bluuebunny/crossref_metadata_2025 huggingface dataset:
A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity.
Authors: Isacchi A , Statuto M , Chiesa R , Bergonzoni L , Rusnati M , Sarmientos P , Ragnotti G , Presta M
Published: May 2006

A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.
Conversion of the interleukin 1 receptor antagonist into an agonist by site-specific mutagenesis.
Authors: Ju G , Labriola-Tompkins E , Campen C A , Benjamin W R , Karas J , Plocinski J , Biondi D , Kaffka K L , Kilian P L , Eisenberg S P
Published: May 2006

Interleukin 1 (IL-1) receptor antagonist (IL-1ra) is a naturally occurring protein that binds to the IL-1 receptor present on T cells, fibroblasts, and other cell types and acts to block IL-1-induced responses. IL-1ra is a pure antagonist and has no agonist activity in in vitro or in vivo systems. By site-specific mutagenesis, an analog of IL-1ra was created that contained a substitution of a single amino acid, Lys-145----Asp. This analog, IL-1ra K145D, exhibited partial agonist activity in the D10.G4.1 cell proliferation assay. The newly acquired agonist activity could not be neutralized by antisera to IL-1 alpha or IL-1 beta, but it could be blocked by a monoclonal antibody to the T-cell IL-1 receptor. The analog also showed agonist activity as assayed by increased prostaglandin E2 synthesis from CHO cells expressing recombinant mouse IL-1 receptor. These results with IL-1ra K145D demonstrate the importance of the region surrounding the corresponding Asp-145 residue in IL-1 beta for triggering the biological response to IL-1.
Biosynthetic threonine deaminase gene of tomato: isolation, structure, and upregulation in floral organs.
Authors: Samach A , Hareven D , Gutfinger T , Ken-Dror S , Lifschitz E
Published: May 2006

The gene encoding the plant biosynthetic threonine deaminase (Td; EC 4.2.1.16) has been cloned as a result of its unusual upregulation in tomato flowers. The Td gene of tomato encodes a polypeptide of 595 residues, the first 80 of which comprise a putative two-domain transit peptide cleaved at position 51. Comparison of the amino acid sequence with the corresponding enzymes from yeast and bacteria reveals a near identity of the important catalytic regions and greater than 40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is greater than 50-fold higher in sepals and greater than 500-fold higher in the rest of the flower than in leaves or roots. Its overexpression, however, is strictly confined to the parenchymal cells of the floral organs. In young tomato leaves, the chloroplast-bound enzyme is found almost exclusively in the subepidermal spongy mesophyll cells.
Localization of carbonic anhydrase IV in a specific capillary bed of the human eye.
Authors: Hageman G S , Zhu X L , Waheed A , Sly W S
Published: May 2006

Carbonic anhydrase (CA) activity plays an important role in controlling aqueous humor production in the eye and in regulating intraocular pressure. Prior studies identified the soluble isozymes CA II and CA I in the human eye and also suggested a distinct membrane-associated CA. We used an antibody to CA IV, the membrane-anchored isozyme from human lung, to study CA IV in eye tissues and to compare its distribution with that of CA II. We found intense immunostaining for CA IV associated with endothelial cells of one specific uveal capillary bed, the choriocapillaris. CA IV was not detected in endothelial cells of the contiguous capillaries of the iris or in endothelial cells of other vessels. Immunoreactivity for CA IV was also intense in epithelial and fiber cells of the lens but was not detectable in the neuroretina, the ciliary process (except for capillaries), and the cornea, all sites where immunostaining with anti-CA II antibody was intense. These studies indicate that the membrane-associated CA in human eye, which was suspected from histochemical studies, is CA IV. Defining the physiological role of this ocular isozyme remains a challenge.
Single K+ channels in membrane patches of arterial chemoreceptor cells are modulated by O2 tension.
Authors: Ganfornina M D , López-Barneo J
Published: May 2006

Type I cells of the carotid body are known to participate in the detection of O2 tension in arterial blood but the primary chemotransduction mechanisms are not well understood. Here we report the existence in excised membrane patches of type I cells of a single K+ channel type modulated by changes in PO2. Open probability of the O2-sensitive K+ channel reversibly decreased by at least 50% on exposure to hypoxia but single-channel conductance (approximately 20 pS) was unaltered. In the range between 70 and 150 mmHg (1 mmHg = 133 Pa) the decrease of single-channel open probability was proportional to the PO2 measured in the vicinity of the membrane patch. The inhibition of K+ channel activity by low PO2 was independent of the presence of non-hydrolyzable guanine triphosphate analogues at the internal face of the membrane. The results indicate that the O2 sensor of type I cells is in the plasma membrane and suggest that environmental O2 interacts directly with the K+ channels.
Structure and promoter activity of the gene for the erythroid transcription factor GATA-1.
Authors: Hannon R , Evans T , Felsenfeld G , Gould H
Published: May 2006

We have cloned the gene for the chicken erythroid transcription factor GATA-1 (formerly Eryf1, NF-E1, or GF-1). The gene is composed of six exons, two of which encode the two finger domains of the protein. Transcription of GATA-1 in chicken embryonic erythroid cells initiates from multiple sites clustered approximately 200 base pairs upstream from the start of protein-coding sequence. A number of sequence motifs for known DNA-binding proteins are found to be protected in DNase I-footprinting experiments by either erythroid or brain nuclear extracts or by both. Notably, a cluster of three GATA-1 sites is protected by the erythroid extract, as well as by purified GATA-1. We find that the upstream region of the gene functions as a powerful promoter when transfected into embryonic erythroid cells. In primary chicken embryo fibroblasts the promoter exhibits lower activity, which is increased when the cells are cotransfected with a second plasmid expressing the GATA-1 cDNA. The results suggest that GATA-1 protein plays an autoregulatory role in its own expression.
Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene.
Authors: Kim S J , Lee H D , Robbins P D , Busam K , Sporn M B , Roberts A B
Published: May 2006

Transforming growth factor beta (TGF-beta) isoforms inhibit the growth of many cell types and block progression of the cell cycle by inhibiting events in late G1 phase. The retinoblastoma gene product, RB, also has properties of a cell-cycle regulatory factor. It remains underphosphorylated in the presence of TGF-beta and has been shown to repress the activity of the c-fos promoter, resulting in inhibition of transit through the cell cycle. These observations led us to examine effects of human RB on the expression of the human TGF-beta 1 gene. Using chimeric TGF-beta 1 promoter-chloramphenicol acetyltransferase gene constructs, we show that RB induces TGF-beta 1 gene expression in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma cells but represses its expression in NIH 3T3 and AKR-2B mouse cells. Several sequences homologous to the c-fos RB control element were identified in the TGF-beta 1 promoter. These results demonstrate that human RB can regulate TGF-beta 1 gene expression negatively or positively depending on the cell type.
Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterization from rat brain.
Authors: Theibert A B , Estevez V A , Ferris C D , Danoff S K , Barrow R K , Prestwich G D , Snyder S H
Published: May 2006

High-affinity, membrane-associated inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6) binding proteins were solubilized and isolated utilizing a heparin-agarose resin followed by an IP4 affinity resin. The IP6 receptor comprises a protein complex of 115-, 105-, and 50-kDa subunits, all of which comigrate under native conditions. The Kd of the receptor for IP6 is 12 nM, whereas inositol 1,3,4,5,6-pentakisphosphate (IP5), IP4, and inositol 1,4,5-trisphosphate (IP3) are 50%, 30%, and 15%, respectively, as potent. Two protein complexes copurify with the IP4 receptor fraction. A 182/123-kDa complex elutes first from the affinity column followed by a 174/84-kDa protein complex, which elutes at higher salt. Both complexes show high affinity for IP4 (Kd = 3-4 nM). IP5, IP6, and IP3 display approximately 25%, 10%, and 0.1%, respectively, the affinity of IP4. Ligand binding to IP6 and IP4 receptors is inhibited 50% by heparin at 0.1 microgram/ml. IP4 receptor proteins are stoichiometrically phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, whereas negligible phosphorylation is observed for the IP6 receptor.
Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor.
Authors: Lin H Y , Kaji E H , Winkel G K , Ives H E , Lodish H F
Published: May 2006

By screening a cDNA library derived from the A10 rat vascular smooth muscle cell line for functional expression in COS cells, we have isolated a high-affinity receptor for endothelin 1 (Kd = 476 pM) and endothelin 2. The affinity of the cloned endothelin receptor for endothelin 3 is greater than 100 times less in A10 cells and in a CHO cell line stably transformed by the endothelin receptor cDNA. The 426-amino acid receptor polypeptide has seven putative hydrophobic transmembrane domains and is presumed to be a member of the family of guanine nucleotide-binding regulatory (G) protein-coupled receptors. Microinjection of in vitro transcripts of the cloned cDNA into CHO cells confers a transient increase in intracellular calcium in response to endothelin 1, indicating that the receptor is functional and couples to the appropriate G protein(s). RNA analysis reveals high expression in rat lung and heart, tissues known to exhibit binding to iodinated endothelin 1.
Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis.
Authors: Deutscher M P , Reuven N B
Published: May 2006

The rapid synthesis and breakdown of mRNA in prokaryotes can impose a significant energy drain on these cells. Previous in vivo studies [Duffy, J. J., Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 565-579; Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 581-591] indicated that while RNA turnover in Escherichia coli was hydrolytic, it was nonhydrolytic in Bacillus subtilis. Here we provide an explanation for these observations based on enzymatic analysis of extracts of these two organisms. RNA degradation to the mononucleotide level in E. coli extracts is due solely to two active ribonucleases, RNase II and polynucleotide phosphorylase, which act hydrolytically and phosphorolytically, respectively. RNase II activity represents close to 90% of the total activity of the extract, as expected for predominantly hydrolytic degradation in this organism. In contrast, RNase II is absent from B. subtilis extracts, and the primary mode of RNA degradation is phosphorolytic, employing the Bacillus equivalent of polynucleotide phosphorylase and releases nucleoside diphosphates as products. A low level of a Mn2(+)-stimulated, hydrolytic ribonuclease is also detectable in B. subtilis extracts. Overall, E. coli and B. subtilis extracts differ by about 20- to 100-fold, depending on the substrate, in their relative use of hydrolytic and phosphorolytic routes of RNA degradation. The relation of the mode of mRNA degradation to the environment of the cell is discussed.
Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.
Authors: Malik A , Egan J E , Houghten R A , Sadoff J C , Hoffman S L
Published: May 2006

Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria.
Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.
Authors: Ogata N , Alter H J , Miller R H , Purcell R H
Published: May 2006

Patient H is an American patient who was infected with hepatitis C virus (HCV) in 1977. The patient became chronically infected and has remained so for the past 13 years. In this study, we compared the nucleotide and predicted amino acid sequences of the HCV genome obtained from plasma collected in 1977 with that collected in 1990. We find that the two HCV isolates differ at 123 of the 4923 (2.50%) nucleotides sequenced. We estimate that the mutation rate of the H strain of HCV is approximately 1.92 x 10(-3) base substitutions per genome site per year. The nucleotide changes were exclusively base substitutions and were unevenly distributed throughout the genome. A relatively high rate of change was observed in the region of the HCV genome that corresponds to the non-structural protein 1 gene region of flaviviruses, where 44 of 960 (4.6%) nucleotides were different. Within this region there was a 39-nucleotide domain in which 28.2% of the nucleotides differed between the two isolates. In contrast, relatively few nucleotide substitutions were observed in the 5' noncoding region, where only 2 of 276 (0.7%) nucleotides were different. Our results suggest that the mutation rate of the HCV genome is similar to that of other RNA viruses and that genes appear to be evolving at different rates within the virus genome.
Three-dimensional structure of human basic fibroblast growth factor.
Authors: Eriksson A E , Cousens L S , Weaver L H , Matthews B W
Published: May 2006

The three-dimensional structure of human basic fibroblast growth factor (bFGF) has been determined by x-ray crystallography and refined to a crystallographic residual of 17.4% at 2.2-A resolution. The structure was initially solved at a nominal resolution of 2.8 A by multiple isomorphous replacement using three heavy-atom derivatives. Although the map clearly showed the overall fold of the molecule, electron density was not observed for the first 19 amino-terminal and the last 3 carboxyl-terminal amino acids, suggesting that they are disordered. The bFGF crystals were grown from 2.0 M ammonium sulfate at pH 8.1 in space group P1 with cell dimensions a = 30.9 A, b = 33.4 A, c = 35.9 A, alpha = 59.5 degrees, beta = 72.0 degrees, and gamma = 75.6 degrees. There is one molecule per unit cell and the crystals diffract to spacings beyond 1.9 A. The overall structure of bFGF can be described as a trigonal pyramid with a fold very similar to that reported for interleukin 1 beta, interleukin 1 alpha, and soybean trypsin inhibitor. An apparent sulfate ion is bound within a basic region on the surface of the molecule and has a ligands the main-chain amide of Arg-120 and the side chains of Asn-27, Arg-120, and Lys-125. This is suggested as the presumed binding site for heparin. Residues 106-115, which are presumed to bind to the bFGF receptor [Baird, A., Schubert, D., Ling, N. & Guillemin, R. (1988) Proc. Natl. Acad. Sci. USA 85, 2324-2328], include an irregular loop that extends somewhat from the surface of the protein and is about 25 A from the presumed heparin binding site. The backbone structure of this putative receptor-binding loop is very similar, although not identical, to the corresponding region of interleukin 1 beta.
Subtraction inhibition combined with a spiking threshold accounts for cortical direction selectivity.
Authors: Maex R , Orban G A
Published: May 2006

We have modeled simple-cell direction selectivity by a nonlinearity consisting of a subtraction inhibition followed by half-wave rectification and compared the performance of this model to that of different versions of the elaborated Reichardt detector for similar inputs and parameter settings. Not only does the subtraction model fit the experimental data more closely than the elaborated Reichardt detector, but the subtraction model also is more plausible from a physiological and anatomical point of view. Moreover, the subtraction model operates optimally at plausible spatiotemporal parameter settings. Therefore, we conclude that there is no need to invoke specific synaptic interactions, such as implied in the Reichardt detector, to account for simple-cell direction selectivity.
Interleukin 7 receptor ligation stimulates tyrosine phosphorylation, inositol phospholipid turnover, and clonal proliferation of human B-cell precursors.
Authors: Uckun F M , Dibirdik I , Smith R , Tuel-Ahlgren L , Chandan-Langlie M , Schieven G L , Waddick K G , Hanson M , Ledbetter J A
Published: May 2006

Functional interleukin 7 (IL-7) receptors are expressed on the surface of multiphenotypic, biphenotypic, and immature B-lineage human lymphoid precursor cells with germ-line immunoglobulin heavy-chain genes but not on more mature B-lineage lymphoid cells with rearranged and/or expressed immunoglobulin heavy-chain genes. Thus, IL-7 may have an important regulatory role during the earliest stages of human B-cell ontogeny. The engagement of the surface IL-7 receptors on immature B-cell precursor cells with recombinant human IL-7 (rhIL-7) results in enhanced tyrosine phosphorylation of multiple phosphoproteins, stimulates inositol phospholipid turnover and DNA synthesis, and promotes their clonal proliferation. These effects are (i) specific for rhIL-7, since rhIL-3, rhIL-4, rhIL-5, rhIL-6, and recombinant human granulocyte colony-stimulating factor do not elicit similar activities on IL-7 receptor-positive human pro-B cells; and (ii) mediated by IL-7 receptors, since they are not observed in IL-7 receptor-negative B-lineage lymphoid cell populations. rhIL-7-induced tyrosine phosphorylation on the 35-, 53-, 55-, 62-, 69-, 76-, 94-, 150-, 170-, and 190-kDa substrates as well as rhIL-7-induced stimulation of inositol phospholipid turnover are abrogated by the tyrosine kinase inhibitor genistein. These results demonstrate that the IL-7 receptor on immature human B-cell precursor populations is intimately linked to a functional tyrosine kinase pathway and tyrosine phosphorylation is an important and perhaps mandatory step in the generation of the IL-7 receptor-linked transmembrane signal.
Surface appearance and instability of empty H-2 class I molecules under physiological conditions.
Authors: Ortiz-Navarrete V , Hämmerling G J
Published: May 2006

Recent evidence suggests that endogenously produced antigenic peptides are required for assembly of major histocompatibility complex class I chains with beta 2-microglobulin and transport to the cell surface. The RMA-S mutant cells are thought to be defective in intracellular peptide loading to class I molecules and, therefore, devoid of class I surface expression. Here we report that at physiological temperature (37 degrees C) "empty" class I molecules appear at the cell surface of RMA-S cells where they can be trapped with H-2 antibodies. In the absence of the stabilizing ligand, the class I molecules rapidly alter their conformation but remain at the cell surface as demonstrated with a rabbit antiserum. Such denatured H-2 molecules can also be found on normal wild-type RMA cells. However, their amount is strongly reduced after culture of RMA cells with a class I binding peptide. These findings indicate that empty class I molecules appear at the surface not only on mutant but also on normal cells, suggesting that in normal cells the supply with peptides is limited.
Thymic targets for Abelson murine leukemia virus are early gamma/delta T lymphocytes.
Authors: Holland G D , Ito K , Kaehler D A , Tonegawa S , Risser R
Published: May 2006

Molecular analysis has shown that the majority of Abelson murine leukemia virus (Ab-MuLV)-induced primary thymomas represent transformed gamma/delta thymocytes. Many of these thymomas are of monoclonal origin as judged by provirus integration pattern and contain rearranged genes encoding T-cell receptor (TCR) gamma and delta chains but germ-line immunoglobulin heavy-chain genes. Some of the monoclonal tumors contain multiple rearranged alleles encoding TCR gamma, delta, and beta chains. Further, one Ab-MuLV thymoma cell line contained germ-line-configuration TCR gamma- and delta-chain genes, which became rearranged after in vitro propagation. Clones of this cell line were observed to rearrange these genes after intrathymic passage. Also, some subclones of this cell line underwent rearrangement of their immunoglobulin heavy-chain genes in culture. These observations suggest that the thymic targets for Ab-MuLV transformation are early gamma/delta thymocytes, some of which continue to rearrange their TCR gamma- and delta-chain genes.
Niemann-Pick disease: a frequent missense mutation in the acid sphingomyelinase gene of Ashkenazi Jewish type A and B patients.
Authors: Levran O , Desnick R J , Schuchman E H
Published: May 2006

Although the A and B subtypes of Niemann-Pick disease (NPD) both result from the deficient activity of acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) and the lysosomal accumulation of sphingomyelin, they have remarkably distinct phenotypes. Type A disease is a fatal neurodegenerative disorder of infancy, whereas type B disease has no neurologic manifestations and is characterized primarily by reticuloendothelial involvement and survival into adulthood. Both disorders are more frequent among individual of Ashkenazi Jewish ancestry than in the general population. The recent isolation and characterization of cDNA and genomic sequences encoding ASM has facilitated investigation of the molecular lesions causing the NPD subtypes. Total RNA was reverse-transcribed, and the ASM cDNA from an Ashkenazi Jewish type A patient was specifically amplified by the polymerase chain reaction (PCR). Molecular analysis of the PCR products revealed a G----T transversion of nucleotide 1487, which occurred at a CpG dinucleotide and predicted an Arg----Leu substitution in residue 496. Hybridization of PCR-amplified genomic DNA with allele-specific oligonucleotides indicated that the proband was homoallelic for the Arg----Leu substitution and that both parents and several other relatives were heterozygous. This mutation was detected in 32% (10 of 31) of the Ashkenazi Jewish NPD type A alleles studied and occurred in only 5.6% (2 of 36) of ASM alleles from non-Jewish type A patients. Of interest, the Arg----Leu substitution occurred in one of the ASM alleles from the two Ashkenazi Jewish NPD type B patients studied and in none of the ASM alleles of 15 non-Jewish type B patients. In contrast, the mutation was not present in 180 ASM alleles from normal individuals of Ashkenazi Jewish descent. These findings identify a frequent missense mutation among NPD patients of Ashkenazi Jewish ancestry that results in neuronopathic type A disease when homoallelic and can result in the nonneuronopathic type B phenotype when heteroallelic. The identification of this ASM mutation in Ashkenazi Jewish patients should facilitate the prevention of NPD in this population by carrier detection with molecular diagnostic techniques.
Anti-Müllerian hormone Bruxelles: a nonsense mutation associated with the persistent Müllerian duct syndrome.
Authors: Knebelmann B , Boussin L , Guerrier D , Legeai L , Kahn A , Josso N , Picard J Y
Published: May 2006

The persistent Müllerian duct syndrome (PMDS) is characterized by the persistence of Müllerian derivatives, uterus and tubes, in otherwise normally virilized males. In a previous study, we showed that this syndrome is heterogeneous, with lack of production of anti-Müllerian hormone (AMH) by testicular tissue accounting for only some, AMH-negative, cases of this disorder. We have characterized the point mutation responsible for an AMH-negative PMDS in three siblings: a guanine to thymine transversion at position 2096 in the fifth exon changes a GAA triplet, coding for glutamic acid, to a TAA stop codon. The mutation could also be recognized, using the polymerase chain reaction, on RNA produced in trace amounts by a lymphoblastic cell line. The translation product, although undetectable in testicular tissue, could be visualized in culture medium of cells transfected with the mutant gene.
DL-2-amino-4-phosphonobutyric acid does not eliminate "ON" responses in the visual system of goldfish.
Authors: DeMarco P J , Bilotta J , Powers M K
Published: May 2006

DL-2-Amino-4-phosphonobutyric acid (APB) suppresses activity in retinal ON pathways. It is generally assumed that loss of the ON pathway would result in loss of ON responses in the visual system. We tested this assumption by recording activity from the optic nerves of intact goldfish (Carassius auratus) before and after intraocular injection of APB. Whole-nerve responses to increments and decrements of light were compared to electroretinogram responses and to tectal evoked potentials. APB severely reduced the amplitude of the electroretinogram b-wave but left ON and OFF responses from the optic nerve and tectum intact, although decreased in sensitivity. We conclude that APB does not completely eliminate ON responses in the visual system, at least in goldfish. The selectivity and effectiveness of APB must be evaluated in other species before this agent can be relied upon as a useful tool in understanding the roles of ON and OFF pathways in visual function.
Anticodon-dependent aminoacylation of a noncognate tRNA with isoleucine, valine, and phenylalanine in vivo.
Authors: Pallanck L , Schulman L H
Published: May 2006

An assay based on the initiation of protein synthesis in Escherichia coli has been used to explore the role of the anticodon in tRNA identity in vivo. Mutations were introduced into the initiator tRNA to change the wild-type anticodon from CAU (methionine) to GAU (isoleucine), GAC (valine), and GAA (phenylalanine), where each derivative differs from the preceding by a single base change in the anticodon (underlined). These changes were sufficient to cause the mutant tRNAs to be aminoacylated by the corresponding aminoacyl-tRNA synthetases based on the amino acid inserted into protein from complementary initiation codons. Construction of additional single base anticodon variants (GUU, GGU, GCC, GUC, GCA, and UAA) showed that all three anticodon bases specify isoleucine and phenylalanine identity and that both the middle and the third anticodon bases are important for valine identity in vivo.
On the association of restriction fragment length polymorphisms across species boundaries.
Authors: Fu Y X , Arnold J
Published: May 2006

We study the expected values of gametic-phase disequilibrium in both nuclear and cytonuclear systems in a finite population composed of reproductively isolated subpopulations. Random drift alone within each subpopulation will generate permanent overall non-zero gametic-phase disequilibrium in both a two-locus nuclear system and a cytonuclear system unless the population has an overall initial disequilibrium of zero. We derive formulae for the expected overall disequilibrium when mutation is involved and demonstrate that these values decay to zero at an extremely slow rate compared with the decay of the expected disequilibria within each subpopulation.
Expression cloning of a Na(+)-independent neutral amino acid transporter from rat kidney.
Authors: Tate S S , Yan N , Udenfriend S
Published: May 2006

Uptake of long-chain and aromatic neutral amino acids into cells is known to be catalyzed by the Na(+)-independent system L transporter, which is ubiquitous in animal cells and tissues. We have used a Xenopus oocyte expression system to clone the cDNA of a system L transporter from a rat kidney cDNA library. The 2.3-kilobase cDNA codes for a protein of 683 amino acids. The transporter has four putative membrane-spanning domains and bears no sequence or structural homology to any known animal or bacterial transporter. When transcribed and expressed in Xenopus oocytes, the transporter exhibits many, but not all, of the characteristics of L-system transporters, suggesting that this represents one of several related L-system transporters.
Cellular oxidative modification of low density lipoprotein does not require lipoxygenases.
Authors: Sparrow C P , Olszewski J
Published: May 2006

The oxidative modification of low density lipoprotein (LDL) may play an important role in the pathogenesis of atherosclerosis. LDL can be oxidatively modified in vitro by endothelial cells, mouse peritoneal macrophages, or copper ions. Studies using lipoxygenase inhibitors have suggested that lipoxygenase(s) is required for the cellular modification of LDL [Rankin, S. M., Parthasarathy, S. & Steinberg, D. (1991) J. Lipid Res. 32, 449-456]. We have reexamined the effect of lipoxygenase inhibitors on cellular modification and found that (i) inhibitors specific for 5-lipoxygenase do not block LDL modification; (ii) inhibitors that block lipoxygenase by donating one electron to the enzyme (reductive inactivation) prevent LDL modification by cells and also modification mediated by copper ions, implying that they act as general antioxidants; (iii) the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid blocks 15-lipoxygenase activity in intact macrophages at concentrations 100 times less than those required to block LDL modification by macrophages; and (iv) 5,8,11,14-eicosatetraynoic acid is cytotoxic at concentrations about twice those required to prevent modification. Furthermore, macrophages and the RECB4 line of endothelial cells modify LDL with similar efficiencies despite dramatic differences in 15-lipoxygenase activity. Thus we conclude that neither 5-lipoxygenase nor 15-lipoxygenase is required for modification of LDL by cultured cells.
Targeting of hexokinase 1 to liver and hepatoma mitochondria.
Authors: Gelb B D , Adams V , Jones S N , Griffin L D , MacGregor G R , McCabe E R
Published: May 2006

The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound. This study examined the role of the 15-amino acid N-terminal domain of HK1 in binding to liver and hepatoma mitochondria. A chimeric reporter construct, pCMVHKCAT, encoding this HK1 domain coupled to the chloramphenicol acetyltransferase (CAT) gene was electroporated into mouse Hepa 1-6 hepatoma cells. After digitonin treatment, cell fractions were assayed for HK, lactate dehydrogenase, and CAT activities. Digitonin (75 micrograms/mg of protein) caused cytosolic leak but 70% of HK remained with the pellet. HKCAT, like HK, remained predominantly with the pellet; CAT form the control, pCMVCAT, remained mostly unbound. Binding of membrane-free cell extracts to rat liver mitochondria in vitro showed 91% of the HKCAT bound, whereas only 12% of CAT bound. Specificity of HKCAT binding to mitochondria was demonstrated by competition of HK1 for HKCAT binding sites on rat liver mitochondria as well as by blockage of HKCAT binding by N,N'-dicyclohexylcarbodiimide, which covalently binds to porin and blocks HK1 binding. Deletional mutant constructs of HKCAT showed reduced binding with increasing deletion size. In summary, these studies demonstrate that the 15-amino acid N-terminal domain of HK1 is necessary and sufficient to confer mitochondrial binding properties to CAT and that there is specificity for this binding to the mitochondria.
Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene.
Authors: Miki T , Bottaro D P , Fleming T P , Smith C L , Burgess W H , Chan A M , Aaronson S A
Published: May 2006

Expression cDNA cloning and structural analysis of the human keratinocyte growth factor receptor (KGFR) revealed identity with one of the fibroblast growth factor (FGF) receptors encoded by the bek gene (FGFR-2), except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high-affinity receptor for both KGF and acidic FGF, while FGFR-2 showed high affinity for basic and acidic FGF but no detectable binding by KGF. Genomic analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-2 transcript. Thus, two growth factor receptors with different ligand-binding specificities and expression patterns are encoded by alternative transcripts of the same gene.
Single amino acid change in the helicase domain of the putative RNA replicase of turnip crinkle virus alters symptom intensification by virulent satellites.
Authors: Collmer C W , Stenzler L , Chen X , Fay N , Hacker D , Howell S H
Published: May 2006

The virulent satellite [satellite C (sat C)] of turnip crinkle virus (TCV) is a small pathogenic RNA that intensifies symptoms in TCV-infected turnip plants (Brassica campestris). The virulence of sat C is determined by properties of the satellite itself and is influenced by the helper virus. Symptoms produced in infections with sat C differ in severity depending on the helper virus. The TCV-JI helper virus produces more severe symptoms than the TCV-B helper virus when inoculated with sat C. To find determinants in the TCV helper virus genome that affect satellite virulence, the TCV-JI genome was cloned and the sequence compared to the TCV-B genome. The genomes were found to differ by only five base changes, and only one of the base changes, at nucleotide position 1025, produced an amino acid change, an aspartic acid----glycine in the putative viral replicase. A chimeric TCV genome (TCV-B/JI) containing four of the five base changes (including the base change at position 1025) and a mutant TCV-B genome (TCV-B1025G) containing a single base substitution at position 1025 converted the TCV-B genome into a form that produces severe symptoms with sat C. The base change a position 1025 is located in the helicase of the putative viral replicase, and symptom intensification appears to result from differences in the rate of replication of the satellite supported by the two helper viruses.
Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture.
Authors: Treherne J M , Woodward S K , Varga Z M , Ritchie J M , Nicholls J G
Published: May 2006

The ability of neurons in the central nervous system to grow through a lesion and restore conduction has been analyzed in a developing spinal cord. The preparation consists of the entire central nervous system of the newly born opossum (Monodelphis domestica), isolated and maintained in culture. Cell division, cell migration, and reflexes are maintained in such preparations for up to 8 days in culture. In the present experiments, massive lesions were produced by crushing the spinal cord, which abolished all conduction for a day. By 2-3 days after injury, electrical conduction across the crush could be observed. After 4-5 days, clear recovery had occurred: the amplitude of the conducted volley was comparable to that in acute preparations. In such preparations, the spinal cord had largely regained its normal appearance at the crush site. Axons stained by carbocyanine dyes or horseradish peroxidase had, by 4 days, grown in profusion through the lesion and several millimeters beyond it. These experiments demonstrate that neurons in the central nervous system of newly born mammals, unlike those in adults, can respond to injury by rapid and extensive outgrowth in the absence of peripheral nerve bridges or antibodies that neutralize inhibitory factors of myelin. With rapid and reliable regeneration occurring in vitro, it becomes practicable to assay the effects of molecules that promote or inhibit the restoration of functional connections.
An rbcL sequence from a Miocene Taxodium (bald cypress).
Authors: Soltis P S , Soltis D E , Smiley C J
Published: May 2006

During the past decade, ancient DNAs from both animals and plants have been successfully extracted and analyzed. Recently, the age of DNA that can be recovered and sequenced was increased manyfold by the amplification and sequencing of a DNA fragment from a Magnolia fossil obtained from the Miocene Clarkia deposit (17-20 million yr old). However, the validity of this report has been questioned based on models predicting that DNA should be completely degraded after 4 million yr. We report here the successful amplification, sequencing, and analysis of a 1320-base-pair portion of the chloroplast gene rbcL from a Miocene Taxodium specimen, also from the Clarkia site. These data not only validate the earlier report of sequence data for a Magnolia species from the same site but also suggest that it may be possible to isolate and sequence DNAs routinely from the Clarkia deposit. The ability to recover and sequence DNAs of such age offers enormous research possibilities in the areas of molecular evolution, biogeography, and systematics.
Monoclonal IgM rheumatoid factors bind IgG at a discontinuous epitope comprised of amino acid loops from heavy-chain constant-region domains 2 and 3.
Authors: Artandi S E , Calame K L , Morrison S L , Bonagura V R
Published: May 2006

A combination of site-directed mutagenesis and exon exchange has been used to further define the structure on IgG recognized by monoclonal IgM rheumatoid factors (RFs) from patients with Waldenstrom macroglobulinemia. Most of these RFs bound IgG1, -2, and -4 but not IgG3. For these RFs, His-435 is a critical residue for binding and replacing it with arginine, the residue present in IgG3, destroys or reduces RF binding. However, additional polymorphic sequences in both the heavy-chain constant-region domains (CH) 2 and 3 are important for RF binding. Among the important residues in CH2 are amino acids 252-254 and 309-311, which are conserved among IgG isotypes and comprise two loops of amino acids on the surface of the domain. Therefore, at least three regions, two from CH2 and one from CH3, contribute significantly to the epitope recognized by the RFs. Although this epitope contains many of the same residues as the staphylococcal protein A binding site on IgG, the binding specificities of staphylococcal protein A and monoclonal RFs are not identical. Sera from patients with rheumatoid arthritis contain antibodies directed not only at this epitope but also at other sites on IgG.
Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues.
Authors: Ross C A , Danoff S K , Schell M J , Snyder S H , Ullrich A
Published: May 2006

Three inositol 1,4,5-trisphosphate receptor (IP3R) cDNAs, designated IP3R-II, -III, and -IV, were cloned from a mouse placenta cDNA library. All three display strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains. Levels of mRNA for the three additional IP3Rs in general are substantially lower than for IP3R-I, though in the gastrointestinal tract the levels of IP3R-III may be comparable to IP3R-I. Cerebellar Purkinje cells express at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell.
Complete
15
N and
1
H NMR assignments for the amino-terminal domain of the phage 434 repressor in the urea-unfolded form
Authors: Neri Dario , Wider Gerhard , Wüthrich Kurt
Published: May 2006

The amino-terminal domain of the phage 434 repressor consisting of residues 1-69 forms a globular structure of five tightly packed helices, with nearly identical molecular architectures in crystals and in solution. Upon addition of urea to an aqueous solution of this protein, the NMR spectrum of a second form of the protein appears in addition to the native form, and at a urea concentration of 7 M, this urea-unfolded form is the only species observed. At intermediate urea concentrations, the two forms of the protein inter-convert at a rate that allows the observation of the exchange process by NMR. Starting from the previous assignments for the native protein, we obtained nearly complete sequence-specific
1
H and
15
N NMR assignments for the unfolded form of the protein. For most amino acid residues, the
1
H chemical shifts of the urea-unfolded protein are very similar to the random coil values, but some discrete regions of the polypeptide chain were identified that are likely to retain residual nonrandom spatial structure as evidenced by deviations of
1
H chemical shifts and amide proton exchange rates from the expected random coil values.
Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.
Authors: Kron S J , Drubin D G , Botstein D , Spudich J A
Published: May 2006

The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because it is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act1 mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been significant conservation of actin function, specifically the ability to be moved by interaction with myosin.
Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis.
Authors: van Kesteren R E , Smit A B , Dirks R W , de With N D , Geraerts W P , Joosse J
Published: May 2006

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not clear. To investigate this issue, we cloned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressin, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapeptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalian copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.
Excision of Tn10 from the donor site during transposition occurs by flush double-strand cleavages at the transposon termini.
Authors: Benjamin H W , Kleckner N
Published: May 2006

Tn10 transposition is accomplished without extensive replication of the transposon sequences. Replicative cointegrate formation is precluded by efficient separation of transposon sequences from flanking donor DNA at an early stage in the transposition reaction. We report here that excision of Tn10 from its donor site occurs by a pair of flush double-strand breaks. Breaks occur at each end of the element precisely between the terminal base pair of the element and the first base pair of flanking DNA. This observation provides definitive evidence that cleavage of both strands of the element occurs under the direct control of Tn10 transposase protein. It is highly likely that transposase itself is directly responsible for these cleavages. The implications of this possibility are discussed.