptional activation by the oncoprotein Tax and seems to be independent from the trans-activation through indirect binding to enhancer DNAs.
A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.
Authors: Garel M C , Arous N , Calvin M C , Craescu C T , Rosa J , Rosa R
Published: May 2006

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.
Selective 5-HT1D alpha serotonin receptor gene expression in trigeminal ganglia: implications for antimigraine drug development.
Authors: Rebeck G W , Maynard K I , Hyman B T , Moskowitz M A
Published: May 2006

Pharmacological data suggest that the actions of antimigraine drugs such as sumatriptan may be mediated by 5-HT1D-like serotonin receptors on trigeminovascular nerve endings. We sought molecular evidence for the expression of an mRNA species encoding the 5-HT1D receptor subtype in guinea pig and human trigeminal ganglia, using the polymerase chain reaction with oligonucleotides uniquely homologous to the coding sequences of the 5-HT1B/D family (human 5-HT1D alpha and 5-HT1D beta; rat 5-HT1B). A single band of predicted size was observed in samples from guinea pig trigeminal ganglia; sequence analysis revealed the presence of a single message, which was 85% and 71% identical to the human 5-HT1D alpha and 5-HT1D beta receptor DNA sequences, respectively. Similar analyses of postmortem human trigeminal ganglia revealed the presence of 5-HT1D alpha, but not 5-HT1D beta, receptor message. Inasmuch as one recent report found that mRNA encoding only the 5-HT1D beta receptor subtype was expressed by vascular smooth muscle of the central nervous system, the present findings suggest the importance of developing selective 5-HT1D alpha receptor agonists as a strategy to reduce the risk of myocardial infarction and possibly stroke that complicates the acute treatment of migraine headache.
Parasite virulence and disease patterns in Plasmodium falciparum malaria.
Authors: Gupta S , Hill A V , Kwiatkowski D , Greenwood A M , Greenwood B M , Day K P
Published: May 2006

Heterogeneity in parasite virulence is one of several factors that have been proposed to contribute to the wide spectrum of disease severity in Plasmodium falciparum malaria. We used observed age-structured patterns of disease to define a population structure of P. falciparum, where the latter contains several independently transmitted antigenic types or "strains" that each induce some degree of strain-specific antidisease immunity upon infection. Patterns of incidence of severe and mild disease may be explained by assuming that a majority of these strains are associated with mild disease and that although severe malarial anemia is a complication occurring in a certain proportion of early infections with "mild" parasites, cerebral malaria is caused by a few distinct highly virulent strains. Considerable variation in parasite virulence, as a major factor of disease severity in malaria, is made possible by the absence of competition between the various parasite strains, arising from weak shared immune responses. The theoretical framework presented in this paper can explain other epidemiological observations, such as the results of interventions with insecticide-impregnated bednets.