bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of biologically active human interleukin 1 subpeptides by transfected simian COS cells.
Authors: Rosenwasser L J , Webb A C , Clark B D , Irie S , Chang L , Dinarello C A , Gehrke L , Wolff S M , Rich A , Auron P E
Published: May 2006

"Interleukin 1" (IL-1) is a term used to describe the family of macrophage-derived proteins that mediate many immune and inflammatory reactions. We have previously described the molecular cloning and sequencing of the cDNA encoding the predominant (neutral) form of human IL-1, which has been designated IL-1 beta. We report here that transfection of simian COS cells with this cDNA clone results in the transcription of IL-1 mRNA and the synthesis of antibody-neutralizable intracellular IL-1 biological activity. In addition, selective deletion of regions of the IL-1 cDNA judged not to be essential for function, on the basis of conserved sequence homology, resulted in localization of a "core" region responsible for a majority of the biological activity. These results permit mapping the active site of IL-1 to a peptide of 6970 molecular weight located within the carboxyl third (between Met-136 and Gln-197) of the IL-1 precursor.
Classical conditioning using stimulation of the inferior olive as the unconditioned stimulus.
Authors: Mauk M D , Steinmetz J E , Thompson R F
Published: May 2006

We show that conditioned eyelid responses develop when the unconditioned stimulus is electrical stimulation of the dorsal accessory nucleus of the inferior olive. When compared to conditioning using a standard unconditioned stimulus (air puff), the conditioning produced in this manner appears quite normal: the responses develop at a similar rate, are of comparable magnitude and topography, and demonstrate a steep interstimulus interval function; and response topography varies according to the interstimulus interval. These data indicate that activation of neurons in the dorsal accessory olive is a sufficient condition for a stimulus to be an effective unconditioned stimulus. Previous experiments indicate the dorsal accessory olive is necessary in that lesions have effects functionally equivalent to removal of the unconditioned stimulus. These data indicate that the dorsal accessory olive forms a portion of the pathway conveying information about the occurrence of an unconditioned stimulus to sites of synaptic plasticity responsible for conditioning.
Inducible overproduction of the mouse c-myc protein in mammalian cells.
Authors: Wurm F M , Gwinn K A , Kingston R E
Published: May 2006

We have made Chinese hamster ovary (CHO) cell lines that contain up to 2000 copies of the coding region of the mouse c-myc gene fused to the promoter of the Drosophila gene (hsp70) encoding a Mr 70,000 heat shock protein. Incubation of these cells at 43 degrees C results in an estimated 100-fold induction of c-myc mRNA. Translation of this mRNA occurs when the cells are returned to 37 degrees C, and during the first 3 hr of recovery at 37 degrees C, the c-myc protein is one of the most abundantly synthesized proteins in the cells. The products of the induced c-myc gene are phosphoproteins of apparent Mr 64,000, 66,000, and 75,000. Induced cells die, suggesting that elevated levels of c-myc are cytotoxic. Amplification of genes placed under control of the Drosophila hsp70 promoter may provide a general method for inducibly over expressing proteins in mammalian cells.
Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.
Authors: Lewis M E , Sherman T G , Burke S , Akil H , Davis L G , Arentzen R , Watson S J
Published: May 2006

Synthetic oligonucleotide probes can be easily obtained and used, in contrast to cDNA cloning to develop probes, and thus the present study was carried out to determine whether such probes could also be useful for in situ hybridization. A 24-base synthetic oligonucleotide complementary to part of the alpha-melanocyte-stimulating hormone (alpha-MSH) coding region of proopiomelanocortin (POMC) mRNA was 5'-end-labeled by using [gamma-32P]ATP with T4 polynucleotide kinase or was 3' tailed by using [alpha-32P]dATP or [3H]dCTP with terminal deoxynucleotidyltransferase. Blot analysis of pituitary poly(A)+ RNA showed that the oligonucleotide hybridized to a single species with a molecular size of approximately 1200 nucleotides, consistent with that determined previously for POMC mRNA. The oligonucleotide, regardless of labeling method, hybridized to cells in the pituitary intermediate lobe, but not in the posterior lobe. Only the 3H-labeled probe gave resolution of individual pituitary anterior lobe cells. The specificity of the hybridization was determined by showing that the intermediate lobe signal was blocked by prehybridization of the tissue with unlabeled alpha-MSH oligonucleotide probe. Furthermore, the hybridized probe exhibited a sharp sigmoid curve when melted off. Finally, the oligonucleotide probe detected, in situ, the haloperidol-induced elevation of intermediate lobe POMC mRNA. Thus, the oligonucleotide probe exhibited hybridization in an anatomically and biochemically specific manner, and it detected a tissue-specific change in mRNA levels in situ.
Decreased c-myc expression is an early event in retinoic acid-induced differentiation of F9 teratocarcinoma cells.
Authors: Griep A E , DeLuca H F
Published: May 2006

Retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells is known to be accompanied by changes in gene expression. We examined the expression of the c-myc gene during retinoic acid-induced differentiation of F9 cells. Retinoic acid caused a 50% reduction in the level of c-myc mRNA after 3 hr of treatment and a 90% reduction after 12 hr. We have also examined several features of c-myc genomic structure in F9 cells, PYS2 (differentiated parietal yolk sac) cells, and liver--namely, methylation, amplification, and gross organization. Comparison of Hpa II and Msp I digests of DNAs from F9, PYS2, and liver showed that the c-myc gene in F9 cells is hypomethylated relative to that in PYS2 cells and in liver. The Hpa II sites that undergo methylation during differentiation were found to be in the second intron. Gross structural changes of the c-myc gene have not occurred in F9 or PYS2, and the c-myc gene is not amplified.
Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Authors: Basson M E , Thorsness M , Rine J
Published: May 2006

We have isolated two genes from yeast encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase [hydroxymethylglutaryl-coenzyme A reductase (NADPH); HMG-CoA reductase; EC 1.1.1.34], the rate-limiting enzyme of sterol biosynthesis. These genes, HMG1 and HMG2, were identified by hybridization to a cDNA clone encoding hamster HMG-CoA reductase. DNA sequence analysis reveals homology between the amino acid sequence of the proteins encoded by the two yeast genes and the carboxyl-terminal half of the hamster protein. Cells containing mutant alleles of both HMG1 and HMG2 are unable to undergo spore germination and vegetative growth. However, cells containing a mutant allele of either HMG1 or HMG2 are viable but are more sensitive to compactin, a competitive inhibitor of HMG-CoA reductase, than are wild-type cells. Assays of HMG-CoA reductase activity in extracts from hmg1- and hmg2- mutants indicate that HMG1 contributes at least 83% of the activity found in wild-type cells.
Double-stranded gap repair in the photosynthetic prokaryote
Synechococcus
R2
Authors: Kolowsky Karen S. , Szalay Aladar A.
Published: May 2006

The photosynthetic cyanobacterium
Synechococcus
R2 is transformed by chimeric donor molecules lacking a functional replication origin but containing a region of homology to the recipient chromosome. These integrating donor molecules consist of a fragment of
Synechococcus
R2 chromosomal DNA cloned in the
Escherichia coli
vector pBR322 and interrupted by a piece of foreign DNA. During integration, this interrupting DNA is often lost by nonreciprocal exchange between homologous regions of donor and recipient. When transformed with donor molecules containing
in vitro
-generated double-stranded gaps or deletions as large as 20 kilobase pairs in the fragment homologous to the recipient chromosome,
Synechococcus
R2 can repair these lesions by using recipient information. Chromosomal DNA of the resulting transformants contains direct repeats of the recipient copy on either side of integrated pBR322 DNA. Homologous recombination between these repeats generates a circular molecule that can be recovered by transformation to
E. coli
. Plasmids recovered in
E. coli
contain the entire copy of information initially present in the region of the
Synechococcus
recipient corresponding to the donor gap or deletion. We suggest applications of this mechanism for cloning of genes identified by transposon mutagenesis.
Changes in genetic diversity in the red winter wheat regions of the United States
Authors: Cox T. S. , Murphy J. P. , Rodgers D. M.
Published: May 2006

Pedigree and acreage data were utilized to determine trends in genetic diversity of soft red winter (SRW) and hard red winter (HRW) wheats. Four uniformity estimates were computed: (
a
)
r̄
1
, the mean relationship among all cultivars grown in a given year; (
b
)
r̄
2
, the mean relationship among primary cultivars; (
c
)
r̄
3
, the mean relationship of primary cultivars weighted by acreage; and (
d
)
r̄
4
, the mean relationship of primary cultivars grown in different years, weighted by acreage. In the SRW region, there has been a slow but steady increase in relationship among cultivars (
r̄
1
and
r̄
2
). There was a dramatic increase in field uniformity (
r̄
3
) during the 1970s, but
r̄
3
had sharply decreased by 1984 to its lowest point ever (0.22). All uniformity estimates decreased sharply for HRW wheats from 1919 to 1949 and have decreased gradually since. Uniformity is higher in HRW than in SRW wheats, primarily because of the persistence of a core of HRW germ plasm from cultivar `Turkey,' but the difference is diminishing. Both classes appear to be entering a new era of increasing diversity.
Predicted conformations for the immunodominant region of the circumsporozoite protein of the human malaria parasite Plasmodium falciparum.
Authors: Gibson K D , Scheraga H A
Published: May 2006

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum contains multiple tandem repeats of the amino acid sequence Asn-Ala-Asn-Pro. The repeated sequence encompasses the immunodominant region of the protein, and antibodies raised against it are potent inhibitors of invasion and development of sporozoites in cultured hepatocytes. Using a modified build-up procedure, we have explored a large number of possible helical and near-helical conformations of a terminally blocked tetraicosapeptide, consisting of six repeats of the sequence Asn-Ala-Asn-Pro, and conclude that two helical conformations are energetically favored to the exclusion of all others. One of these conformations is longer, thinner, and left-handed and is likely to be adopted in nonpolar environments, while the other is shorter, broader, and right-handed and should be favored in aqueous solutions. We propose that the immunodominant region of the circumsporozoite protein of P. falciparum adopts one of these conformations in vivo.
An inhibitor of SOS induction, specified by a plasmid locus in Escherichia coli.
Authors: Bagdasarian M , Bailone A , Bagdasarian M M , Manning P A , Lurz R , Timmis K N , Devoret R
Published: May 2006

Plasmid R6-5 contains a locus whose product inhibits induction of sfiA and prophage lambda in a recA441 mutant at 42 degrees C and in a recA+ host after treatment with nalidixic acid. This plasmidic SOS-inhibition locus (psi) is situated on an 8.1-kilobase DNA fragment near oriT, the origin of plasmid R6-5 conjugational transfer. Loss of the Psi function, resulting from the insertion of Tn3 into psi+, greatly reduced the synthesis of two proteins, designated PsiA (Mr 24,500) and PsiB (Mr 12,500). Using host cells in which there was an inactive LexA repressor, we found that Psi function does not act by interfering with the expression of the SOS pathway. The Psi function may affect the generation of an SOS signal. We postulate that during the course of evolution, the Psi function has been selected in some conjugative plasmids so as to permit them to transfer single-stranded DNA without generating an SOS signal.
Lithium dampens neurotransmitter response in smooth muscle: relevance to action in affective illness.
Authors: Menkes H A , Baraban J M , Freed A N , Snyder S H
Published: May 2006

Lithium, by inhibiting inositol phosphate metabolism, interferes with the phosphatidylinositol ("phosphoinositide") cycle, which is stimulated by numerous hormones and neurotransmitters. To examine the relevance of this action to neurotransmission, we evaluated effects of lithium treatment on smooth muscle responses to transmitters. In lithium-pretreated tracheal muscle, the relaxation following carbachol or histamine contractions is retarded. Lithium does not affect relaxation following contractions elicited by treatment with KCl and phorbol 12,13-diacetate in combination, which bypasses receptor stimulation of the phosphatidylinositol cycle. Half-maximal effects of lithium occur at 1 mM, corresponding to therapeutic concentrations. Dampening of neurotransmitter responses by lithium treatment may explain the unique ability of lithium to relieve and prevent both mania and depression.
Immunochemical and pharmacological distinctions between curaremimetic neurotoxin binding sites of central, autonomic, and peripheral origin.
Authors: Lukas R J
Published: May 2006

Comparative pharmacological and immunochemical studies were conducted on alpha-bungarotoxin binding sites from rat brain or muscle, Torpedo electric tissue, or the TE671 or PC12 clonal cell lines. Characteristic distinctions were observed in the pharmacological profile of drugs competing for toxin binding to different tissues. Differences also were found in the proportion of toxin binding sites (membrane-bound or detergent-solubilized) that are immunologically reactive with either monoclonal antibodies directed against nicotinic acetylcholine receptors from the electric organ of Torpedo or polyclonal antisera raised against nicotinic receptors from the electric organ of Electrophorus. These results suggest that toxin binding sites are structurally heterogeneous. Structural heterogeneity of nicotinic acetylcholine receptors, neurotoxin binding sites, or both, may contribute to the manifestation of nicotinic receptor functional heterogeneity and may explain the apparent discrepancy at some sites between toxin binding activity and toxin functional potency.
Interpretation of dose-response curves for luteinizing hormone release by gonadotropin-releasing hormone, related peptides, and leukotriene C4 according to a hormone/receptor/effector model.
Authors: Leiser J , Conn P M , Blum J J
Published: May 2006

The dose-response curves for pituitary luteinizing hormone (LH) release in response to gonadotropin-releasing hormone and its agonists are unusually broad. It appears, however, that these ligands bind to a single class of receptors. It is shown that these dose-response data can be explained by either of two models in which ligand-receptor complexes stimulate LH secretion by interacting with either of two different effector systems or by interacting with a single effector system but forming monomeric and dimeric active effector complexes. A combination of these two basic models can account for the very broad, biphasic dose-response curve reported for LH release in response to leukotriene C4.
Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain.
Authors: Covarrubias L , Nishida Y , Mintz B
Published: May 2006

Insertional mutagenesis in a transgenic mouse strain (HUGH/3) was caused by integration of plasmid DNA containing the human growth hormone gene and pBR322 plasmid sequences. From this study, which includes another instance of mutagenesis, and from other reports, it is apparent that insertional mutagenesis occurs fairly frequently during DNA integration in the mouse egg and that it is not specific for the exogenous DNA employed. The mutation in HUGH/3 is recessive and results in death of embryos homozygous for the donor sequences shortly after implantation, at the egg cylinder stage on days 4-5 of gestation. Restriction mapping of the insert and of the flanking DNA regions indicates that integration must have involved a series of complex events. Approximately five copies of plasmid sequences are arrayed in tandem but are interrupted at least twice by mouse cellular sequences. In addition, the mouse flanking DNA shows extensive rearrangements, probably including a deletion of at least 10 kilobases. The rearrangements may reflect an initially unstable DNA structure followed by attainment of a more stable conformation.
The specific direct interaction of helper T cells and antigen-presenting B cells.
Authors: Kupfer A , Swain S L , Janeway C A , Singer S J
Published: May 2006

Cell couples have been formed by mixing an antigen- and Ia-specific cloned helper T-cell line with a B-cell hybridoma presenting the antigen. By immunofluorescence observations, we have shown that the microtubule-organizing center (MTOC) inside the helper T cell, but not in the bound antigen-presenting cell, becomes oriented to face the area of specific cell-cell contact. This MTOC orientation is antigen- and Ia-specific, and thus provides direct evidence for the specific interaction of a helper T cell with a B cell. It is presumed that the function served by this MTOC orientation, which is accompanied by the coordinate reorientation of the Golgi apparatus, is to target Golgi apparatus-derived secretory vesicles, containing putative lymphokines and/or growth factors, from the helper T cell directly to the antigen-presenting cell.
Alternative protein products with different carboxyl termini from a single class I gene, H-2Kb.
Authors: Lew A M , Margulies D H , Maloy W L , Lillehoj E P , McCluskey J , Coligan J E
Published: May 2006

In addition to mRNA encoding the canonical form of the murine class I antigen H-2Kb (348 amino acid residues), mRNA that would encode a shortened form of H-2Kb (missing 9 amino acids from the C-terminus) has been identified in C57BL/6 spleen cells by RNase-protection studies. The alternative transcripts of H-2Kb arise through the use of different AG acceptor splice sites for exon VIII. The existence of a shortened H-2Kb protein was demonstrated by sequential immunoprecipitation. Lysates of spleen cells that had been labeled with [35S]methionine and [3H]histidine were precleared with rabbit anti-peptide serum reactive with the C-terminus of the canonical H-2Kb. The shortened form of H-2Kb was immunoprecipitated from this lysate with H-2Kb alloantiserum. Both forms of H-2Kb were isolated by NaDodSO4/PAGE. Tryptic peptide mapping confirmed that these molecules differed only at their C-terminus. The shortened form of H-2Kb is also found in a B-cell line (R8) but not in three cloned T-cell lines or in a T-cell lymphoma (EL4), suggesting that regulatory events are involved in producing the two forms of H-2Kb. Putative lariat branch points involved in these alternative splicing events for the 3' coding region of H-2 class I pre-mRNAs are proposed.
Plasmodium falciparum polypeptides associated with the infected erythrocyte plasma membrane.
Authors: Stanley H A , Reese R T
Published: May 2006

Plasmodium falciparum proteins associated with plasma membranes of infected erythrocytes were identified by using three techniques: isolated plasma membranes from infected and uninfected erythrocytes were compared by gel electrophoresis and silver staining; isolated plasma membranes from cells metabolically labeled with [35S]methionine were assayed by gel electrophoresis; and uninfected and infected intact erythrocytes were surface-labeled by lactoperoxidase iodination, and the labeled polypeptides were compared by gel electrophoresis. The results from these experiments indicate that at least six parasite-derived polypeptides (Mr = greater than 240,000, 150,000, 55,000, 45,000, 35,000, and 20,000) are associated with the infected erythrocyte plasma membrane. At least four of these peptides (Mr = 55,000, 45,000, 35,000, and 20,000) may be exposed on the surface of the infected erythrocytes.
Perturbation of the human T-cell antigen receptor-T3 complex leads to the production of inositol tetrakisphosphate: evidence for conversion from inositol trisphosphate.
Authors: Stewart S J , Prpic V , Powers F S , Bocckino S B , Isaacks R E , Exton J H
Published: May 2006

Antibodies directed against the T-cell antigen receptor-T3 complex mimic antigen and lead to cellular changes consistent with activation. When cells of the human T-cell line Jurkat were stimulated with a monoclonal antibody directed against T3, inositol phosphates were produced. In addition to inositol trisphosphate, which is the product of phosphatidylinositol bisphosphate cleavage, a second inositol polyphosphate was formed. This compound was more polar than inositol trisphosphate but less polar than inositol pentakisphosphate. It cochromatographed with inositol tetrakisphosphate from ostrich erythrocytes. In permeabilized Jurkat cells, this compound was shown to be formed from inositol 1,4,5-trisphosphate, but only in the presence of ATP, and 32P was incorporated into it from [gamma-32P]ATP. There also was coincident formation of inositol 1,3,4-trisphosphate. We conclude that the more polar compound is inositol tetrakisphosphate, which is formed by phosphorylation of inositol 1,4,5-trisphosphate and may be the precursor of inositol 1,3,4-trisphosphate.
Biosynthesis and maturation of peroxisomal beta-oxidation enzymes in fibroblasts in relation to the Zellweger syndrome and infantile Refsum disease.
Authors: Schram A W , Strijland A , Hashimoto T , Wanders R J , Schutgens R B , van den Bosch H , Tager J M
Published: May 2006

The biosynthesis of the peroxisomal enzymes acyl-CoA oxidase, 3-oxoacyl-CoA thiolase (acetyl-CoA acyl-transferase, EC 2.3.1.16), and catalase (EC 1.11.1.6) was studied in cultured skin fibroblasts from a control subject and from patients with Zellweger syndrome and the infantile form of Refsum disease, inherited disorders in which peroxisomes are deficient and certain peroxisomal functions are impaired. The results of continuous labeling and pulse-chase experiments indicate that in control fibroblasts, as in rat liver, acyl-CoA oxidase is synthesized as a 72-kDa percursor that is converted to two polypeptides of 52 and 20 kDa and 3-oxoacyl-CoA thiolase is synthesized as a 44-kDa precursor that is converted to the 41-kDa mature protein. In fibroblasts from the patients the precursors of the two enzymes are formed but their maturation is impaired, and they are rapidly degraded. In contrast, the biosynthesis of catalase is not impaired. We conclude that functional peroxisomes are required for the maturation and stability of acyl-CoA oxidase and 3-oxoacyl-CoA thiolase but not for catalase.
Protein sequencing by tandem mass spectrometry.
Authors: Hunt D F , Yates J R , Shabanowitz J , Winston S , Hauer C R
Published: May 2006

Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.
Coculture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells.
Authors: Levi-Schaffer F , Austen K F , Gravallese P M , Stevens R L
Published: May 2006

The heparin-containing mast cells that reside in the connective tissue of the mouse, but not the chondroitin sulfate-containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse bone marrow-derived mast cells (BMMC), the probable in vitro counterparts of in vivo mucosal mast cells, were cultured for 14 days with mouse skin-derived 3T3 fibroblasts in RPMI 1640 medium containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin 3 in the conditioned medium. The mast cells remained viable throughout the period of coculture, since they failed to release lactate dehydrogenase and because they increased their histamine content approximately 15-fold. After 12-14 days of coculture, greater than 50% of the BMMC changed histochemically to become safranin+; 30-40% of the 35S-labeled glycosaminoglycans on the proteoglycans synthesized by these cocultured mast cells were heparin, whereas heparin was not detected in the initial BMMC. In the absence of WEHI-3 conditioned medium, BMMC adhered to the fibroblast monolayer, and after 8 days of coculture, the number of mast cells did not change and their histamine content remained the same. However, these mast cells also became safranin+ and synthesized 40% heparin glycosaminoglycans. Thus, coculture of BMMC with fibroblasts induces a phenotypic change so that the resulting mast cells stain safranin+ and synthesize heparin proteoglycans, whereas the presence of WEHI-3 conditioned medium stimulates proliferation and an increase in histamine content.
Construction of two Escherichia coli amber suppressor genes: tRNAPheCUA and tRNACysCUA.
Authors: Normanly J , Masson J M , Kleina L G , Abelson J , Miller J H
Published: May 2006

Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering. The genes for either tRNAPheGAA or tRNACysGCA both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor (tRNAPheCUA) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACysCUA) is 17-50% efficient. To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence. NH2-terminal sequence analysis of the resultant proteins revealed that tRNAPheCUA and tRNACysCUA insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, tRNAPheCUA and tRNACysCUA have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.
The human c-ros gene (ROS) is located at chromosome region 6q16----6q22.
Authors: Nagarajan L , Louie E , Tsujimoto Y , Balduzzi P C , Huebner K , Croce C M
Published: May 2006

The human homolog, c-ros, of the transforming gene, v-ros, of the avian sarcoma virus, UR2, has been isolated from a human genomic library. A single-copy fragment from the human c-ros genomic clone has been used to map the human c-ros homolog (ROS) to human chromosome region 6q16----6q22 by somatic cell hybrid analysis and chromosomal in situ hybridization. Thus, the c-ros gene joins the c-myb oncogene, which is distal to the c-ros gene on the long arm of human chromosome 6, as a candidate for involvement in chromosome 6q deletions and rearrangements seen in various malignancies.
Synthesis of the complete trans-activation gene product of human T-lymphotropic virus type III in Escherichia coli: demonstration of immunogenicity in vivo and expression in vitro.
Authors: Aldovini A , Debouck C , Feinberg M B , Rosenberg M , Arya S K , Wong-Staal F
Published: May 2006

Human T-lymphotropic virus type III (HTLV-III) contains a gene (tat-III) the product of which activates the expression of viral genes in trans. We have expressed in Escherichia coli the complete tat-III-encoded protein as well as a truncated form that lacks three amino acids from the amino terminus. These proteins are recognized by sera of many, but not all, infected individuals including patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex, as well as asymptomatic seropositive persons. Seropositivity for the tat-III protein does not appear to correlate with the clinical stage of HTLV-III-related disease. Antibodies raised in rabbits against the E. coli-produced protein detect the native protein (apparent molecular mass, 14.5 kDa) in a virus-producing cell line. A second protein (26 kDa), of unknown origin but viral related, is also specifically recognized by the immune serum.
Chemical reactivity at an antibody binding site elicited by mechanistic design of a synthetic antigen.
Authors: Tramontano A , Janda K D , Lerner R A
Published: May 2006

Monoaryl phosphonate esters, designated as analogs of the transition state in the hydrolysis of carboxylic esters, were synthesized and used as haptens to generate specific monoclonal antibodies. Some of these antibodies react with cognate aryl carboxylic esters to release a fluorescent alcohol. The reaction appears to be stoichiometric; however, the activity is slowly regenerated under alkaline conditions or by treatment with hydroxylamine. Specificity is rigorous for esters of p-trifluoroacetamidophenylacetic acid, demonstrating a structural correspondence with the phosphonate hapten. Saturation kinetics are observed and kinetic parameters (kmax, Vmax, and Km) are reported. The haptenic phosphonate is a competitive inhibitor of the reaction (Ki, 35 nM); whereas the carboxylate product of ester hydrolysis is a less effective inhibitor (Ki, ca. 7500 nM). Chemical modification of side chain groups in the protein show a partial reduction in activity on acylation of lysine or nitration of tyrosine and a dramatic quenching upon modification of histidine. The evidence is discussed in terms of a mechanism in which amino acids of the antibody combining site participate in nucleophilic and/or general base catalysis. The properties of this system suggest that it is an example of enzymic transacylation where a deacylation step is not catalyzed. The possibility of deriving enzymic function from immunological specificity through this approach is advanced.
Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions.
Authors: Oppenheim A , Peleg A , Fibach E , Rachmilewitz E A
Published: May 2006

Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%.
Sequences of Ly-5 cDNA: isoform-related diversity of Ly-5 mRNA.
Authors: Saga Y , Tung J S , Shen F W , Boyse E A
Published: May 2006

The Ly-5 system of the mouse is expressed exclusively by hematopoietic cells and comprises a series of glycoprotein isoforms that typify different hematopoietic cell lineages. The 200-kDa isoform of T cells and the 220-kDa isoform of B cells are known to differ in peptide composition. The complete 1152 amino acid sequence of the 200-kDa isoform protein deduced from cDNA sequence appears to comprise a leader sequence of some 30 residues, an external N-terminal domain of 370 residues, a probably single transmembrane domain of 22 residues, and an unusually large cytoplasmic domain of 730 residues. Both the external and cytoplasmic domains include regions of internal homology suggestive of evolution from a smaller ancestral gene. RNA transfer blotting has previously shown that B-cell mRNA for Ly-5 is larger than T-cell mRNA. S1 nuclease protection mapping with Ly-5 cDNA probes suggests that this difference can be ascribed to interpolation of an extra B-cell sequence located at the 5' end of B-cell mRNA, probably immediately following the leader sequence. From restriction mapping of overlapping Ly-5 genomic clones spanning 60 kilobases it is concluded that Ly-5 isoforms are generated by differential processing of transcripts of a single gene, rather than from a family of linked Ly-5 genes.
The growth cones of
Aplysia
sensory neurons: Modulation by serotonin of action potential duration and single potassium channel currents
Authors: Belardetti F. , Schacher S. , Kandel E. R. , Siegelbaum S. A.
Published: May 2006

Serotonin (5-HT) closes a specific K channel (“S”) in the cell body of
Aplysia
sensory neurons, resulting in a slow excitatory postsynaptic potential and spike broadening. To determine whether the S channel is present and can be modulated in processes of the neuron other than the cell body, we studied the effects of 5-HT on growth cones of sensory neurons in culture by using the patch-clamp technique. Simultaneous application of 5-HT to the cell body and to the growth cones of sensory neurons produced, in both, a slow depolarization of ∼5 mV. Also, 5-HT produced a lengthening of the duration of action potential in the growth cone and cell body by 20-30%. Similar effects were observed in isolated growth cones that had been severed from the rest of the neuron, implying that the growth cones contain all the molecular components (i.e., receptors, channels, cAMP cascade) necessary for 5-HT action. Cell-attached patch-clamp recordings demonstrated the presence of S channels in sensory neuron growth cones. Application of serotonin to the bath produced long-lasting all-or-none closures of these channels in a manner identical to the previously characterized action of 5-HT in the cell body. Thus, channel modulation is not restricted to the cell body and probably occurs throughout the sensory neuron. This strengthens the view that S-channel modulation may also occur at the sensory neuron presynaptic terminal, where it could play a role in the presynaptic facilitation produced by 5-HT.
ATP-independent type II topoisomerase from trypanosomes.
Authors: Douc-Rasy S , Kayser A , Riou J F , Riou G
Published: May 2006

We have characterized in Trypanosoma cruzi a DNA topoisomerase capable of decatenating complex trypanosomal kinetoplast DNA networks in the absence of ATP. The enzymatic activity requires Mg2+ and K+. Using a defined DNA topoisomer we showed that the linking number changes by steps of 2, which characterizes the enzyme as a type II topoisomerase. The enzyme can catenate supercoiled DNA molecules, unknot DNA, and cleave double-stranded DNA. The enzyme has no ATPase activity. The native enzyme has an Mr of about 200,000. Crude extracts and partially purified fractions contain an aggregating factor that can substitute spermidine in catenating reactions. Because of the presence of this factor, the kinetoplast DNA can only be decatenated by purified fractions. The enzyme is inhibited by certain drugs and provides a potential target for chemotherapy. Such an enzyme was also characterized in Trypanosoma equiperdum.
Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis.
Authors: Mandecki W
Published: May 2006

A DNA double-strand break can be efficiently repaired in Escherichia coli if an oligodeoxyribonucleotide is provided to direct the repair. The oligonucleotide must be at least 20 residues long and have a sequence identical to sequences flanking the break. The phenomenon can be used to introduce defined mutations into DNA in the area of a double-strand break. To obtain mutants, the oligonucleotide that carries a mutation and the denatured linearized plasmid DNA are introduced into E. coli by transformation. No enzymatic manipulation in vitro is required. The mutants can constitute up to 98% of the total number of transformants obtained. The efficiency of mutagenesis decreases as the distance between the mutation and the plasmid cleavage site increases. The universality of the method was tested by introducing mutations into four genes, using four plasmids and three E. coli strains, as well as eight restriction enzymes to linearize DNA. Several models of the oligonucleotide-directed DNA double-strand break repair are discussed.
Distinct transcription factors bind specifically to two regions of the human histone H4 promoter.
Authors: Dailey L , Hanly S M , Roeder R G , Heintz N
Published: May 2006

Two proteins specifically binding to separate regions of the human histone H4 promoter were identified in nuclear extracts prepared from synchronized S-phase HeLa cells. Competition experiments with H4 promoter mutants and DNase protection assays ("footprinting") demonstrate that these factors bind to regions of the H4 promoter that are essential for maximal expression in vitro. One of these factors (H4TF-1) binds to sequences between -80 and -110 base pairs upstream of the H4 cap site, whereas the other (H4TF-2) binds to the H4 subtype-specific sequence element immediately upstream from the "TATA" homology. Neither of these activities can efficiently bind to any of the other histone gene subtypes or simian virus 40 DNA. Binding of H4TF-1 to the distal region of the pHu4A histone H4 promoter is inhibited competitively with varying efficiency by four of six human histone H4 genes cloned in this laboratory, whereas efficient competition for binding of H4TF-2 is exhibited by five of the six H4 genes. Since both of these factors bind to significant regions of the pHu4A histone H4 promoter and can be bound by several different human H4 genes, we believe that they are important for maximal transcription of the gene and that they may be involved in its regulated expression during the cell cycle.
A cell surface desmosome-associated component: identification of tissue-specific cell adhesion molecule.
Authors: Jones J C , Yokoo K M , Goldman R D
Published: May 2006

Autoantibodies in the serum of patients suffering the blistering skin disease pemphigus vulgaris recognize a 140-kDa glycoprotein (GP) present in enriched fractions of bovine tongue epidermal desmosomes. Immunofluorescence observations of cryostat sections of bovine tongue epidermis reveal that affinity-purified rabbit antibodies to the 140-kDa GP generate a punctate intercellular stain that is similar to that generated by antibodies directed against a desmosome plaque component (desmoplakin). In cultured mouse keratinocytes, the antibodies against 140-kDa GP recognize desmosomes along areas of cell-cell contact. Double immunofluorescence of cultured keratinocytes with these antibodies and a desmoplakin antiserum reveals that the antibodies against the 140-kDa GP stain a single fluorescent line along areas of cell-cell contact. This single fluorescent line lies between double fluorescent lines generated by the desmoplakin antiserum. Immunogold ultrastructural localization reveals that the 140-kDa antigen is localized not only along the intercellular area of the desmosome but also is found along the whole epidermal cell surface. The antibodies to the 140-kDa GP are able to induce a disruption of cell-cell contact in cultured keratinocytes that possess desmosomes. We propose that the 140-kDa GP is a cell adhesion molecule (CAM). Furthermore we discuss the heterogeneity of desmosomes in the light of our findings that antibodies against the 140-kDa GP recognize specific stratified squamous epithelial tissues.
Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: comparison of the individual contributions of the F and G glycoproteins to host immunity.
Authors: Olmsted R A , Elango N , Prince G A , Murphy B R , Johnson P R , Moss B , Chanock R M , Collins P L
Published: May 2006

A cDNA clone representing the mRNA coding sequence of the fusion glycoprotein (F) gene of human respiratory syncytial virus (RSV) was constructed and inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of a vaccinia virus promoter. The resulting recombinant vaccinia virus, vaccinia F, expressed the F1 and F2 cleavage products (48 and 20 kDa, respectively) of the F glycoprotein in cell culture. F1 and F2 were indistinguishable from their authentic RSV counterparts with respect to glycosylation, disulfide linkage, electrophoretic mobility, cell-surface expression, and antigenic specificity. Cotton rats infected intradermally with vaccinia F developed a high titer of serum F-specific antibodies, which neutralized infectivity of RSV. This neutralizing antibody response exceeded that induced by infection of the respiratory tract with RSV and was 6-fold higher than that induced by vaccinia G, a recombinant vaccinia virus that expressed the RSV G glycoprotein gene. Immunization with vaccinia F stimulated almost complete resistance to replication of RSV in the lower respiratory tract as well as significant resistance in the upper respiratory tract. The degree of resistance conferred by vaccinia F exceeded that induced by vaccinia G.
Survival, organization, and function of microcarrier-attached hepatocytes transplanted in rats.
Authors: Demetriou A A , Levenson S M , Novikoff P M , Novikoff A B , Chowdhury N R , Whiting J , Reisner A , Chowdhury J R
Published: May 2006

Hepatocytes harvested by collagenase perfusion of rat liver were attached to collagen-coated microcarriers and injected intraperitoneally into congeneic or allogeneic bilirubin-UDP-glucuronosyltransferase (EC 2.4.1.17)-deficient (Gunn) rats or allogeneic analbuminemic (NAR) rats. Five days later, the microcarriers were observed to have formed conglomerates chiefly on the anterior surface of the pancreas. Scanning electron microscopy showed hepatocytes attached to the granular collagen-coated surface of the microcarriers and newly formed connective tissue. Light microscopy revealed that the microcarriers formed a lattice with the collagen tissue; hepatocytes were seen within this lattice or on the surface of the microcarriers. Hepatocyte plasma membranes were nucleoside-diphosphatase (NDPase)-positive. Newly formed blood islands, blood vessels containing erythrocytes and leukocytes and NDPase-positive endothelium were observed in close proximity to the hepatocytes and fibroblasts. Transmission electron microscopic examination showed hepatocytes with microvilli and nucleoid-containing peroxisomes with catalase activity. Hepatocytes were present for up to 2 months in congeneic recipients, the longest period of observation after transplantation. After normal microcarrier-attached hepatocytes were transplanted into allogeneic Gunn rats, bilirubin glucuronides were present in bile for 6 days. When congeneic Gunn rat recipients were used, bilirubin glucuronides were present in bile throughout the study (28 days); this was accompanied by reduction of serum bilirubin concentrations to nearly normal levels. After injection of normal hepatocytes into allogeneic NAR rats, plasma albumin concentration progressively increased for 6 days and then declined. In NAR recipients which were immunosuppressed with cyclosporin A, peak plasma albumin levels were reached in 14 days and persisted nearly at that level throughout the study (28 days).
On the automorphism group of the conformal superalgebra
Authors: Sternberg Shlomo
Published: May 2006

There is no automorphism of the conformal superalgebra that induces PT (parity and time reversal) on space time. Those conformal transformations that induce PT must change the sign of the odd brackets. In particular, in any implementation of conformal supersymmetry, CP can preserve but CPT must (at best) reverse the sign of the odd brackets (C, charge conjugation).
Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator.
Authors: Ebright R H
Published: May 2006

Glutamine-18 of the lac repressor (lacR) has been substituted by glycine, by serine, and by leucine. The specificities of wild-type lacR and of the three substituted lacR variants have been analyzed with respect to base pairs 5, 6, 7, 8, 9, and 10 of the lac operator (lacO). The data indicate that [Gly18]lacR, [Ser18]lacR, and [Leu18]lacR lose the ability to distinguish between the O+ base pair G . C and the Oc base pairs T . A and A . T at position 7 of lacO (KdOc/KdO+ approximately equal to 1). In contrast, the three substituted variants retain the ability to discriminate O+ from Oc at each other position, by factors of 9 to 37. Therefore, I propose that glutamine-18 contacts base pair 7 of lacO. These data suggest that the interaction between the helix-turn-helix motif and DNA may be very similar or identical in lacR and the catabolite gene activator protein.
The mitotic stability of deletion derivatives of chromosome III in yeast.
Authors: Surosky R T , Newlon C S , Tye B K
Published: May 2006

We have constructed a series of deletion derivatives of chromosome III in yeast. Two telocentric chromosomes, one with a deletion of about 100 kilobases (kb) from the left arm and another with a deletion of about 240 kb from the right arm, are mitotically stable, showing only a 2- to 3-fold decrease in stability compared to a normal chromosome III. Chromosomes as small as 100 kb with deletions on both the left and right arms show only slight decreases in mitotic stability. Slight decreases in size in chromosomes smaller than 100 kb produce dramatic decreases in mitotic stability. In general, deletion chromosomes of similar size but different structure display similar stabilities. We find no evidence for the existence of any new cis-acting elements [besides the centromere, autonomously replicating sequences (ARS elements) and telomeres] essential for the stabilization of chromosome III.
Immunoglobulin J chain gene from the mouse.
Authors: Matsuuchi L , Cann G M , Koshland M E
Published: May 2006

During a primary immune response, murine B lymphocytes are induced to express the gene for the immunoglobulin J chain. As a first step in determining the mechanism of induction, genomic DNA clones encoding the murine J chain were obtained from cell lines representative of B lymphocytes before and after J chain expression. Analysis of the coding regions showed that the J chain gene has a different structure from the other immunoglobulin genes. It consists of four exons organized in a simple 7.3-kilobase transcription unit that does not require DNA rearrangement or alternative processing for expression. These structural properties indicate that transcription of the J chain gene is initiated by changes in chromatin conformation, probably involving a J chain-specific DNA-binding factor. Analysis of the 5' flanking sequences of the J chain gene, on the other hand, showed that the promoter region contains two conserved elements that have been implicated in the lymphocyte-specific expression of the light chain genes. The sharing of these elements suggests that, once the J chain gene is activated, its transcription is regulated by mechanisms similar to those controlling the light chain genes.