bluuebunny/crossref_metadata_2025 huggingface dataset:
A1 adenosine-receptor antagonists activate chloride efflux from cystic fibrosis cells.
Authors: Eidelman O , Guay-Broder C , van Galen P J , Jacobson K A , Fox C , Turner R J , Cabantchik Z I , Pollard H B
Published: May 2006

A1 adenosine-receptor-antagonist drugs such as 8-cyclopentyl-1,3-dipropylxanthine (CPX) and xanthine amine congener (XAC) are found to activate the efflux of 36Cl- from CFPAC cells. These cells are a pancreatic adenocarcinoma cell line derived from a cystic fibrosis (CF) patient homozygous for the common mutation, deletion of Phe-508. The active concentrations for these compounds are in the low nanomolar range, consistent with action on A1 adenosine receptors. In addition, drug action can be blocked by exogenous agonists such as 2-chloroadenosine and also can be antagonized by removal of endogenous agonists by treatment with adenosine deaminase. Cells lacking the CF genotype and phenotype, such as HT-29 and T84 colon carcinoma cell lines, appear to be resistant to activation of chloride efflux by either drug. CFPAC cells transfected with the CF transmembrane regulator gene, CFTR, are also resistant to activation by CPX. We conclude that, since these antagonists are of relatively low toxicity and appear to act somewhat selectively, they might be considered as promising therapeutic candidates for CF.
In vitro mutagenesis of biologically active transcripts of beet necrotic yellow vein virus RNA 2: evidence that a domain of the 75-kDa readthrough protein is important for efficient virus assembly.
Authors: Schmitt C , Balmori E , Jonard G , Richards K E , Guilley H
Published: May 2006

RNA 2 of the multipartite genome of beet necrotic yellow vein virus carries the cistron for 21-kDa viral coat protein at its 5' extremity. The amber termination codon of the coat protein cistron undergoes suppression approximately 10% of the time so that translation continues into an adjacent 54-kDa open reading frame, yielding a 75-kDa readthrough protein. The roles of coat protein and the readthrough protein in infection were investigated with biologically active transcripts of RNA 2. Much of the coat protein cistron of the RNA 2 transcript could be deleted without interfering with viral replication and local lesion formation on leaves, although formation of the rod-shaped virions did not occur. Mutants in which the amber coat protein termination codon was replaced with an ochre codon or a tyrosine codon were also viable. The ochre codon was suppressed both in vitro and in planta. The mutant containing the tyrosine substitution produced only the 75-kDa read-through protein and was deficient in viral assembly. Deletions in the 54-kDa readthrough domain were also viable in planta but had different effects on virus assembly. A deletion in the C-terminal portion of the readthrough domain did not interfere with RNA packaging but, unexpectedly, deletions in the N-terminal portion were assembly deficient, although 21-kDa coat protein was produced in planta. Thus, the 75-kDa protein can apparently intervene in virion assembly even though it has not been detected in purified virions.
In vivo production of a stable single-stranded cDNA in Saccharomyces cerevisiae by means of a bacterial retron.
Authors: Miyata S , Ohshima A , Inouye S , Inouye M
Published: May 2006

Gram-negative bacteria such as Myxococcus xanthus, Stigmatella aurantiaca, and Escherichia coli contain retroelements called retrons. Retrons consist of the msr-msd region and the gene for reverse transcriptase (RT), which are essential for the production of the branched RNA-linked ms-DNA (multicopy single-stranded DNA). In this study, we attempted to produce msDNA in the yeast Saccharomyces cerevisiae. Retron Ec67 from E. coli, which is responsible for the production of msDNA-Ec67, was cloned under the GAL10 promoter in a 2-microns-based plasmid. msDNA thus produced was detected by extending the 3' end of the msDNA by avian myeloblastosis virus RT. This yielded a main product of 117 nucleotides. Treatment of this product with RNase A resulted in a DNA of 105 nucleotides. These results are in good agreement with the structure of msDNA-Ec67. The production of msDNA-Ec67 was further confirmed by Southern blot hybridization. The msDNA production was dependent upon the bacterial RT gene in the clone and was increased severalfold when the RT gene of retron Ec67 was placed in front of the msr-msd region. The potential of msDNA as a eukaryotic vector producing a stable single-stranded DNA as well as RNA is discussed.
Changing patterns in cytoskeletal mRNA expression and protein synthesis during murine erythropoiesis in vivo.
Authors: Peters L L , White R A , Birkenmeier C S , Bloom M L , Lux S E , Barker J E
Published: May 2006

The major cytoskeletal proteins alpha-spectrin, beta-spectrin, and ankyrin are synthesized and assembled into a supportive membrane skeleton during erythroid differentiation. Information on the temporal appearance of mRNA and protein species is essential for understanding both the cytoskeletal assembly process and the function of various isoforms. We have isolated highly enriched populations of fetal erythroid cells at various stages of maturation. mRNAs for erythroid ankyrin, alpha-spectrin, and beta-spectrin were expressed at all stages but there were differences in transcript types and levels. The ratio of 9-kilobase (kb) to 7.5-kb erythroid ankyrin transcripts decreased markedly during differentiation, but there was no change in the ratio of the 10.1-kb and 9.3-kb erythroid beta-spectrin transcripts. The relative amounts of ankyrin, alpha-spectrin, and beta-spectrin mRNA increased during yolk sac cell differentiation, whereas only alpha-spectrin mRNA increased during differentiation of the fetal liver cells. The amounts of beta-spectrin mRNA exceeded the amounts of alpha-spectrin mRNA in the early precursors from both yolk sac and fetal liver; protein synthetic levels showed the same pattern. The 16-day fetal peripheral reticulocytes, on the other hand, had the adult mRNA and protein synthetic ratios with alpha/beta greater than 1. The data indicate that at least two mechanisms exist to meet changing erythroid membrane cytoskeletal requirements during development in utero: (i) stage-specific processing of the mRNA for the major cytoskeletal linker protein ankyrin and (ii) developmentally regulated alpha/beta-spectrin protein synthetic rates.
Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins.
Authors: Chiang C M , Ustav M , Stenlund A , Ho T F , Broker T R , Chow L T
Published: May 2006

We have shown that E1 and E2 proteins of human papillomavirus type 11 (HPV-11) were essential to support the replication of the homologous viral origin (ori) in a transient replication assay, similar to reports on bovine papillomavirus type 1 (BPV-1). Unexpectedly, matched or even mixed combinations of E1 and E2 proteins from HPV-11 or BPV-1 replicated either ori in human, monkey, and rodent cell lines of epithelial or fibroblastic lineage, albeit with varied efficiencies. Either set of viral proteins was also able to initiate replication of ori-containing plasmids from many other human and animal papillomaviruses. Thus the interactions among the cis elements and trans factors of papillomaviruses are more conserved than expected from the other members of the papovavirus family, simian virus 40 and polyomavirus, for which large tumor antigen does not replicate a heterologous ori in either permissive or nonpermissive cells. We infer that the stringent species and tissue specificities observed for papillomaviruses in vivo are not entirely due to direct restrictions on viral DNA replication. Rather, transcriptional control of viral gene expression must play a dominant role.
Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells.
Authors: Ginocchio C , Pace J , Galán J E
Published: May 2006

Penetration of intestinal epithelial cells is an important step in the pathogenesis of Salmonella infections. We have characterized a gene, invE, that is necessary for Salmonella invasion of cultured epithelial cells. The predicted amino acid sequence of InvE showed significant homology to the Yersinia outer membrane protein YopN (LcrE). Strains of Salmonella carrying mutations in invE were unable to penetrate Henle-407 human intestinal cells and Madin-Darby canine kidney cells, although they were fully capable of attaching to the same cells. Unlike wild-type Salmonella typhimurium, invE mutants failed to change the intracellular free calcium levels or the distribution of polymerized actin in cultured epithelial cells; neither did they alter the normal architecture of the microvilli of polarized Madin-Darby canine kidney cells. Wild-type S. typhimurium was able to rescue the invasive phenotype of the invE mutants in simultaneous infections of cultured epithelial cells although it did not rescue the Escherichia coli strain RDEC-1. We hypothesize that invE mutants are deficient in triggering the intracellular events that lead to bacterial internalization.
Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate.
Authors: Matsue H , Rothberg K G , Takashima A , Kamen B A , Anderson R G , Lacey S W
Published: May 2006

The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor increases the efficiency of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.
Changes in intracellular calcium during the development of epithelial polarity and junctions.
Authors: Nigam S K , Rodriguez-Boulan E , Silver R B
Published: May 2006

The "Ca2+ switch" model with cultured Madin-Darby canine kidney (MDCK) cells is useful in studying the biogenesis of epithelial polarity and junction formation and provides insight into early steps in the morphogenesis of polarized epithelial tissues. When extracellular Ca2+ in the medium is changed from less than 5 microM to 1.8 mM, MDCK cells rapidly change from a nonpolarized state exhibiting little cell-cell contact (with the apical membrane and junctional proteins largely within the cell) to a polarized state with well-formed tight junctions and desmosomes. To examine the role of intracellular Ca2+ in the development of polarity and junctions, we made continuous spectrofluorimetric measurements of intracellular Ca2+ during the "switch," using the fluorescent indicator fura-2. Intracellular Ca2+ increased greater than 10-fold during the switch and gave a complex pattern of increase, decrease, and stabilization. In contrast, intracellular pH [monitored with 2',7'-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF)] did not change during the period studied. When intracellular Ca2+ curves in several cells were compared, considerable heterogeneity in the rate of increase of intracellular Ca2+ levels and in peak levels was evident, perhaps reflecting the heterogeneity among cells in establishing junctions and polarity. The heterogeneity of the process was confirmed by digital imaging of intracellular Ca2+ and was present even in a "clonal" line of MDCK cells, indicating the heterogeneity was intrinsic to the process and not simply a function of slight genetic variation within the population of MDCK cells. In pairs of cells that had barely established cell-cell contact, often one cell exhibited a much greater increase in intracellular Ca2+ than the other cell in the pair. At the site of cell-cell contact, an apparent localized change (an increase over the basal level) in intracellular Ca2+ was frequently present and occasionally appeared to extend beyond the point of cell-cell contact. Since the region of cell-cell contact is also the site where junctions form and where vesicles containing apical membranes fuse during the development of polarity, we postulate a role for global and local changes in intracellular Ca2+ in these events.
Sensitivity to vanadate and isoforms of subunits A and B distinguish the osteoclast proton pump from other vacuolar H+ ATPases.
Authors: Chatterjee D , Chakraborty M , Leit M , Neff L , Jamsa-Kellokumpu S , Fuchs R , Baron R
Published: May 2006

Analysis of proton (H+) transport by inside-out vesicles derived from highly purified chicken osteoclast (OC) membranes has revealed the presence of a newly discovered type of vacuolar H+ ATPase (V-ATPase). Unlike vesicles derived from any other cell type or organelle, H+ transport in OC-derived vesicles is sensitive to V-ATPase inhibitors (N-ethylmaleimide and Bafilomycin A1) and vanadate (IC50, 100 microM), an inhibitor previously found to affect only P-type ATPases. The OC H+ ATPase contains several V-like subunits (115, 39, and 16 kDa) but subunits A and B of the catalytic domain of the enzyme differ from that of other V-ATPases. In OCs, subunit A has a mass of 63 kDa instead of the 67-70 kDa expressed in monocytes, macrophages, and kidney microsomes, which contain a vanadate-insensitive H+ ATPase. Moreover, two types of 57- to 60-kDa B subunits are also found: one is expressed predominantly in OCs and the other is expressed in kidney microsomes. The OC H+ pump may therefore constitute a class of H+ ATPase with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme. This H+ ATPase is involved in resorption of bone and may be expressed in a cell-specific manner, thereby opening possibilities for therapeutic intervention.
Fast sequencing of oligosaccharides: the reagent-array analysis method.
Authors: Edge C J , Rademacher T W , Wormald M R , Parekh R B , Butters T D , Wing D R , Dwek R A
Published: May 2006

A method of oligosaccharide analysis involving controlled fragmentation resulting from enzymatic digestion is presented. The principle involves generating a set of fragments from the original oligosaccharides, characterizing them in terms of their hydrodynamic volumes, determining their molar proportions, and identifying the oligosaccharides by comparison with a computer-generated data base. Experimentally, this technique involves incubation of aliquots of a sample with a set of defined mixtures of exoglycosidases followed by pooling of the products and a single analysis on the product pool. This method has several practical advantages over current techniques, including speed and the ability to use smaller amounts of starting material. The detection of the intensity-versus-hydrodynamic volume profile is limited only by the specific activity of the labeling method. The ability to perform the enzyme digestions is limited by the individual Km values of the enzymes.
Gene order comparisons for phylogenetic inference: evolution of the mitochondrial genome.
Authors: Sankoff D , Leduc G , Antoine N , Paquin B , Lang B F , Cedergren R
Published: May 2006

Detailed knowledge of gene maps or even complete nucleotide sequences for small genomes leads to the feasibility of evolutionary inference based on the macrostructure of entire genomes, rather than on the traditional comparison of homologous versions of a single gene in different organisms. The mathematical modeling of evolution at the genomic level, however, and the associated inferential apparatus are qualitatively different from the usual sequence comparison theory developed to study evolution at the level of individual gene sequences. We describe the construction of a database of 16 mitochondrial gene orders from fungi and other eukaryotes by using complete or nearly complete genomic sequences; propose a measure of gene order rearrangement based on the minimal set of chromosomal inversions, transpositions, insertions, and deletions necessary to convert the order in one genome to that of the other; report on algorithm design and the development of the DERANGE software for the calculation of this measure; and present the results of analyzing the mitochondrial data with the aid of this tool.
Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.
Authors: Yoshimura M , Cooper D M
Published: May 2006

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+.
Stable association between the bovine papillomavirus E5 transforming protein and activated platelet-derived growth factor receptor in transformed mouse cells.
Authors: Petti L , DiMaio D
Published: May 2006

The 44-amino acid E5 transforming protein of bovine papillomavirus is the shortest protein known to induce tumorigenic transformation of fibroblasts. We showed previously that expression of the E5 protein activates the cellular beta receptor for platelet-derived growth factor (PDGF) and proposed that the activated receptor transmits the transforming signal to the cell. Here we use coimmunoprecipitation analysis to show that the E5 protein and the activated PDGF receptor exist in a stable complex in transformed mouse C127 cells. These results suggest a distinct mechanism of growth factor receptor activation and provide further evidence that the PDGF receptor is an important target of the E5 protein.
Detection of DNA domains in Drosophila, human, and plant chromosomes possessing mainly 50- to 150-kilobase stretches of DNA.
Authors: Tchurikov N A , Ponomarenko N A
Published: May 2006

We have used pulsed-field gel electrophoresis of undigested DNA prepared by cell lysis in agarose with proteinase K detergent treatment and found a resolvable DNA fraction, denoted forum DNA (f-DNA). By changing the pulsed-field gel pulse length from 25 to 4500 sec, to obtain optimal separation in different ranges, we have found f-DNA to occupy a rather broad zone from 2 megabases to 10 kilobases (kb), but mainly at a range between 50 and 150 kb. f-DNA seems to appear as a result of nonrandom spontaneous degradation during cell treatment. The terminal regions of f-DNA segments have been cloned by using a jumping library. The molecular analysis of unique DNA sequence from an anonymous Drosophila DNA segment led to the conclusion that f-DNA appears as a result of nonrandom chromosomal DNA cleavage within sensitive regions that occupy a few kilobases. This conclusion was confirmed by detection of rather discrete hybridization bands on pulsed-field gel Southern blots in a region of good separation of undigested f-DNA after hybridization with different unique and repetitive probes. We propose that f-DNA segments may correspond to some regular higher-order structures in the eukaryotic chromosomes.
Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion.
Authors: Goldshleger R , Tal D M , Moorman J , Stein W D , Karlish S J
Published: May 2006

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+, K(+)-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K(+)-ATPase--the so-called "19-kDa membranes"--containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the alpha chain, and a largely intact beta chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (approximately 9 kDa) unidentified peptide(s) are labeled by [14C]DCCD in a Rb(+)-protectable fashion. Rb(+)-protected [14C]DCCD incorporation into the "19 kDa membranes" and into native Na+,K(+)-ATPase is linearly correlated with inactivation of Rb+ occlusion. Similar linear correlations are observed when Rb(+)-protected [14C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per alpha chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the alpha chain. After cyanogen bromide digestion of purified, labeled alpha chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) approximately 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb(+)-protected [14C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.
Glutaredoxin homolog encoded by vaccinia virus is a virion-associated enzyme with thioltransferase and dehydroascorbate reductase activities.
Authors: Ahn B Y , Moss B
Published: May 2006

Glutaredoxins (GRXs), also known as thioltransferases, use glutathione as a cofactor for reduction of disulfides in prokaryotes and eukaryotes. We demonstrate that the vaccinia virus O2L open reading frame encodes a functional GRX, as predicted by Johnson et al. [Johnson, G. P., Goebel, S. J., Perkus, M. E., Davis, S. W., Winslow, J. P. & Paoletti, E. (1991) Virology 181, 378-381] from sequence homology. The 12-kDa protein product of the O2L open reading frame was synthesized after viral DNA replication, coincident with a major increase in cytoplasmic glutathione-dependent thioltransferase activity. The protein was associated with purified vaccinia virions and was not released by treatment with a nonionic detergent unless dithiothreitol was added. The virion-derived protein, as well as a recombinant form expressed in Escherichia coli, exhibited thioltransferase and dehydroascorbate reductase activities indicative of a functional GRX. The postreplicative synthesis of vaccinia virus GRX and its association with virions suggest that the enzyme may have novel roles in the virus growth cycle.
Dopamine transporter mRNA content in human substantia nigra decreases precipitously with age.
Authors: Bannon M J , Poosch M S , Xia Y , Goebel D J , Cassin B , Kapatos G
Published: May 2006

The dopamine transporter is the primary means of inactivating synaptic dopamine as well as a major site of action for psychostimulants (such as cocaine and amphetamine) and for neurotoxins that induce parkinsonism. In the present study, a human dopamine transporter partial cDNA clone obtained by polymerase chain reaction exhibited 87% and 89% identity at the nucleic acid and amino acid levels, respectively, with transmembrane domains 3-5 of the rat homolog. This clone was used to quantitate human dopamine transporter mRNA by nuclease protection assay. The postmortem content of dopamine transporter mRNA in the substantia nigrae of 18- to 57-yr-old subjects was relatively constant, while in subjects greater than 57 yr old, a precipitous (greater than 95%) decline in substantia nigra dopamine transporter mRNA was evident. In contrast, tyrosine hydroxylase mRNA in the same samples declined in a linear manner with increasing age. In situ hybridization experiments confirmed the profound loss of dopamine transporter gene expression in melanin-positive (presumptive dopamine) nigral neurons. These data may begin to shed light on compensatory changes occurring in human dopamine neurons during normal aging.
The A alpha mating locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins.
Authors: Stankis M M , Specht C A , Yang H , Giasson L , Ullrich R C , Novotny C P
Published: May 2006

The A alpha mating locus is one of four multiallelic loci that govern sexual development in the basidiomycete fungus Schizophyllum commune. We have determined the nucleotide sequence encoding three A alpha mating types, A alpha 1, A alpha 3, and A alpha 4. We have found that the locus for A alpha 3 and A alpha 4 consists of two genes: Y and Z. The locus for A alpha 1 encodes only one gene, Y. The Z polypeptides encoded by different alleles exhibit 42% identity. The Y polypeptides exhibit 49-54% identity. The finding that the deduced Z and Y polypeptides have homeodomain motifs suggests that these polypeptides may be DNA-binding regulatory proteins that control the expression of developmental genes. The deduced Z polypeptide also has acidic regions that might be functionally analogous to the acidic regions in yeast GAL4 and GCN4 that activate transcription. The Y polypeptide has a serine-rich region and a basic region that shows some identity to the lysine-rich region of H1 histones.
Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis.
Authors: Soutar A K , Hawkins P N , Vigushin D M , Tennent G A , Booth S E , Hutton T , Nguyen O , Totty N F , Feest T G , Hsuan J J
Published: May 2006

A mutation in the gene for apolipoprotein AI (apoAI) was identified in an English family with autosomal dominant non-neuropathic systemic amyloidosis. The plasma of all affected individuals contained a variant apoAI with one additional charge, as well as normal apoAI. The propositus was heterozygous; the coding region of his apoAI gene contained both the normal sequence and a single-base substitution changing the codon for residue 60 of the mature protein from CTG (leucine) to CGG (arginine). Allele-specific oligonucleotide hybridization showed that the other affected individuals were also heterozygotes and that there was concordance of the mutant allele with the presence of variant plasma apoAI. Amyloid fibrils isolated from the spleen of the propositus consisted of proteins that ran as a doublet with an apparent mass of approximately 10 kDa in SDS/PAGE and a trace band at 28 kDa. Electrospray mass spectrometry of the purified 10-kDa material revealed components with mass corresponding to the N-terminal 88, 92, 93, and 94 residues of apoAI each with substitution of arginine for leucine. These observations were confirmed by direct protein sequencing and laser desorption time-of-flight mass analysis. No material with the normal apoAI sequence was detected. The trace band at 28 kDa yielded the N-terminal sequence of mature apoAI, indicating that intact or minimally degraded apoAI was also present in the fibril preparation. Discovery of this mutation and the detailed characterization of the apoAI fragments that form the amyloid fibrils open additional avenues for investigation of amyloidogenesis.
Abnormal sodium pump distribution during renal tubulogenesis in congenital murine polycystic kidney disease.
Authors: Avner E D , Sweeney W E , Nelson W J
Published: May 2006

Congenital polycystic kidney disease is characterized by the formation of large fluid-filled cysts in kidney tubules. It has been postulated that increased epithelial cell proliferation and altered transtubular fluid transport are necessary for cyst formation. To address the latter problem, we have studied the plasma membrane distribution of the alpha 1 and beta 1 subunits of Na+/K(+)-ATPase during progressive stages of proximal and collecting tubular cyst formation in the CPK mouse, a murine model of autosomal recessive polycystic kidney disease. In both control and cystic proximal tubules, Na+/K(+)-ATPase distribution was restricted to the basal-lateral membrane of cells. However, in newborn through day 5 kidney tissue, 16% of control vs. 47% of cystic outer cortical, 6% of control vs. 46% of cystic inner cortical, and 2% of control vs. 63% of cystic medullary collecting tubules demonstrated apical and lateral membrane distribution of Na+/K(+)-ATPase. In all nephrogenic zones, the percentage of control or cystic collecting tubules demonstrating apical membrane distribution of Na+/K(+)-ATPase decreased over time, but the percentage of cystic collecting tubules with apical membrane Na+/K(+)-ATPase remained significantly greater than in developmentally matched controls. No alterations in the normal distributions of other apical or basal-lateral membrane marker proteins were noted at any stage of control or cystic proximal or collecting tubule development. We conclude that apical-lateral membrane Na+/K(+)-ATPase expression is a normal transient feature of early collecting tubule development. However, apical membrane Na+/K(+)-ATPase persists in cystic kidneys, suggesting that such expression may be a manifestation of the relatively undifferentiated phenotype of epithelial cells lining collecting tubule cysts. The persistence of apical membrane Na+/K(+)-ATPase, if the enzyme is functional, may have pathogenic important in abnormal transtubular fluid transport in polycystic kidney disease.
Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.
Authors: Stamler J S , Jaraki O , Osborne J , Simon D I , Keaney J , Vita J , Singel D , Valeri C R , Loscalzo J
Published: May 2006

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately 1 microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.
The intrinsic helix-forming tendency of L-alanine.
Authors: Vila J , Williams R L , Grant J A , Wójcik J , Scheraga H A
Published: May 2006

Conformational energy calculations have been carried out for three hexadecapeptides in water--namely, a copolymer with the sequence acetyl-AAAAKAAAA-KAAAAKA-amide, 3K(I), in both the charged and neutral forms; a neutral peptide with the sequence acetyl-AAQAAAAQAAAAQAAY-amide, AQY; and a 16-residue L-alanine homopolymer with acetyl and amide terminal groups. The conformational energy was a sum of the empirical conformational energy program for peptides (ECEPP/2) potential energy plus continuum hydration free energy. An empirical (JRF) parameter set was used for the hydration free energy, together with an electrostatic contribution to the solvent effect from charged lysines. The computed relatively high helix content of the most probable conformation of charged 3K(I) and the intermediate helix content of AQY agree reasonably well with experimental values. The computed very low helix content of the alanine homopolymer agrees with experiments on block copolymers and on host-guest random copolymers. The calculations suggest that the high helix content computed for 3K(I) is due to the sum of internal and hydration free energies of the lysine residues rather than to a high intrinsic helix-forming tendency of alanine. The principal component lowering the computed helix contents of AQY and the alanine copolymer relative to 3K(I) is hydration.
The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family involved in signal transduction.
Authors: Fu X Y , Schindler C , Improta T , Aebersold R , Darnell J E
Published: May 2006

ISGF-3 is a multiprotein transcription factor that is very quickly activated in the cell cytoplasm only after attachment of interferon alpha to the cell surface. To understand the specific cytoplasmic activation of proteins that move to the nucleus and direct increased transcription of specific genes, we have purified and now report completion of the cloning of cDNA encoding the four proteins of ISGF-3. With all of the sequences available, it is clear that three of these proteins are encoded by members of a previously unrecognized gene family. We suggest that proteins encoded by this gene family serve the function of interpreting the fact that a cell surface receptor has bound its ligand so that specific signal transduction to the nucleus can occur.
Extensive variation in evolutionary rate of rbcL gene sequences among seed plants.
Authors: Bousquet J , Strauss S H , Doerksen A H , Price R A
Published: May 2006

Extensive variation in synonymous and nonsynonymous rates of substitution was observed among 50 sequences of the gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) representing bryophyte, conifer, dicot, and monocot taxa. Relative rate tests revealed rate differences of up to 138% for nonsynonymous substitutions and up to 85% for synonymous ones. Within angiosperms, the annual forms evolved more rapidly, on average, than perennial forms. This rate heterogeneity was more extensive at nonsynonymous sites than synonymous ones, and it resulted primarily from a recent acceleration of substitution rate in many groups of angiosperms.
Phosphorylation of the retinoblastoma protein by cdk2.
Authors: Akiyama T , Ohuchi T , Sumida S , Matsumoto K , Toyoshima K
Published: May 2006

The retinoblastoma gene product (the RB protein) is phosphorylated in a cell cycle-dependent manner and this modification is believed to be important for cells to progress through the cell cycle. We found that purified cdk2 (cyclin-dependent kinase/cell division kinase 2) can phosphorylate the RB protein in vitro at the sites phosphorylated in the cell. The timing of activation of cdk2 in the cell cycle was similar to that of the onset of phosphorylation of the RB protein. The kinase coprecipitated with the RB protein also exhibited a similar substrate specificity to cdk2 and a similar time course of activation during the cell cycle. We further showed that cdk2 formed a complex with the RB protein in vitro and that its formation was not competitively inhibited by the simian virus 40 large T antigen. These observations suggest that cdk2 or a cdk2-related protein is involved in the cell cycle-dependent phosphorylation of the RB protein.
Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle.
Authors: Wagner E , Plank C , Zatloukal K , Cotten M , Birnstiel M L
Published: May 2006

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.
Expression of a family of complementary-strand transcripts in Epstein-Barr virus-infected cells.
Authors: Karran L , Gao Y , Smith P R , Griffin B E
Published: May 2006

A major family of polyadenylylated cytoplasmic transcripts are expressed from the BamHI A-I region of the Epstein-Barr virus genome, off the strand complementary to that encoding several functions associated with viral replication and the lytic cycle, including the DNA polymerase (BALF-5). These complementary-strand transcripts (the main one is about 4.8 kilobases long), expressed in all cell types associated with Epstein-Barr virus, are present at high levels in nasopharyngeal carcinoma tumors. Sequence analysis of clones that correspond to spliced transcripts in a cDNA library from such a tumor, C15, generates a profile of the main complementary mRNA. It contains at least three AUG-initiated open reading frames, the largest of which could be translated to give a polypeptide of about 20 kDa. Evidence from several types of experiments suggests that conditions which support the up (or down) regulation of transcriptional expression from one viral DNA strand within the relevant region of the genome produce the opposite effect on transcripts from the other strand. The capacity for interference between complementary Epstein-Barr viral transcripts offers a mechanism for control of gene expression that may be related to maintenance of viral latency.
Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity.
Authors: Von Hoff D D , McGill J R , Forseth B J , Davidson K K , Bradley T P , Van Devanter D R , Wahl G M
Published: May 2006

Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malignancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as double-minute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.
Retinoic acid-induced duplication of the zebrafish retina.
Authors: Hyatt G A , Schmitt E A , Marsh-Armstrong N R , Dowling J E
Published: May 2006

Exogenous treatment of zebrafish embryos with retinoic acid induces a duplication of the retinas during development. These effects occur only when retinoic acid is applied within a 2-hr period prior to and during the initial formation of the optic primordia, and they are concentration-dependent. Light microscopic examination reveals that the second retina derives from cells in the ventral region of the developing eyecup that normally become pigment epithelial cells. Two distinct ganglion cell fields are usually observed in eyes with duplicated retinas. Bundles of axons from each ganglion cell field join as they leave the eye and innervate the contralateral tectum.
Endocytosis by antigen presenting cells: dendritic cells are as endocytically active as other antigen presenting cells.
Authors: Levine T P , Chain B M
Published: May 2006

Although dendritic cells are the most potent of all antigen presenting cells, they have paradoxically been regarded as having only a minimal capacity for endocytosis, which is a crucial step in antigen processing prior to presentation. Previous studies of dendritic cells, which are only available in small numbers, have been restricted to measurement of long-term endocytosis and so have stressed lysosomal accumulation. Measurement of traffic through late endosomes, which are closely related to the organelle in which antigen processing occurs, has, to date, required large numbers of cells and therefore has not been possible for dendritic cells. To resolve the paradox for dendritic cells, we have developed a flow cytometric assay of fluid-phase endocytosis that assesses late endosomal traffic by kinetic analysis of exocytosis in small numbers of cells. Using this assay, we show that fluid-phase endocytosis--in particular, traffic through late endosomes--is as active in dendritic cells as in other antigen presenting cells.
Serum proteases alter the antigenicity of peptides presented by class I major histocompatibility complex molecules.
Authors: Falo L D , Colarusso L J , Benacerraf B , Rock K L
Published: May 2006

Any effect of serum on the antigenicity of peptides is potentially relevant to their use as immunogens in vivo. Here we demonstrate that serum contains distinct proteases that can increase or decrease the antigenicity of peptides. By using a functional assay, we show that a serum component other than beta 2-microglobulin enhances the presentation of ovalbumin peptides produced by cyanogen bromide cleavage. Three features of this serum activity implicate proteolysis: it is temperature dependent, it results in increased antigenicity in a low molecular weight peptide fraction, and it is inhibited by the protease inhibitor leupeptin. Conversely, presentation of the synthetic peptide OVA-(257-264) is inhibited by serum. This inhibition is unaffected by leupeptin but is blocked by bestatin, a protease inhibitor with distinct substrate specificities. Implications for peptide-based vaccine design and immunotherapy are discussed.
Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition.
Authors: Zhang W , Young A C , Imarai M , Nathenson S G , Sacchettini J C
Published: May 2006

To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.
Engineered iron oxide-adhesion mutants of the Escherichia coli phage lambda receptor.
Authors: Brown S
Published: May 2006

Escherichia coli able to specifically adhere to iron oxide and not adhere to other metal oxides were constructed by genetic engineering. Concatamers of random oligonucleotides were introduced into a portion of a plasmid-borne lamB gene encoding an external domain of the phage lambda receptor. Bacteria able to adhere to iron oxide were selected by serial enrichment from the population of plasmid transformants. The concatameric nature of the inserted DNA allows a genetic analysis analogous to exons shuffling. Results of this genetic analysis indicate that in some isolates, part of the binding site is encoded by flanking vector sequences. This strategy may prove generally useful for identifying protein sequences able to recognize specific surfaces.