bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.
Authors: Gamba G , Saltzberg S N , Lombardi M , Miyanoshita A , Lytton J , Hediger M A , Brenner B M , Hebert S C
Published: May 2006

Electroneutral Na+:Cl- cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl- cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic agents and can be distinguished from Na+:K+:2Cl- cotransporters based on a lack of K+ requirement and insensitivity to sulfamoylbenzoic acid diruetics like bumetanide. We report here the isolation of a cDNA encoding a thiazide-sensitive, electroneutral sodium-chloride cotransporter from the winter flounder urinary bladder using an expression cloning strategy. The pharmacological and kinetic characteristics of the cloned cotransporter are consistent with the properties of native thiazide-sensitive sodium-chloride cotransporters in teleost urinary bladder and mammalian renal distal tubule epithelia. The nucleotide sequence predicts a protein of 1023 amino acids (112 kDa) with 12 putative membrane-spanning regions, which is not related to other previously cloned sodium or chloride transporters. Northern hybridization shows two different gene products: a 3.7-kb mRNA localized only to the urinary bladder and a 3.0-kb mRNA present in several non-bladder/kidney tissues.
High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells.
Authors: St Geme J W , Falkow S , Barenkamp S J
Published: May 2006

Nontypable Haemophilus influenzae are Gram-negative bacilli that represent a common cause of human disease. These organisms initiate infection by colonizing the upper respiratory tract. Despite the essential role of colonization, the bacterial determinants of this process remain poorly defined. We recently identified a family of surface-exposed high-molecular-weight proteins of nontypable H. influenzae that are related to filamentous hemagglutinin, a critical adherence factor of Bordetella pertussis. The genes encoding the two such high-molecular-weight proteins (HMW-1 and HMW-2) expressed by a prototypic nontypable H. influenzae strain have now been cloned and sequenced. In this study we examined the role of the HMWs in adherence to human epithelial cells. We found that loss of expression of HMW-1 by the prototypic strain and a HMW-1-like protein in a heterologous nontypable H. influenzae strain markedly decreased the capacity to adhere. The absence of expression of both HMW-1 and HMW-2 in the prototypic strain or their homologs in the second strain was associated with a further decrease in adherence. Expression of either HMW-1 or HMW-2 in nonadherent laboratory strains of Escherichia coli resulted in acquisition of the adherence phenotype. These results indicate that both HMW-1 and HMW-2 and the homologous proteins from a second strain can mediate attachment. We speculate that these proteins and the related proteins in other nontypable H. influenzae isolates are important colonization factors.
Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo.
Authors: Padgett R W , Wozney J M , Gelbart W M
Published: May 2006

The type beta transforming growth factor family is composed of a series of processed, secreted growth factors, several of which have been implicated in important regulatory roles in cell determination, inductive interactions, and tissue differentiation. Among these factors, the sequence of the DPP protein from Drosophila is most similar to two of the vertebrate bone morphogenetic proteins, BMP2 and BMP4. Here we report that the human BMP4 ligand sequences can function in lieu of DPP in Drosophila embryos. We introduced the ligand region from human BMP4 into a genomic fragment of the dpp gene in place of the Drosophila ligand sequences and recovered transgenic flies by P-element transformation. We find that this chimeric dpp-BMP4 transgene can completely rescue the embryonic dorsal-ventral patterning defect of null dpp mutant genotypes. We infer that the chimeric DPP-BMP4 protein can be processed properly and, by analogy with the action of other family members, can activate the endogenous DPP receptor to carry out the events necessary for dorsal-ventral patterning. Our evidence suggests that the DPP-BMP4 signal transduction pathway has been functionally conserved for at least 600 million years.
Chromosomal localization of glutamate receptor genes: relationship to familial amyotrophic lateral sclerosis and other neurological disorders of mice and humans.
Authors: Gregor P , Reeves R H , Jabs E W , Yang X , Dackowski W , Rochelle J M , Brown R H , Haines J L , O'Hara B F , Uhl G R
Published: May 2006

Receptors for the major excitatory neurotransmitter glutamate may play key roles in neurodegeneration. The mouse Glur-5 gene maps to chromosome 16 between App and Sod-1. The homologous human GLUR5 gene maps to the corresponding region of human chromosome 21, which contains the locus for familial amyotrophic lateral sclerosis. This location, and other features, render GLUR5 a possible candidate gene for familial amyotrophic lateral sclerosis. In addition, dosage imbalance of GLUR5 may have a role in the trisomy 21 (Down syndrome). Further characterization of the murine glutamate receptor family includes mapping of Glur-1 to the same region as neurological mutants spasmodic, shaker-2, tipsy, and vibrator on chromosome 11; Glur-2 near spastic on chromosome 3; Glur-6 near waltzer and Jackson circler on chromosome 10; and Glur-7 near clasper on chromosome 4.
Dating the genetic bottleneck of the African cheetah.
Authors: Menotti-Raymond M , O'Brien S J
Published: May 2006

The cheetah is unusual among fields in exhibiting near genetic uniformity at a variety of loci previously screened to measure population genetic diversity. It has been hypothesized that a demographic crash or population bottleneck in the recent history of the species is causal to the observed monomorphic profiles for nuclear coding loci. The timing of a bottleneck is difficult to assess, but certain aspects of the cheetah's natural history suggest it may have occurred near the end of the last ice age (late Pleistocene, approximately 10,000 years ago), when a remarkable extinction of large vertebrates occurred on several continents. To further define the timing of such a bottleneck, the character of genetic diversity for two rapidly evolving DNA sequences, mitochondrial DNA and hypervariable minisatellite loci, was examined. Moderate levels of genetic diversity were observed for both of these indices in surveys of two cheetah subspecies, one from South Africa and one from East Africa. Back calculation from the extent of accumulation of DNA diversity based on observed mutation rates for VNTR (variable number of tandem repeats) loci and mitochondrial DNA supports a hypothesis of an ancient Pleistocene bottleneck that rendered the cheetah depauperate in genetic variation for nuclear coding loci but would allow sufficient time for partial reconstitution of more rapidly evolving genomic DNA segments.
Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.
Authors: Reddy M K , Weitzel S E , von Hippel P H
Published: May 2006

The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43.
Clonal ordering of 17p and 5q allelic losses in Barrett dysplasia and adenocarcinoma.
Authors: Blount P L , Meltzer S J , Yin J , Huang Y , Krasna M J , Reid B J
Published: May 2006

Both 17p and 5q allelic losses appear to be involved in the pathogenesis or progression of many human solid tumors. In colon carcinogenesis, there is strong evidence that the targets of the 17p and 5q allelic losses are TP53, the gene encoding p53, and APC, respectively. It is widely accepted that 5q allelic losses precede 17p allelic losses in the progression to colonic carcinoma. The data, however, supporting this proposed order are largely based on the prevalence of 17p and 5q allelic losses in adenomas and unrelated adenocarcinomas from different patients. We investigated the order in which 17p and 5q allelic losses developed during neoplastic progression in Barrett esophagus by evaluating multiple aneuploid cell populations from the same patient. Using DNA content flow cytometric cell sorting and polymerase chain reaction, 38 aneuploid cell populations from 14 patients with Barrett esophagus who had high grade dysplasia, cancer or both were evaluated for 17p and 5q allelic losses. 17p allelic losses preceded 5q allelic losses in 7 patients, both 17p and 5q allelic losses were present in all aneuploid populations of 4 patients, and only 17p (without 5q) allelic losses were present in the aneuploid populations of 3 patients. In no patient did we find that a 5q allelic loss preceded a 17p allelic loss. Our data suggest that 17p allelic losses typically occur before 5q allelic losses during neoplastic progression in Barrett esophagus.
The alpha aneurism: a structural motif revealed in an insertion mutant of staphylococcal nuclease.
Authors: Keefe L J , Sondek J , Shortle D , Lattman E E
Published: May 2006

The x-ray crystal structure of a mutant of staphylococcal nuclease that contains a single glycine residue inserted in the C-terminal alpha-helix has been solved to 1.67 A resolution and refined to a crystallographic R value of 0.170. This inserted glycine residue is accommodated in the alpha-helix by formation of a previously uncharacterized bulge, which we term the alpha aneurism. A conformational search of known protein structures has identified the alpha aneurism in a number of protein families, including the histocompatibility antigens and hemoglobins.
Odor-induced increases in c-fos mRNA expression reveal an anatomical "unit" for odor processing in olfactory bulb.
Authors: Guthrie K M , Anderson A J , Leon M , Gall C
Published: May 2006

Expression of the immediate-early gene c-fos was used to evaluate the coordinate activation of olfactory bulb neurons by brief exposure to specific odors in the alert rat. In situ hybridization to c-fos mRNA was compared to regional increases in 2-deoxy-D-[14C]glucose incorporation in an adjacent section analysis. Levels of c-fos mRNA in olfactory bulb were high in rats recently removed from their home cage but were low in animals placed in a relatively odor-free chamber for 30 min. Presentation of specific odors to alert rats for as little as 5 min increased c-fos mRNA in radially distributed neuronal ensembles that spanned the lamina of the main olfactory bulb. The complementary RNA (cRNA)-labeled neuronal collectives consisted of cells in the glomerular layer that precisely defined the borders of individual glomeruli and underlying tufted, mitral, and granule cells. The activated fields were much broader in the granule cell layer than in the overlying glomerular layer and thus exhibited a flask-like, as opposed to a columnar, contour. The bulbar distribution of cRNA-labeled cell arrays differed with different odors and, in the glomerular layer, corresponded to focal regions of high 2-deoxy-D-[14C]glucose uptake. Administration of the noncompetitive N-methyl-D-aspartate receptor antagonist MK801 did not attenuate the odor induction of c-fos but, instead, increased c-fos mRNA levels throughout the bulb. We propose that the neuronal ensembles expressing increased c-fos mRNA with odor stimulation represent principal functional units of sensory processing in the main olfactory bulb of the behaving rat.
Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites.
Authors: Grigoriev M , Praseuth D , Guieysse A L , Robin P , Thuong N T , Hélène C , Harel-Bellan A
Published: May 2006

Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner. We have recently designed psoralen-oligonucleotide conjugates, which, upon binding to double-stranded DNA sequences via triple helix formation, may be cross-linked in vitro to both strands of the DNA following UV irradiation. A psoralen-oligonucleotide conjugate was targeted to the promoter of the alpha subunit of the interleukin 2 receptor (IL-2R alpha) gene. The triple helix site overlaps the binding site for the transcription factor NF-kappa B, which activates transcription from the IL-2R alpha promoter. After UV irradiation, the oligonucleotide conjugate becomes cross-linked to the target site and inhibits transcription of reporter plasmids transfected in live cells. Inhibition is observed when UV-induced cross-linking occurs both in vitro (before transfection) and in vivo (after transfection). We directly demonstrate that this inhibitory effect is due to triple helix formation at the target site, since a mutant of the promoter, to which oligonucleotide binding was inhibited, was not affected by the psoralen-oligonucleotide conjugate after UV irradiation. In addition, we demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.
A graded representation model for Macdonald's polynomials.
Authors: Garsia A M , Haiman M
Published: May 2006

We define doubly graded Sn modules Rmu for which we conjecture that the multiplicities of irreducible representations in various bi-degrees are given by the Macdonald coefficients Klambdamu. Assuming one fundamental conjecture, the modules Rmu can be given several equivalent definitions, which we discuss. We prove the conjectures in various special cases.
Two cell-density domains within the Myxococcus xanthus fruiting body.
Authors: Sager B , Kaiser D
Published: May 2006

Myxococcus xanthus, one of the simplest of multicellular organisms, develops into an organized, multicellular aggregate, called a fruiting body. Examination of the internal structure of the nascent fruiting body showed it to consist of a hemispherical outer domain of densely packed and ordered cells. Inside this dense shell is an inner domain of less ordered cells at 3-fold lower cell density. Single cells move in a bidirectional stream in the outer domain, orbiting the fruiting body throughout development, whereas in the inner domain, cell movement ceases as the fruiting body matures. The fruiting body thus consists of two domains, distinguished from each other by differential cell density, cell arrangements, and cell movements.
Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts.
Authors: Tuaillon N , Taylor L D , Lonberg N , Tucker P W , Capra J D
Published: May 2006

We (N.L. and L.D.T.) have introduced a human heavy-chain minilocus into mice transgenically. Constructs contain 2 heavy-chain variable (VH; psi VH3-105 and VH5-251), 10 diversity (D), 6 heavy-chain joining (JH), and either constant (C)mu or C mu and C gamma gene segments. Several founder lines were established and studied before immunization. Seventy heavy-chain transcripts were cloned and sequenced from murine splenic B lymphocytes, and gene-segment use was assessed before and after class-switching. In general, the repertoire was "fetal" in appearance with little evidence of somatic mutation in any gene segment. The two VH gene segments were found rearranged to mu- and gamma-chain C segments, with a preference of VH5-251. We observed a preponderance of the most-J-proximal D gene (DHQ52) segments among the mu transcripts (44%). The JH gene-segment use mimics most patterns seen in human antibodies. Diversification in CDR3 was extensive and included clear examples of D inversions and D-D fusions. These data suggest that a human immunoglobulin minilocus can undergo recombinatorial processes in a manner analogous to that seen in the human fetal/preimmune repertoire. This model, in addition to providing a potential source of human monoclonal antibodies, is ideal for the study of further questions concerning immunoglobulin gene-segment recombination.
Molecular diversity of Ca2+ channel alpha 1 subunits from the marine ray Discopyge ommata.
Authors: Horne W A , Ellinor P T , Inman I , Zhou M , Tsien R W , Schwarz T L
Published: May 2006

In many neurons, transmitter release from presynaptic terminals is triggered by Ca2+ entry via dihydropyridine-insensitive Ca2+ channels. We have looked for cDNAs for such channels in the nervous system of the marine ray Discopyge ommata. One cDNA (doe-2) is similar to dihydropyridine-sensitive L-type channels, and two cDNAs (doe-1 and doe-4) are similar to the subfamily of dihydropyridine-insensitive non-L-type channels. doe-4, which encodes a protein of 2326 aa, most closely resembles a previously cloned N-type channel. doe-1, which encodes a protein of 2223 aa, is a member of a separate branch of the non-L-type channels. Northern blot analysis reveals that doe-1 is abundant in the forebrain. doe-4 is more plentiful in the electric lobe and, therefore, may control neurotransmitter release in motor nerve terminals. These results show that the familial pattern of Ca(2+)-channel genes has been preserved from a stage in evolution before the divergence of higher and lower vertebrates > 400 million years ago. The cloning of these channels may be a useful starting point for elucidating the role of the Ca2+ channels in excitation-secretion coupling in nerve terminals.
CD43, the major sialoglycoprotein of human leukocytes, is proteolytically cleaved from the surface of stimulated lymphocytes and granulocytes.
Authors: Bazil V , Strominger J L
Published: May 2006

CD43, the major sialoglycoprotein of human leukocytes, whose expression is defective in patients with the Wiskott-Aldrich syndrome, was down-regulated by phorbol 12-myristate 13-acetate (PMA) on granulocytes but not on lymphocytes. However, CD43 expressed on both of these leukocyte subpopulations was down-regulated after crosslinking by anti-CD43 monoclonal antibodies, a stimulation that may simulate the effect of a natural CD43 ligand. Soluble, labeled CD43 molecules were isolated from culture supernatants of both surface-iodinated granulocytes activated by PMA and lymphocytes stimulated with anti-CD43 antibodies. Thus, in this case down-regulation represents release from the cell surface into the culture medium, rather than internalization. The apparent molecular masses of the released molecules and of soluble CD43 isolated from human serum were identical. Importantly, PMA-induced down-regulation of CD43 on granulocytes was markedly blocked both by the metalloprotease inhibitor 1,10-phenanthroline and by the serine protease inhibitors N alpha-(p-tosyl)-L-lysine chloromethyl ketone and Pefabloc SC, which inhibit two different classes of proteases, thus indicating that the release is proteolytic.
Atomic force microscopy of biochemically tagged DNA.
Authors: Murray M N , Hansma H G , Bezanilla M , Sano T , Ogletree D F , Kolbe W , Smith C L , Cantor C R , Spengler S , Hansma P K
Published: May 2006

Small fragments of DNA of known length were made with the polymerase chain reaction. These fragments had biotin molecules covalently attached at their ends. They were subsequently labeled with a chimeric protein fusion between streptavidin and two immunoglobulin G-binding domains of staphylococcal protein A. This tetrameric species was expected to bind up to four DNA molecules via their attached biotin moieties. The DNA-protein complex was deposited on mica and imaged with an atomic force microscope. The images revealed the protein chimera at the expected location at the ends of the strands of DNA as well as the expected dimers, trimers, and tetramers of DNA bound to a single protein.
Assembly of cartilage collagen fibrils is disrupted by overexpression of normal type II collagen in transgenic mice.
Authors: Garofalo S , Metsäranta M , Ellard J , Smith C , Horton W , Vuorio E , de Crombrugghe B
Published: May 2006

Cartilage collagen fibrils, which are characterized by their thin, uniform diameters, are formed of a multicomponent system of three collagen types (II, IX, and XI) and interacting proteoglycans. We have used a genetic approach to test whether the proper assembly of this multiprotein structure was altered by overexpression of one of its normal components. Here we show that in transgenic mice in which the normal mouse alpha 1(II) collagen is overexpressed, thick abnormal collagen fibrils are generated. Mice that showed the highest expression of the transgene also displayed a larger proportion of abnormal fibrils and died at birth. We propose that an imbalance among the constituents of the cartilage collagen fibrils disrupts the mechanism that controls their assembly. The results show the applicability of the transgenic mice system to studies of complex multicomponent protein assemblies in intact animals.
Tandem double CC-->TT mutations are produced by reactive oxygen species.
Authors: Reid T M , Loeb L A
Published: May 2006

Oxidative damage to DNA is mutagenic and thus may play a role in carcinogenesis. Because of the large number of different DNA lesions formed by oxidative species, no genetic alteration so far identified is exclusively associated with oxygen damage. Tandem double CC-->TT mutations are known to occur via UV damage to DNA and are thought to be a specific indicator of UV exposure. Using a sensitive reversion assay that can detect both single and double mutations within the same codon of the M13-encoded lacZ alpha gene, we show that treatments that produce reactive oxygen species can also produce tandem double CC-->TT mutations. The frequency at which these mutations occur is less than that for single base mutations by a factor of approximately 30. The induction of these mutations is inhibited by treatment that scavenges hydroxyl radicals. This unique mutation provides a marker of oxygen free radical-induced mutagenesis in cells that are not exposed to UV-irradiation and an indicator for assessing the involvement of oxidative damage to DNA in aging and tumor progression.
Common elements in interleukin 4 and insulin signaling pathways in factor-dependent hematopoietic cells.
Authors: Wang L M , Keegan A D , Li W , Lienhard G E , Pacini S , Gutkind J S , Myers M G , Sun X J , White M F , Aaronson S A
Published: May 2006

Interleukin 4 (IL-4), insulin, and insulin-like growth factor I (IGF-I) efficiently induced DNA synthesis in the IL-3-dependent murine myeloid cell lines FDC-P1 and FDC-P2. Although these factors could not individually sustain long-term growth of these lines, a combination of IL-4 with either insulin or IGF-I did support continuous growth. The principal tyrosine-phosphorylated substrate observed in FDC cells stimulated with IL-4, previously designated 4PS, was of the same size (170 kDa) as the major substrate phosphorylated in response to insulin or IGF-I. These substrates had phosphopeptides of the same size when analyzed by digestion with Staphylococcus aureus V8 protease, and each tightly associated with the 85-kDa component of phosphatidylinositol 3-kinase after factor stimulation. IRS-1, the principal substrate phosphorylated in response to insulin or IGF-I stimulation in nonhematopoietic cells, is similar in size to 4PS. However, anti-IRS-1 antibodies failed to efficiently precipitate 4PS, and some phosphopeptides generated by V8 protease digestion of IRS-1 were distinct in size from the phosphopeptides of 4PS. Nevertheless, IL-4, insulin, and IGF-I were capable of stimulating tyrosine phosphorylation of IRS-1 in FDC cells that expressed this substrate as a result of transfection. These findings indicate that (i) IL-4, insulin, and IGF-I use signal transduction pathways in FDC lines that have at least one major feature in common, the rapid tyrosine phosphorylation of 4PS, and (ii) insulin and IGF-I stimulation of hematopoietic cell lines leads to the phosphorylation of a substrate that may be related to but is not identical to IRS-1.
Transcriptional activation of low density lipoprotein receptor gene by angiotensin-converting enzyme inhibitors and Ca(2+)-channel blockers involves protein kinase C isoforms.
Authors: Block L H , Keul R , Crabos M , Ziesche R , Roth M
Published: May 2006

The pharmacological potency of angiotensin-converting enzyme (ACE) inhibitors (lisinopril and enalaprilat) on the transcription of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase genes was examined in human vascular smooth muscle cells and compared with the action of Ca(2+)-channel blockers (manidipine, verapamil, and diltiazem). Analogous to Ca(2+)-channel blockers, nanomolar concentrations of enalaprilat or lisinopril stimulated the synthesis of low density lipoprotein receptor mRNA and amplified the transcription induced by recombinant platelet-derived growth factor BB. In contrast to Ca(2+)-channel blockers, ACE inhibitors did not alter the transcription of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. Platelet-derived growth factor BB stimulated the translocation of delta and epsilon isoforms of protein kinase C. Similar to Ca(2+)-channel blockers, ACE inhibitors reduced the translocation of delta and epsilon isoforms of protein kinase C. Furthermore, ACE inhibitors and Ca(2+)-channel blockers inhibited platelet-derived growth factor BB-induced transcription of c-fos and c-jun genes. The findings suggest that increased de novo synthesis of mRNA low density lipoprotein receptor apparently involves the participation of delta and epsilon isoforms of protein kinase C and transcription factors c-Fos and c-Jun.
Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA.
Authors: Baron C , Heider J , Böck A
Published: May 2006

The SELB protein from Escherichia coli is a specialized elongation factor required for the UGA-directed insertion of the amino acid selenocysteine into selenopolypeptides. Discrimination of the UGA codon requires the presence of a recognition element within the mRNA, which is located at the 3' side of the UGA codon; a hairpin structure can be formed within this mRNA region. By gel shift assays, a specific interaction between SELB and the mRNA recognition element could be demonstrated. Footprinting experiments, using nucleases or iodine as cleaving agents, showed that SELB binds to the loop region of the hairpin structure. In the presence of selenocysteinyl-tRNA, SELB formed a complex with the charged tRNA and the mRNA. The results indicate that targeted insertion of selenocysteine is accomplished by the binding of the SELB protein to this mRNA recognition element, resulting in the formation of a selenocysteinyl-tRNA.SELB complex at the mRNA in the immediate neighborhood of the UGA codon.
Proposed atomic structure of a truncated human immunodeficiency virus glycoprotein gp120 derived by molecular modeling: target CD4 recognition and docking mechanism.
Authors: Gabriel J L , Mitchell W M
Published: May 2006

The atomic structure of a truncated glycoprotein gp120 from human immunodeficiency virus 1 (HIV-1) that contains the principal neutralizing antigenic sites and the CD4 binding domain has been derived by molecular dynamics and calculation of potential energy using the DREIDING force field. The resultant N-glycosylated molecular model is consistent with known properties of gp120 and docks with CD4 with a substantial reduction in the sum of the internal potential energies of the individual proteins (delta E = -200 kcal/mol). The primary mechanism of recognition and binding is the insertion of the solvent-accessible Phe-43 of CD4 into a gp120 solvent-accessible acceptor pit formed by Trp-427, Tyr-435, and the high-mannose oligosaccharide N-linked to Asn-230. delta E for the nonglycosylated complex is reduced significantly (-75 kcal/mol). Binding is by pi-pi* interactions of the aromatic groups forming a hydrophobic, thermodynamically stable environment for these functional noncovalent bonding participants. This model for gp120 provides a theoretical basis for the evaluation of HIV molecular pathogenesis involving the env proteins, the analysis of conformation on functional immune response of the host, and the design of nonproteinaceous inhibitors specific for the CD4 binding site on gp120.
Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein.
Authors: Israel D I , Kaufman R J
Published: May 2006

A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [3H]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.
Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene.
Authors: Croizier G , Croizier L , Argaud O , Poudevigne D
Published: May 2006

Recombinant baculoviruses obtained by coinfection of insect cells with Autographa californica and Bombyx mori nuclear polyhedrosis viruses (AcNPV and BmNPV, respectively) possess a wider in vitro host range than either parent virus. To localize the DNA sequences responsible for this species specificity, we used a two-step method of production and selection of recombinant viruses with altered specificity. Sf9 cells, which are permissive for AcNPV, were first cotransfected with genomic AcNPV DNA and a complete or incomplete set of BmNPV restriction fragments. AcNPV-BmNPV recombinants from the Sf9 supernatant were then selected on the basis of ability to replicate in B. mori Bm5 cells, which are not permissive for AcNPV. Cotransfection of AcNPV DNA with the 7.6-kbp BmNPV Sma I-C fragment was sufficient to produce recombinants able to infect both Sf9 and Bm5 cells. A series of cotransfections with subclones of this fragment defined a 79-nt sequence within the p143 helicase gene capable of extending AcNPV host range in vitro. In this 79-nt region, BmNPV and AcNPV differ at six positions, corresponding to four amino acid substitutions. The involvement of the 79-nt region in species specificity control was confirmed by cotransfecting AcNPV DNA and gel-purified polymerase chain reaction products derived from the BmNPV p143 gene. Replacement in the AcNPV genome of three AcNPV-specific amino acids by the three corresponding BmNPV-specific amino acids at positions 556, 564, and 577 of the p143 protein extends AcNPV host range to B. mori larvae.
Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.
Authors: Dontsova O , Tishkov V , Dokudovskaya S , Bogdanov A , Döring T , Rinke-Appel J , Thamm S , Greuer B , Brimacombe R
Published: May 2006

A DNA fragment containing the Escherichia coli 5S rDNA sequence linked to a T7 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with T7 polymerase using a mixture of [alpha-32P]UTP and 4-thio-UTP, yielding a transcript in which approximately 18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.
Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.
Authors: Reedquist K A , Fukazawa T , Druker B , Panchamoorthy G , Shoelson S E , Band H
Published: May 2006

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.
Further characterization of retinoblastoma gene-mediated cell growth and tumor suppression in human cancer cells.
Authors: Zhou Y , Li J , Xu K , Hu S X , Benedict W F , Xu H J
Published: May 2006

We have transfected the osteosarcoma cell line Saos2 and the bladder carcinoma cell line 5637 with additional retinoblastoma (RB) expression plasmids. The RB-reconstituted Saos2 and 5637 cells showed only slightly lower ratios of cells undergoing DNA synthesis compared to their parental RB- tumor cells, and there were no noticeable changes in cell morphology. Furthermore, we have isolated long-term RB+ clones from Saos2, 5637, and the retinoblastoma cell line WERI-Rb27 after transfection/transduction with a RB expression plasmid or retrovirus. These clones were similar to their parental cell lines in terms of morphology and growth rates, and they all expressed functional RB protein (p110RB) as evidenced by its potential of phosphorylation, simian virus 40 large tumor antigen binding, and nuclear tethering. No mutation or deletion of the exogenous RB gene was detectable by PCR and single-strand conformation polymorphism analysis. In addition, either the individual or pooled RB+ clones did form malignant tumors in nude mice but usually with a longer latency period and lower frequency. Such tumors also retained normal RB expression, suggesting that at least a portion of the RB-reconstituted tumor cells were still tumorigenic. This phenomenon is referred to by us as tumor suppressor resistance (TSR).
Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes.
Authors: Marsh T L , Reich C I , Whitelock R B , Olsen G J
Published: May 2006

The first step in transcription initiation in eukaryotes is mediated by the TATA-binding protein, a subunit of the transcription factor IID complex. We have cloned and sequenced the gene for a presumptive homolog of this eukaryotic protein from Thermococcus celer, a member of the Archaea (formerly archaebacteria). The protein encoded by the archaeal gene is a tandem repeat of a conserved domain, corresponding to the repeated domain in its eukaryotic counterparts. Molecular phylogenetic analyses of the two halves of the repeat are consistent with the duplication occurring before the divergence of the archael and eukaryotic domains. In conjunction with previous observations of similarity in RNA polymerase subunit composition and sequences and the finding of a transcription factor IIB-like sequence in Pyrococcus woesei (a relative of T. celer) it appears that major features of the eukaryotic transcription apparatus were well-established before the origin of eukaryotic cellular organization. The divergence between the two halves of the archael protein is less than that between the halves of the individual eukaryotic sequences, indicating that the average rate of sequence change in the archael protein has been less than in its eukaryotic counterparts. To the extent that this lower rate applies to the genome as a whole, a clearer picture of the early genes (and gene families) that gave rise to present-day genomes is more apt to emerge from the study of sequences from the Archaea than from the corresponding sequences from eukaryotes.
Interleukin 1 beta and corticotropin-releasing factor inhibit pain by releasing opioids from immune cells in inflamed tissue.
Authors: Schäfer M , Carter L , Stein C
Published: May 2006

Local analgesic effects of exogenous opioid agonists are particularly prominent in painful inflammatory conditions and are mediated by opioid receptors on peripheral sensory nerves. The endogenous ligands of these receptors, opioid peptides, have been demonstrated in resident immune cells within inflamed tissue of animals and humans. Here we examine in vivo and in vitro whether interleukin 1 beta (IL-1) or corticotropin-releasing factor (CRF) is capable of releasing these endogenous opioids and inhibiting pain. When injected into inflamed rat paws (but not intravenously), IL-1 and CRF produce antinociception, which is reversible by IL-1 receptor antagonist and alpha-helical CRF, respectively, and by the immunosuppressant cyclosporine A. In vivo administration of antibodies against opioid peptides indicates that the effects of IL-1 and CRF are mediated by beta-endorphin and, in addition, by dynorphin A and [Met]enkephalin, respectively. Correspondingly, IL-1 effects are inhibited by mu-, delta-, and kappa-opioid antagonists, whereas CRF effects are attenuated by all except a kappa-antagonist. Finally, IL-1 and CRF produce acute release of immunoreactive beta-endorphin in cell suspensions freshly prepared from inflamed lymph nodes. This effect is reversible by IL-1 receptor antagonist and alpha-helical CRF, respectively. These findings suggest that IL-1 and CRF activate their receptors on immune cells to release opioids that subsequently occupy multiple opioid receptors on sensory nerves and result in antinociception. beta-Endorphin, mu- and delta-opioid receptors play a major role, but IL-1 and CRF appear to differentially release additional opioid peptides.
Alpha 1(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and homology with type XV collagen.
Authors: Rehn M , Pihlajaniemi T
Published: May 2006

We report on the isolation of mouse cDNA clones which encode a collagenous sequence designated here as the alpha 1 chain of type XVIII collagen. The overlapping clones cover 2.8 kilobases and encode an open reading frame of 928 amino acid residues comprising a putative signal peptide of 25 residues, an amino-terminal noncollagenous domain of 301 residues, and a primarily collagenous stretch of 602 residues. The clones do not cover the carboxyl-terminal end of the polypeptide, since the translation stop codon is absent. Characteristic of the deduced polypeptide is the possession of eight noncollagenous interruptions varying in length from 10 to 24 residues in the collagenous amino acid sequence. Other features include the presence of several putative sites for both N-linked glycosylation and O-linked glycosaminoglycan attachment and homology of the amino-terminal noncollagenous domain with thrombospondin. It is of particular interest that five of the eight collagenous sequences of type XVIII show homology to the previously reported type XV collagen, suggesting that the two form a distinct subgroup among the diverse family of collagens. Northern blot hybridization analysis revealed a striking tissue distribution for type XVIII collagen mRNAs, as the clones hybridized strongly with mRNAs of 4.3 and 5.3 kilobases that were present only in lung and liver of the eight mouse tissues studied.
An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3.
Authors: Durbin H , Young S , Stewart L M , Wrba F , Rowan A J , Snary D , Bodmer W F
Published: May 2006

The monoclonal antibody PR1A3 has been used successfully for in vivo imaging of colorectal cancers, and several properties associated with this antibody, including minimal reactions of the antibody with circulating antigen in patients' sera, differentiate it from anti-carcinoembryonic antigen (CEA) antibodies used in similar studies. However, the antigen bound by PR1A3 was identified as CEA by analysis of somatic cell hybrids and by antigen expression from yeast artificial chromosomes, cosmids, and cDNA clones. The molecular weight, presence of a glycosyl-phosphatidylinositol anchor, elevation of surface expression by gamma-interferon, and N-terminal amino acid sequence all confirmed the antigen identification as CEA. A series of biliary glycoprotein-CEA hybrid proteins was produced which demonstrated that the epitope bound by the antibody was at the site of membrane attachment and involved parts of the glycosyl-phosphatidylinositol anchor and the B3 domain of CEA to form a conformational epitope. Access to this epitope, although possible when the antigen was on the cell surface, appeared to be blocked when CEA was released from the cell. The nature and location of the epitope on CEA are proposed to be responsible for the unique properties of the antibody.
SRY protein enhances transcription of Fos-related antigen 1 promoter constructs.
Authors: Cohen D R , Sinclair A H , McGovern J D
Published: May 2006

In mammals, testis determination is under the control of the chromosome Y-linked SRY gene. Sry is expressed in the fetal mouse just before development of the testis and shows germ-cell-dependent expression in the adult mouse. SRY protein contains a high-mobility-group (HMG)-box DNA-binding domain, and potential target sequences have been identified. The fos-related antigen 1 (fra-1) gene is closely related to the protooncogene c-fos and encodes a component of transcription factor AP-1. Fra-1 is expressed during spermatogenesis, and the promoter of the rat fra-1 gene contains several potential binding sites for members of the HMG-box family of DNA-binding proteins. We demonstrate that purified SRY protein binds strongly to one of the putative fra-1 HMG-box response elements and that SRY enhances the transcription of rat fra-1 promoter constructs in cotransfection experiments. These results suggest that the function of HMG-box transcription factors may be mediated, in part, by activation of members of the AP-1 transcription factor family.
Delineation of a region in the B2 bradykinin receptor that is essential for high-affinity agonist binding.
Authors: Nardone J , Hogan P G
Published: May 2006

We have made mutations in the predicted sixth transmembrane segment of a rat B2 bradykinin receptor and analyzed the variant proteins by expressing them in COS-1 cells. Two amino acid substitutions reduced the affinity of the receptor for bradykinin (Phe261-->Val by 1600-fold; Thr265-->Ala by 700-fold) with comparatively little effect on the affinity for the bradykinin antagonists NPC17731 and D-Arg-[Hyp3,D-Phe7]bradykinin (where Hyp is hydroxyproline). Three other substitutions (Gln262-->Ala, Asp268-->Ala, and Thr269-->Ala) modestly reduced the affinity for bradykinin and for the antagonist D-Arg-[Hyp3,D-Phe7]bradykinin. Even the most dramatically affected mutated receptors were still able to couple, after bradykinin binding, to phosphatidylinositol turnover. The data suggest that bradykinin directly contacts the face of the sixth transmembrane helix formed by the residues Phe261, Gln262, Thr265, Asp268, and Thr269 or that this face of the helix is the site of intraprotein contacts that serve to stabilize the agonist-binding conformation of the receptor.
Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family.
Authors: Jefferies H B , Reinhard C , Kozma S C , Thomas G
Published: May 2006

The immunosuppressant rapamycin blocks p70s6k/p85s6k activation and phosphorylation of 40S ribosomal protein S6 in Swiss 3T3 cells. The same net result is obtained when the macrolide is added 3 hr after serum stimulation. In stimulated cells p70s6k/p85s6k inactivation is achieved within minutes, whereas S6 dephosphorylation requires 1-2 hr, supporting the concept that S6 dephosphorylation results from kinase inactivation. In parallel, rapamycin treatment causes a small, but significant, reduction in the initiation rate of protein synthesis, as measured both by [35S]methionine incorporation into protein and by recruitment of 80S ribosomes into polysomes. More striking, analysis of individual mRNA transcripts revealed that rapamycin selectively suppresses the translation of a family of mRNAs that is characterized by a polypyrimidine tract immediately after their N7-methylguanosine cap, a motif that can act as a translational modulator. This family includes transcripts for ribosomal proteins, elongation factors of protein synthesis, and proteins of as-yet-unknown function. The results imply that (i) 40S ribosomes containing phosphorylated S6 may selectively recognize this motif or proteins which bind to it and (ii) rapamycin may inhibit cell growth by blocking S6 phosphorylation and, thus, translation of these mRNAs.