bluuebunny/crossref_metadata_2025 huggingface dataset:
Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle.
Authors: Takahashi M , Seagar M J , Jones J F , Reber B F , Catterall W A
Published: May 2006

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: alpha (167 kDa), beta (54 kDa), and gamma (30 kDa). Cleavage of disulfide bonds reveals two distinct alpha polypeptides and an additional component, delta. The alpha 1 subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). alpha 2, a 143-kDa glycoprotein, has none of the properties characteristic of alpha 1 but binds lectins and contains about 25% N-linked carbohydrate. alpha 2 is disulfide-linked to delta, a 24- to 27-kDa glycopeptide. beta (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. gamma (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purified anti-alpha 1 antibodies or alpha 2-specific lentil lectin-agarose demonstrated that alpha 1 alpha 2 beta gamma delta behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the alpha 2 delta complex dissociates from alpha 1 beta gamma in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.
Isolation of a rearranged human transforming gene following transfection of Kaposi sarcoma DNA.
Authors: Delli Bovi P , Basilico C
Published: May 2006

By transfecting high molecular weight DNA from a Kaposi sarcoma lesion into murine NIH 3T3 cells, we have identified and molecularly cloned a set of human DNA sequences capable of inducing focus formation, growth in agar, and tumorigenicity in these cells. The human DNA sequences present in primary, secondary, and tertiary NIH 3T3 transformants encompass about 32 kilobases (kb) and contain four rearrangements with respect to normal human DNA and a portion of the c-fms protooncogene (FMS in human gene nomenclature). However, the minimal transforming region (6.6 kb) identified in our cloned DNA borders on the c-fms DNA region but does not contain c-fms coding sequences. The fms sequences are also not represented in the two transcripts (approximately equal to 1.2 and 3.5 kb) detected in NIH 3T3 transformants; however, they might provide elements regulating expression. Hybridization to several known oncogene probes and preliminary sequencing data indicate that we have identified a previously unrecognized "activated" oncogene. Since the rearrangements present in our cloned DNA sequences are not detectable in the original Kaposi tumor DNA used for transfection, it is possible that this oncogene was generated during gene transfer.
Splice site selection in the proteolipid protein (PLP) gene transcript and primary structure of the DM-20 protein of central nervous system myelin.
Authors: Nave K A , Lai C , Bloom F E , Milner R J
Published: May 2006

Proteolipid protein (PLP) is the major myelin membrane protein of the central nervous system. We have isolated a copy of an alternatively spliced PLP gene transcript from a mouse brain cDNA library that was screened for PLP-related sequences. The encoded 241-amino acid protein differs from PLP by an internal deletion of 35-amino acid residues (116-150) from the major hydrophilic domain. This PLP variant is identical with the DM-20 protein of myelin, previously described as a brain-specific myelin component and known to be related to PLP. We determined the corresponding nucleotide sequence of the rat PLP gene and found that DM-20 mRNA results when a second 5' splice site, located 105 nucleotides within the third exon of the primary PLP transcript, is utilized in precursor mRNA (pre-mRNA) splicing. This demonstrates that alternative 5' splice site selection can determine the protein product of a cellular gene. DM-20 mRNA is expressed in rat brain with approximately 50% abundance relative to PLP mRNA and appears to be developmentally coregulated.
Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor.
Authors: Moscatelli D , Joseph-Silverstein J , Manejias R , Rifkin D B
Published: May 2006

A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentrations of 0.1-10 ng/ml, the same range that was effective for guinea pig and human Mr 18,000 bFGFs. The binding of human 125I-labeled bFGF to baby hamster kidney cells is inhibited equally by the Mr 25,000 guinea pig protein and the Mr 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the Mr 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the Mr 25,000 and Mr 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the Mr 25,000 guinea pig bFGF was converted to a Mr 18,000 protein. These results show that the two molecules are closely related and suggest that the Mr 25,000 protein shares substantial homology with the Mr 18,000 bFGF.
Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts.
Authors: Shaham M , Adler B , Ganguly S , Chaganti R S
Published: May 2006

Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). We report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental or Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 micrograms/ml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEBr) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEBr colonies independently derived from transfection of human DNA and one of three DEBr colonies independently derived from transfection of Chinese hamster DNA. In addition, Alu-equivalent hamster sequences were detected in three of seven additional independently derived colonies from transfection of Chinese hamster DNA. The DEBr phenotype of these colonies was stably maintained over several subcultures. Our results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA.
P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia.
Authors: Matteson K J , Phillips J A , Miller W L , Chung B C , Orlando P J , Frisch H , Ferrandez A , Burr I M
Published: May 2006

Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. We have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, we conclude that the P450XXIA2 gene "deletions" widely reported in CAH patients probably represent gene conversions, unequal crossovers, or polymorphisms rather than simple gene deletions.
RNA splicing permits expression of a maize gene with a defective Suppressor-mutator transposable element insertion in an exon.
Authors: Kim H Y , Schiefelbein J W , Raboy V , Furtek D B , Nelson O E
Published: May 2006

The bz-m13CS9 allele of the bronze-1 gene in maize contains a 902-base-pair defective Suppressor-mutator (dSpm) transposable element in the second exon. Nevertheless, 40-50% of the enzymatic activity conditioned by a nonmutant allele at the bronze-1 locus is routinely recovered in crude extracts prepared from plants carrying bz-m13CS9 in the absence of an autonomous Suppressor-mutator element. Analyses of RNAs produced by such plants show that transcription proceeds through the dSpm. The dSpm sequence of the messenger RNA precursor is then removed by RNA splicing using the donor site of the single bronze-1 intron and an acceptor site within the inverted terminal repeat of the dSpm. This results in a messenger RNA with the proper reading frame that could produce a functional enzyme. These data demonstrate that this dSpm insertion in an exon of a structural gene has produced a functional allele with a novel intron consisting, in part, of the dSpm. This mechanism appears to allow dSpm elements to reduce the impact of their insertions on gene expression.
Expression of herpes simplex virus 1 glycoprotein B by a recombinant vaccinia virus and protection of mice against lethal herpes simplex virus 1 infection.
Authors: Cantin E M , Eberle R , Baldick J L , Moss B , Willey D E , Notkins A L , Openshaw H
Published: May 2006

The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.
The interleukin 3 gene is located on human chromosome 5 and is deleted in myeloid leukemias with a deletion of 5q.
Authors: Le Beau M M , Epstein N D , O'Brien S J , Nienhuis A W , Yang Y C , Clark S C , Rowley J D
Published: May 2006

The gene IL-3 encodes interleukin 3, a hematopoietic colony-stimulating factor (CSF) that is capable of supporting the proliferation of a broad range of hematopoietic cell types. By using somatic cell hybrids and in situ chromosomal hybridization, we localized this gene to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, IL-3 was found to be deleted in the 5q-chromosome of one patient with refractory anemia who had a del(5)(q15q33.3), of three patients with refractory anemia (two patients) or acute nonlymphocytic leukemia (ANLL) de novo who had a similar distal breakpoint [del(5)(q13q33.3)], and of a fifth patient, with therapy-related ANLL, who had a similar distal breakpoint in band q33 [del(5)(q14q33.3)]. Southern blot analysis of somatic cell hybrids retaining the normal or the deleted chromosome 5 from two patients with the refractory anemia 5q- syndrome indicated that IL-3 sequences were absent form the hybrids retaining the deleted chromosome 5 but not from hybrids that had a cytologically normal chromosome 5. Thus, a small segment of chromosome 5 contains IL-3, GM-CSF (the gene encoding granulocyte-macrophage-CSF), CSF-1 (the gene encoding macrophage-CSF), and FMS (the human c-fms protooncogene, which encodes the CSF-1 receptor). Our findings and earlier results indicating that GM-CSF, CSF-1, and FMS were deleted in the 5q-chromosome, suggest that loss of IL-3 or of other CSF genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).
Antioxidant activity of albumin-bound bilirubin.
Authors: Stocker R , Glazer A N , Ames B N
Published: May 2006

Bilirubin, when bound to human albumin and at concentrations present in normal human plasma, protects albumin-bound linoleic acid from peroxyl radical-induced oxidation in vitro. Initially, albumin-bound bilirubin (Alb-BR) is oxidized at the same rate as peroxyl radicals are formed and biliverdin is produced stoichiometrically as the oxidation product. On an equimolar basis, Alb-BR successfully competes with uric acid for peroxyl radicals but is less efficient in scavenging these radicals than vitamin C. These results show that 1 mol of Alb-BR can scavenge 2 mol of peroxyl radicals and that small amounts of plasma bilirubin are sufficient to prevent oxidation of albumin-bound fatty acids as well as of the protein itself. The data indicate a role for Alb-BR as a physiological antioxidant in plasma and the extravascular space.
Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing.
Authors: Akeson A L , Wiginton D A , States J C , Perme C M , Dusing M R , Hutton J J
Published: May 2006

Adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, we synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNAs showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These changes do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.
Molecular cloning of the cDNA and chromosomal gene for vitamin D-dependent calcium-binding protein of rat intestine.
Authors: Darwish H M , Krisinger J , Strom M , DeLuca H F
Published: May 2006

A cDNA encoding the vitamin D-dependent rat intestinal calcium-binding protein has been isolated by screening a rat intestinal cDNA library. The cDNA is 406 nucleotides long and appears to contain all the sequences of the mRNA. The cDNA includes the entire protein coding region. It consists of 237 nucleotides coding for 79 amino acids, including the starting methionine, flanked by 62 and 107 noncoding nucleotides at the 5' and 3' ends, respectively. Using the cloned cDNA, we have isolated a genomic clone from a rat liver genomic library. Restriction mapping and Southern analysis using synthetic oligonucleotides localized the gene to a 4.0-kilobase-pair HindIII fragment.
Cytoplasmic Z-RNA.
Authors: Zarling D A , Calhoun C J , Hardin C C , Zarling A H
Published: May 2006

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.
The erg gene: a human gene related to the ets oncogene.
Authors: Reddy E S , Rao V N , Papas T S
Published: May 2006

We have isolated a cDNA clone representing the complete coding sequence of a human gene named erg, related to the ets oncogene. Nucleotide sequence analysis of this cDNA (4.6 kilobases long) revealed that this gene encodes a 363-residue protein whose predicted amino acid sequence showed a homology of approximately equal to 40% and approximately equal to 70% to two domains corresponding to the 5' and 3' regions of v-ets oncogene, respectively. A 3.2- to 3.6-kilobase and approximately equal to 5-kilobase transcript of the erg gene, which differ in size from those of the previously described Hu-ets 1 and Hu-ets 2 genes, were observed in different cells. These results suggest that the erg gene is a member of the ets oncogene family.
Allelic variation in the DR subregion of the human major histocompatibility complex.
Authors: Bell J I , Denney D , Foster L , Belt T , Todd J A , McDevitt H O
Published: May 2006

Allelic variation in the DR subregion of the human major histocompatibility complex has been analyzed by nucleic acid sequencing of cDNA clones obtained from cell lines homozygous by consanguinity for all the common serological types DR1-9. Two expressed loci were identified in the haplotypes DR2, -3, -4, -7, and -9; one locus being present at a much lower frequency (4-7%) than the other. The low-frequency allele was highly conserved between each of the DRw53 (DR4, -7, -9) and the DRw52 (DR3, -5, -6) haplotypes. Analysis of the variation between alleles confirms the presence of three allelic hypervariable regions. At each variable residue, a limited range of amino acid substitutions are found, distinguishing them from immunoglobulin hypervariable regions. Dinucleotide substitutions are extremely common. Individual hypervariable regions are often shared between haplotypes. Much of the variation in these alleles can be attributed to the shuffling of these regions between haplotypes, possibly by the mechanism of gene conversion.
Identity of the V kappa 10-Ars-A gene segments of the A/J and BALB/c strains.
Authors: Meek K , Sanz I , Rathbun G , Nisonoff A , Capra J D
Published: May 2006

To characterize the light chain gene segments involved in the murine immune response to keyhole limpet hemocyanin p-azophenylarsonate (Ars), we have determined the amino acid and/or nucleotide sequences of several anti-arsonate antibodies of the Ars-A family in the A/J, C.AL-20, and BALB/c strains. These structures have been compared to certain BALB/c anti-phenyloxazolone and anti-levan antibodies previously sequenced and to the A/J V kappa 10-Ars-A genomic sequence (where V kappa = kappa chain variable). These primary structural studies were complemented by Southern filter hybridization analyses utilizing V kappa and kappa chain joining (J kappa) molecular probes. We found a surprising uniformity of structure among these antibody light chains derived from different murine strains. Thus, in contrast to the heavy chain variable (Vh) regions of the Ars-A antibody family where the BALB/c strain lacks the VH gene segment utilized in the A/J Ars-A response, the light chain variable region gene segments at the V kappa 10-Ars-A locus appear to be identical between the two strains.
Epstein-Barr virus-containing epithelial cells from nasopharyngeal carcinoma produce interleukin 1 alpha.
Authors: Busson P , Braham K , Ganem G , Thomas F , Grausz D , Lipinski M , Wakasugi H , Tursz T
Published: May 2006

Nasopharyngeal carcinoma (NPC) is a human epithelial cancer that is constantly associated with the Epstein-Barr virus (EBV). Investigations on this tumor have been limited so far by the difficulty of culturing NPC cells for long periods. C15 is an NPC tumor that has been successfully carried in nude mice for greater than 2 yr. C15 cells isolated from the animal were shown to produce a soluble factor with interleukin 1 (IL-1) activity. Its biochemical (Mr, approximately equal to 17,000; pI approximately equal to 5) and immunological properties are identical to those of IL-1 alpha. RNA gel blot analysis showed IL-1 alpha, but not IL-1 beta, transcripts in C15 cells, in sharp contrast to monocytes that express IL-1 beta predominantly. Media from short-term cultures of fresh NPC biopsies also contained a strong IL-1 activity. Several lymphoblastoid cell lines obtained by EBV infection of normal B lymphocytes have been shown to produce IL-1 and use it as an autocrine growth factor. The production of IL-1 by malignant EBV-containing epithelial cells indicates that different types of EBV-infected cells produce IL-1. A relationship might exist between EBV and constitutive production of IL-1. The IL-1 produced by the malignant epithelial cells in vivo could stimulate the development of the pronounced T-cell infiltrate observed in NPC tumors.
Continuous partition lattice
Authors: Björner Anders
Published: May 2006

A continuous analogue to the partition lattices is presented. This is the metric completion of the direct limit of a system of embeddings of the finite partition lattices. The construction is analogous to von Neumann's construction of a continuous geometry over a field
F
from the finite-dimensional projective geometries over
F
.
Influence of altered transcription on the translational control of human ferritin expression.
Authors: Rouault T A , Hentze M W , Dancis A , Caughman W , Harford J B , Klausner R D
Published: May 2006

In this paper, we examine the response of a translational regulatory mechanism when changes in mRNA levels are induced. The gene that encodes the human ferritin heavy chain has been transfected into mouse fibroblasts. Stable transformants that express the human ferritin heavy chain have been isolated. This protein assembles into ferritin polymers and can co-assemble with host mouse ferritin. Biosynthetic rates of the expressed human ferritin varied over a wide range in response to perturbations in iron supply, but total and cytoplasmic messenger RNA levels remained unchanged. When changes in ferritin mRNA levels were induced by treatment with sodium butyrate, proportional changes in the biosynthetic rates of ferritin were observed, but the capacity for modulating biosynthesis in response to alterations in iron availability was preserved. These findings suggest that the final protein biosynthetic rate of a translationally regulated gene depends on both translational regulatory signals and underlying transcription rates.
Unique pathway of expression of an opal suppressor phosphoserine tRNA.
Authors: Lee B J , de la Peña P , Tobian J A , Zasloff M , Hatfield D
Published: May 2006

An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. We have shown that the product of this gene functions as a suppressor in an in vitro assay, and we have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, we show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of any known eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells.
A superactive insulin: [B10-aspartic acid]insulin(human).
Authors: Schwartz G P , Burke G T , Katsoyannis P G
Published: May 2006

The genetic basis for a case of familial hyperproinsulinemia has been elucidated recently. It involves a single point mutation in the proinsulin gene resulting in the substitution of aspartic acid for histidine-10 of the B chain of insulin. We have synthesized a human insulin analogue, [AspB10]insulin, corresponding to the mutant proinsulin and evaluated its biological activity. [AspB10]Insulin displayed a binding affinity to insulin receptors in rat liver plasma membranes that was 534 +/- 146% relative to the natural hormone. In lipogenesis assays, the synthetic analogue exhibited a potency that was 435 +/- 144% relative to insulin, which is statistically not different from its binding affinity. Reversed-phase HPLC indicated that the synthetic analogue is more apolar than natural insulin. We suggest that the observed properties reflect changes in the conformation of the analogue relative to natural insulin, which result in a stronger interaction with the insulin receptor. Thus, a single substitution of an amino acid residue of human insulin has resulted in a superactive hormone.
A calcium regenerative potential controlling ciliary reversal is propagated along the length of ctenophore comb plates.
Authors: Moss A G , Tamm S L
Published: May 2006

We have used the giant ciliary comb plates of ctenophores to record electrical activity directly from cilia. A compound action potential was recorded extracellularly over most of the length of the comb plate cilia in response to electrical stimulation of the ectodermal nerve net. The ciliary action potential was correlated with intracellularly recorded action potentials, selectively blocked by Ca2+-channel antagonists, and correlated with ciliary reorientation and reversed beating. Dual-electrode recording from different sites on the same comb plate showed that, unlike protistan cilia, the approximately 1-mm-long cilia of comb plates are not isopotential. Rather, action potentials are generated 150-200 microns from the base and propagate to the tip of the cilia. These results indicate that voltage-dependent channels that mediate increases in intraciliary Ca2+ concentration are distributed over most of the length of the cilia. Consequently, the Ca2+-sensitive machinery controlling ciliary motor responses is also likely to be located along the length of the axoneme.
Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP.
Authors: Miles K , Anthony D T , Rubin L L , Greengard P , Huganir R L
Published: May 2006

The nicotinic acetylcholine receptor (AcChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the beta and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the alpha subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 microM in the presence of 35 microM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of alpha subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.
Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A.
Authors: Siebert P D , Fukuda M
Published: May 2006

Here we describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector lambda gt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH2-terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. We used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. We established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes.
Role of cytoplasm-specific introgression in the evolution of the polyploid wheats
Authors: Gill Bikram S. , Chen P. D.
Published: May 2006

Studies of N-banded mitotic and meiotic karyotypes of
Triticum turgidum
L. (2
n
= 28; AABB) and
Triticum timopheevii
Zhuk. (2
n
= 28; AAGG) and hybrids between them, along with observations of meiotic pairing between telocentrics of the AB-genome chromosomes and their respective homologues and homeologues in
T. timopheevii
, showed that chromosome 4 (m4) of
Triticum monococcum
L. is present (as 4A
t
) in
T. timopheevii
but is lacking in
T. turgidum
. Neither 4A nor 4B pairs with 4A
t
, but 4A pairs with 4G and, for this reason and because of its banding pattern, must be considered a B-genome chromosome.
T. timopheevii
chromosomes 4A
t
and 3A
t
are involved in a reciprocal translocation, and 2A
t
, 1G, 2G, and 5G are also involved in translocations. Chromosome arm 4BL occasionally pairs with 7G. The satellites are on the short arms of chromosomes 6A
t
and 6G of
T. timopheevii
and 1B and 6B of
T. turgidum
. It is suggested that (
i
)
T. timopheevii
orginated as an allotetraploid of
Aegilops speltoides
Tausch/
T. monococcum
and (
ii
)
T. turgidum
was derived from
T. timopheevii
by introgressive hybridization with an unknown diploid species, which contributed its distinctive cytoplasm, chromosome 4B or a substantial portion of it, and additional chromosome segments. Rapid fixation of 4B in
T. turgidum
was ensured by cytoplasm-specific transmission.
Inhibition of protein synthesis prolongs Ca2+-mediated reduction of K+ currents in molluscan neurons.
Authors: Alkon D L , Bank B , Naito S , Chen C , Ram J
Published: May 2006

Elevated intracellular Ca2+ concentration within the Hermissenda type B cell has previously been shown to cause transient reduction of both the early K+ current IA and the delayed, Ca2+-dependent K+ current ICa2+-K+, a reduction that is more permanent with classical conditioning. Other earlier experiments suggested that Ca2+-mediated reduction of K+ currents initially involves the dual activation of Ca2+/calmodulin-dependent and Ca2+/lipid-dependent protein kinases. In the present study, voltage-clamp conditions that cause substantial increases in intracellular Ca2+ concentration (i.e., a Ca2+ "load") were used to produce IA and ICa2+-K+ reduction with and without the protein synthesis inhibitor anisomycin or cycloheximide or the control substance deacetylanisomycin in the bathing medium. Anisomycin (100 microM) and cycloheximide (100 microM) caused no significant change of resting membrane potential, holding current, or the non-voltage-dependent "leak" current. However, inhibition of protein synthesis prevented recovery from Ca2+-mediated K+-current reduction. This effect resembled the effect of injecting purified Ca2+-dependent kinases and was blocked by the presence of trifluoperazine in the bathing medium. Activation of protein kinase C with a water-soluble phorbol ester caused marked reduction of protein synthesis in Hermissenda neurons as monitored by two-dimensional gel electrophoresis. Synthesis of new proteins therefore may be important for reversal of initial steps during memory storage, and Ca2+-activated phosphorylation pathways may initiate long-term changes by turning off (as well as by turning on) the synthesis of particular proteins.
Polylysine-containing peptides, including the carboxyl-terminal segment of the human c-Ki-ras 2 protein, affect the activity of some key membrane enzymes.
Authors: Gatica M , Allende C C , Antonelli M , Allende J E
Published: May 2006

Polylysine-containing peptides are found to affect membrane protein kinases, phosphatidylinositol kinases, and adenylate cyclase. Poly(L-lysine), poly(D-lysine), random copolymers of lysine and serine or lysine and alanine, and poly(L-ornithine) produced large increases in the in vitro phosphorylation of some membrane proteins present in Xenopus laevis oocyte membranes. Poly(L-arginine) did not cause a similar stimulation. In these membranes the phosphorylation of polydisperse protein of approximately 25 kDa was also greatly increased by 1 mM spermine and spermidine, by 10 microM histone H1, or by 200 microM peptide containing the 14-residue sequence at the carboxyl terminus of the human c-Ki-ras 2 gene product, which has eight lysines. Similar specific stimulation of protein phosphorylation was observed with membranes of NG-108-15 nerve cells in culture. Polylysine peptides, including the c-Ki-ras 2 segment, also stimulate the in vitro phosphorylation of membrane inositolphospholipids, to produce mainly phosphatidylinositol 4-phosphate and less phosphatidylinositol 4,5-bisphosphate. Polylysine also alters the activity of oocyte adenylate cyclase, assayed in the presence of either F- or 5'-guanylyl imidodiphosphate.
Molecular cloning and nucleotide sequence of cDNA for human liver arginase.
Authors: Haraguchi Y , Takiguchi M , Amaya Y , Kawamoto S , Matsuda I , Mori M
Published: May 2006

Arginase (EC 3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, we isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted Mr, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.
Formation of cis-14,15-oxido-5,8,11-icosatrienoic acid from phosphatidylinositol in human platelets.
Authors: Ballou L R , Lam B K , Wong P Y , Cheung W Y
Published: May 2006

Human platelets contain a soluble enzyme or enzyme system that catalyzes the formation of lysophosphatidylinositol and a compound more polar than arachidonic acid (compound A) from 2-arachidonoyl sn-phosphatidylinositol. Arachidonic acid, 2-arachidonoyl sn-phosphatidylcholine, or 2-arachidonoyl sn-phosphatidylethanolamine did not serve as substrate for the production of compound A. The reaction required Ca2+ and was not affected by aspirin, indomethacin, or mepacrin. Enzyme activity was not enhanced in the presence of NADPH, but it was inhibited greater than 90% by CO or N2; inhibition was readily reversible by exposure to atmospheric air. Neither metapyrone (SKF 525A) nor cyanide, inhibitors of cytochrome P-450, inhibited compound A formation, suggesting that a cytochrome P-450 system was not involved. Thrombin stimulated the formation of compound A in whole platelets; ionophore A23187 did so much less effectively; and other agonists such as collagen, ADP, and epinephrine were ineffective. Compound A exhibited a fragmentation pattern by GC/MS identical to that of authentic cis-14,15-oxido-5,8,11-icosatrienoic acid. Collectively, these data indicate that human platelets may contain an enzyme system that catalyzes the epoxidation of the arachidonic acid moiety of phosphatidylinositol and its hydrolysis to liberate cis-14,15-oxido-5,8,11-icosatrienoic acid.
Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.
Authors: Halligan B D , Desiderio S V
Published: May 2006

Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.
3'-Formyl phosphate-ended DNA: high-energy intermediate in antibiotic-induced DNA sugar damage.
Authors: Chin D H , Kappen L S , Goldberg I H
Published: May 2006

Under anaerobic conditions where the nitroaromatic radiation-sensitizer misonidazole substitutes for dioxygen, DNA strand breakage (gaps with phosphate residues at each end) by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS-Chrom) is associated with the generation of a reactive form of formate from the C-5' of deoxyribose of thymidylate residues. Such lesions account for a minority (10-15%) of the strand breakage found in the aerobic reaction without misonidazole. Amino-containing nucleophiles such as tris(hydroxymethyl)aminomethane (Tris) and hydroxylamine act as acceptors for the activated formate. The amount of [3H]formyl hydroxamate produced from DNA labeled with [5'-3H]thymidine is comparable to the spontaneously released thymine. During the course of the reaction, misonidazole undergoes a DNA-dependent reduction and subsequent conjugation with glutathione used to activate NCS-Chrom. From these and earlier results, we propose a possible mechanism in which the carbon-centered radical formed at C-5' by hydrogen atom abstraction by thiol-activated NCS-Chrom reacts anaerobically with misonidazole to form a nitroxyl-radical-adduct intermediate, which fragments to produce an oxy radical at C-5'. beta-Fragmentation results in cleavage between C-5' and C-4' with the generation of 3'-formyl phosphate-ended DNA, a high-energy form of formate, which spontaneously hydrolyzes, releasing formate and creating a 3'-phosphate end, or transfers the formyl moiety to available nucleophiles. A similar mechanism, involving dioxygen addition, is probably responsible for the 10-15% DNA gap formation in the aerobic reaction.
Synergy of interleukin 1 and granulocyte colony-stimulating factor: in vivo stimulation of stem-cell recovery and hematopoietic regeneration following 5-fluorouracil treatment of mice.
Authors: Moore M A , Warren D J
Published: May 2006

The human bladder carcinoma cell line 5637 produces hematopoietic growth factors [granulocyte and granulocyte/macrophage colony-stimulating factors (G-CSF and GM-CSF)] and hemopoietin 1, which synergizes with CSFs to stimulate colony formation by primitive hematopoietic stem cells in 5-fluorouracil-treated mouse bone marrow. Molecular and functional properties of hemopoietin 1 identified it as identical to interleukin 1 alpha (IL-1 alpha). When bone marrow cells from 5-fluorouracil-treated mice were cultured in suspension for 7 days with recombinant human IL-1 alpha and/or G-CSF, it was found that the two factors synergized to enhance recovery of myelopoietic cells and colony-forming cells of both high and low proliferative potential. G-CSF alone did not sustain these populations, but the combination had greater-than-additive stimulating capacity. In vivo, 5-fluorouracil (150 mg/kg) produced profound myelosuppression and delayed neutrophil regeneration for up to 2 weeks in C3H/HeJ mice. Daily administration of recombinant human G-CSF or recombinant human IL-1 alpha accelerated recovery of stem cells, progenitor cells, and blood neutrophils by up to 4 days in 5-fluorouracil-treated C3H/HeJ and B6D2F1 mice. The combination of IL-1 alpha and G-CSF acted synergistically, reducing neutropenia and accelerating recovery of normal neutrophil numbers by up to 7 days. This was accompanied by accelerated regeneration of spleen colony-forming units and erythroid, myeloid, and megakaryocytic progenitor cells in marrow and spleen, with enhanced erythroid and granulocytic differentiation. These results indicate the possible therapeutic potential of combination therapy with IL-1 and hematopoietic growth factors such as G-CSF in the treatment of chemotherapy- or radiation-induced myelosuppression.
DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis.
Authors: Karathanasis S K , Ferris E , Haddad I A
Published: May 2006

The genes coding for apolipoproteins (apo) AI, CIII, and AIV, designated APOA1, APOC3, and APOA4, respectively, are closely linked and tandemly organized in the long arm of the human chromosome 11. A DNA rearrangement involving the genes encoding apoAI and apoCIII in certain patients with premature atherosclerosis has been associated with deficiency of both apoAI and apoCIII in the plasma of these patients. Structural characterization of the genes for apoAI and apoCIII in one of these patients indicates that this rearrangement consists of a DNA inversion containing portions of the 3' ends of the apoAI and apoCIII genes, including the DNA region between these genes. The breakpoints of this DNA inversion are located within the fourth exon of the apoAI gene and the first intron of the apoCIII gene. Thus, this DNA inversion results in reciprocal fusion of the apoAI and apoCIII gene transcriptional units. Expression of these gene fusions in cultured mammalian cells results in stable mRNA transcripts with sequences representing fusions of the apoAI and apoCIII mRNAs. These results indicate that absence of transcripts with correct apoAI and apoCIII mRNA sequences causes apoAI and apoCIII deficiency in the plasma of these patients and suggest that these apolipoproteins are involved in cholesterol homeostasis and protection against premature atherosclerosis.
Characterization of proopiomelanocortin transcripts in human nonpituitary tissues.
Authors: Lacaze-Masmonteil T , de Keyzer Y , Luton J P , Kahn A , Bertagna X
Published: May 2006

Proopiomelanocortin (POMC), the precursor to adrenocorticotropic hormone and other related peptides, was originally identified in the corticotropic cell. Recent evidence shows that POMC products are also normally present in a variety of nonpituitary tissues. To investigate this phenomenon in humans we looked for the presence and characteristics of POMC transcripts in various adult tissues. Blot hybridization analysis of normal adrenal, thymus, and testis RNAs revealed a small RNA species approximately 400 nucleotides shorter than the 1200-nucleotide pituitary species. Primer extension and S1 nuclease mapping studies showed that this small RNA lacked exon 1 and exon 2 of the gene, and it corresponded to a set of at least six molecules starting 41 to 162 nucleotides downstream from the 5' end of exon 3. These RNAs appear to result from heterogeneous transcription initiation sites presumably under the control of "GC box" promoter sequences located in the 3' end of intron 2. They cannot encode a complete POMC molecule, and the only truncated POMC molecules that could be translated would lack a signal peptide necessary for membrane translocation and precursor processing. The use of highly sensitive S1 nuclease mapping techniques with uniformly labeled single-stranded DNA probes allowed the detection of a small but definite amount of the "normal," 1200-nucleotide, mRNA species. It is suggested that it is this POMC mRNA that is responsible for the local production of all the POMC peptides.
Identification of a vaccinia virus gene encoding a type I DNA topoisomerase.
Authors: Shuman S , Moss B
Published: May 2006

Vaccinia virus encapsidates a type I DNA topoisomerase (EC 5.99.1.2). The enzyme was purified from virus cores to apparent homogeneity, yielding a protein of Mr 32,000. The amino-terminal sequence of the isolated Mr 32,000 polypeptide was determined and used to map the putative structural gene for the vaccinia topoisomerase to the H7r open reading frame of the vaccinia genome. This gene encodes a 314-amino acid polypeptide containing a region homologous to a region of the type I topoisomerase from the yeast Saccharomyces cerevisiae.