bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of exocyclic substituents of purine bases on DNA curvature.
Authors: Diekmann S , von Kitzing E , McLaughlin L , Ott J , Eckstein F
Published: May 2006

Complementary oligonucleotides with 5' overhanging deoxyguanosine or deoxycytidine stretches, respectively, of the general form 5'-d(GGGCAARAAC).5'-d(CCCGTTYTTG), where R represents the bases adenine (A), hypoxanthine (base of inosine nucleoside, I), purine (R), 2-aminopurine (n2R), or 2,6-diaminopurine (n2,6(2)R) and where Y represents the pyrimidine bases thymine (T) or cytosine (C), have been chemically synthesized. After hybridization of complementary fragments, they were ligated to form multimers and analyzed by polyacrylamide gel electrophoresis. Anomalous gel migration was observed for the sequences 5'-d(AARAA) when the R.Y base pair was dA.dT, dI.dC, or dR.dT. All of these base pairs lack at least the amino group at position 2 of the purine base. The degree of anomalous gel migration was also related to the substituent at position 6 of the purine base. An amino group at position 6 was more effective than a carbonyl or a hydrogen in inducing anomalous gel migration. Additionally, the fragments 5'-d(GGGCAIAIAC).5'-d(CCCGTCTCTG), 5'-d(GGGCAIIIAC).5'-d(CCCGTCCCTG), and 5'-d(GGGCIIAIIC).5'-d(CCCGCCTCCG) were prepared in which increasing numbers of dA.dT base pairs are replaced by dI.dC base pairs. The degree of gel-migration anomaly of these sequences correlates with the number of dA.dT base pairs left in the five-base purine block. The data support the hypothesis that within the deoxyadenosine tracts, the base pairs fold into the minor groove at position 2 of the base to balance for the NH2 groups at position 6. This hypothesis explains the formation of a B'-form DNA structure for the deoxyadenosine tracts as well as DNA curvature.
Isolation and sequence of a cDNA corresponding to a src-related gene expressed in murine hemopoietic cells.
Authors: Holtzman D A , Cook W D , Dunn A R
Published: May 2006

We have isolated a murine cDNA that shares extensive homology with genes encoding the src (Rous sarcoma virus oncogene)-related family of protein-tyrosine kinases. The cDNA includes an open reading frame of 1509 base pairs, and conceptual translation predicts a protein of 56 kDa. Blot-hybridization analysis indicates that this src-related gene is expressed in normal macrophages and in cell lines representing both the myeloid and lymphoid B-cell lineages and, accordingly, is designated "bmk" (B cell/myeloid kinase). In addition, bmk mRNA levels increase following the induced differentiation of the murine myelomonocytic leukemic cell line WEHI-3B.
Identification of interferon-modulated proliferation-related cDNA sequences.
Authors: Kulesh D A , Clive D R , Zarlenga D S , Greene J J
Published: May 2006

To identify genes mediating the antiproliferative action of interferon (IFN), two cDNA libraries were constructed with mRNA from IFN-treated and untreated human fibrosarcoma (HT1080) cells previously shown to be highly sensitive to the antiproliferative effects of IFN. Differential screening of these two libraries identified cloned sequences whose expression was either induced or repressed with IFN treatment. Rescreening of these sequences with cDNA probes constructed from proliferating or quiescent cells led to the identification of one IFN-induced and three IFN-repressed sequences whose expressions also appeared to be modulated by cell proliferation. Blot-hybridization analysis revealed that RNA levels corresponding to the three repressed genes decreased when HT1080 cells were treated with IFN or when proliferation of normal CUA foreskin fibroblast cells became naturally arrested by contact inhibition. Levels of RNA corresponding to the induced gene increased in HT1080 cells within 24 hr after IFN-treatment but declined below basal levels by 48 hr. Expression of these genes was unaffected or only slightly affected by IFN treatment in variant cells resistant to the antiproliferative effects of IFN. Collectively, these results suggest that the identified cDNAs correspond to genes that are involved in the antiproliferative action of IFN. Moreover, these results also suggest that IFN's antiproliferative action may be exerted through genes that contribute to arresting cell proliferation during contact inhibition.
Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading.
Authors: Fechheimer M , Boylan J F , Parker S , Sisken J E , Patel G L , Zimmer S G
Published: May 2006

Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection of mammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to utilize the salvage pathway of pyrimidine biosynthesis. These loading methods can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and stably. Optimal transfection is observed when the DNA is added to cells in physiological saline lacking divalent cations and containing K+ in place of Na+. DNA molecules 7.1 to 30 kilobases long have been introduced by the scrape loading procedure. In addition, the scrape loading procedure has been employed for cotransfection and subsequent expression of nonselectable genes encoded on DNA molecules added in a mixture with DNA molecules whose expression is selected. Cell lines expressing oncogenes or proteins that are important for regulation of cell growth and division have been obtained by this procedure. The scrape loading procedure is also useful for studies of the cellular changes that occur upon expression of an exogenous gene. As many as 80% of cells scrape loaded with the plasmid pC6, which encodes the simian virus 40 large tumor antigen, contained this protein in the nucleus between 1 and 5 days after transfection. Thus, scrape loading and sonication loading are simple, economical, and reproducible methods for introduction of DNA molecules into adherent and nonadherent cells, and these methods may be useful in the future for experimentation at both fundamental and applied levels.
Zinc (II) and the single-stranded DNA binding protein of bacteriophage T4.
Authors: Gauss P , Krassa K B , McPheeters D S , Nelson M A , Gold L
Published: May 2006

The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single "zinc-finger" sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. We have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. Our results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression.
Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti-peptide antibodies with a protein alpha-helix.
Authors: Fieser T M , Tainer J A , Geysen H M , Houghten R A , Lerner R A
Published: May 2006

Monoclonal antibodies against an alpha-helical region of the iron-containing, oxygen-binding protein myohemerythrin were isolated following immunization of mice with either the whole protein or a peptide homolog of the helix. Three distinct epitopes within the myohemerythrin helix were identified. The individual residues within two of these epitopes that were essential for antibody binding were determined by measuring antibody binding to a set of peptides in which each amino acid of the epitope was replaced in turn by each of the other 19 amino acids. Hydrophilic residues that are exposed in the native conformation and buried, hydrophobic residues were both shown to be irreplaceable, suggesting their direct involvement in antibody binding. The influence of antigen conformation on antibody binding to these amphipathic epitopes was assessed by measuring the relative affinities of the antibodies for peptides, intact protein, and apoprotein. All of the antibodies bound to apoprotein better than to native protein, indicating that relaxation of the native structure by removal of the iron center increases antibody affinity for myohemerythrin. However, not all of the antibodies tested bound to peptides better than to protein, suggesting that increased antigen flexibility is not always sufficient to maximize antibody binding. Antibody binding to peptides appeared to also be influenced by the ability of the peptides to attain secondary structure at the epitopes, either alone or due to carrier influences.
Monoclonal antibody identification of subpopulations of cerebral cortical neurons affected in Alzheimer disease.
Authors: Miller C A , Rudnicka M , Hinton D R , Blanks J C , Kozlowski M
Published: May 2006

Neuronal degeneration is one of the hallmarks of Alzheimer disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, we have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mABs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAB 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1, to the best of our knowledge, do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands on immunoblots of homogenates of normal and AD cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible.
Tissue-specific expression of the RI and RII sodium channel subtypes.
Authors: Gordon D , Merrick D , Auld V , Dunn R , Goldin A L , Davidson N , Catterall W A
Published: May 2006

Anti-peptide antibodies that distinguish between the rat brain sodium channel subtypes referred to as RI and RII were prepared and used to determine their relative expression in nerve and muscle tissues. Sodium channels purified from rat brain are approximately 18% RI and 80% RII. In brain, the RII subtype is preferentially expressed with RI/RII ratios ranging from 0.07 in the hippocampus to 0.17 in the cerebral cortex. The RI subtype is preferentially expressed in more caudal areas of the central nervous system with values of RI/RII of 0.98 for medulla oblongata and 2.2 for spinal cord. Expression of additional unidentified sodium channel subtype(s) is detected in midbrain, medulla, and spinal cord, and expression of unidentified sodium channel subtypes predominates over expression of RI and RII in retina and optic nerve. The RI and RII subtypes are primarily expressed in the central nervous system and are not detected in significant numbers in skeletal or cardiac muscle, sympathetic ganglia, adrenal medulla, sciatic nerve, or cauda equina. The RII subtype appears first in development of both brain and spinal cord but declines in adult spinal cord as the RI subtype increases. The strict regional expression of these two sodium channel subtypes suggests that they may have distinct functional properties or physiological roles.
Immunohistochemical localization of a neuronal nicotinic acetylcholine receptor in mammalian brain.
Authors: Deutch A Y , Holliday J , Roth R H , Chun L L , Hawrot E
Published: May 2006

A monoclonal antibody generated against purified acetylcholine receptor from Torpedo electric organ was used to immunohistochemically localize a neuronal nicotinic acetylcholine receptor. Regions of the rat brain stained with this antibody paralleled those areas of the brain exhibiting [3H]nicotine binding sites and corresponded to areas in which mRNAs encoding for alpha subunits of the neuronal nicotinic acetylcholine receptor are present. Thus, the anteroventral thalamus, cortex, hippocampus, medial habenula, interpeduncular nucleus, and substantia nigra/ventral tegmental area exhibited significant immunoreactivity. Neurons of the medial habenula and substantia nigra were densely stained, and processes were prominently delineated. Furthermore, in the projection areas of the medial habenula (interpeduncular nucleus and median raphe) axons were strongly immunoreactive and were distributed to distinct subdivisions of the target sites. The present data suggest that there are several discrete neuronal systems in which nicotinic acetylcholine receptors have functional importance. These immunohistochemical studies delineate at the single-cell level the localization within the mammalian central nervous system of certain nicotinic acetylcholine receptors.
Modulation of sodium-channel mRNA levels in rat skeletal muscle.
Authors: Cooperman S S , Grubman S A , Barchi R L , Goodman R H , Mandel G
Published: May 2006

Action potentials in many types of excitable cells result from changes in permeability to Na ions. Although these permeability changes in nerve and muscle are mediated by voltage-gated Na channels that are functionally similar, we found that the Na-channel gene expressed in skeletal muscle is different from the genes coding for two Na channels (type I and type II) in brain. Despite the structural differences between muscle and brain Na-channel genes, a cDNA clone derived from rat brain hybridizes to skeletal muscle Na-channel mRNA of approximately 9.5 kilobases. We used this cDNA probe to measure changes in Na-channel mRNA levels in skeletal muscle during development and following denervation. By blot hybridization analysis of electrophoretically fractionated RNA, we found that Na-channel mRNA can be detected as early as embryonic day 17 and that mRNA levels increase 2-fold between birth and postnatal day 35. Denervation of adult muscle causes a further 2- to 3-fold increase in muscle Na-channel mRNA levels, suggesting that expression of Na-channel genes in fast-twitch muscle may be regulated by the state of innervation.
Linear mechanisms of directional selectivity in simple cells of cat striate cortex.
Authors: Reid R C , Soodak R E , Shapley R M
Published: May 2006

The role of linear spatial summation in the directional selectivity of simple cells in cat striate cortex was investigated. The experimental paradigm consisted of comparing the response to drifting grating stimuli with linear predictions based on the response to stationary contrast-reversing gratings. The spatial phase dependence of the response to contrast-reversing gratings was consistent with a high degree of linearity of spatial summation within the receptive fields. Furthermore, the preferred direction predicted from the response to stationary gratings generally agreed with the measurements made with drifting gratings. The amount of directional selectivity predicted was, on average, about half the measured value, indicating that nonlinear mechanisms act in concert with linear mechanisms in determining the overall directional selectivity.
Regulation of the protein glycosylation pathway in yeast: structural control of N-linked oligosaccharide elongation.
Authors: Gopal P K , Ballou C E
Published: May 2006

The yeast Saccharomyces cerevisiae X2180 strain with the mnn1 mnn2 mnn9 mutations, all of which affect mannoprotein glycosylation, synthesizes N-linked oligosaccharides having the following structure: (Formula: see text) whereas the mnn1 mnn2 mutant extends the alpha 1----6-linked backbone of some of the core oligosaccharides by adding 20-30 mannose units. Membrane fractions from the mnn1 mnn2 and mnn1 mnn2 mnn9 mutants are equally effective in catalyzing transfer from GDP-[3H]mannose to add mannose in both alpha 1----2 and alpha 1----6 linkages to an oligosaccharide having the following structure: (Formula: see text) but neither membrane preparation can utilize the homologous mnn1 mnn2 mnn9 oligosaccharide as an acceptor. Thus, addition of the alpha 1----2-linked mannose side chain to the terminal alpha 1----6-linked mannose in oligosaccharides of the mnn9 mutant inhibits the elongation reaction and may serve as an important structural control of mannoprotein glycosylation. The mnn9 mutation also increases the transit time for invertase secretion, meaning that this mutation could affect the processing machinery in the Golgi apparatus.
Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae.
Authors: Lue N F , Kornberg R D
Published: May 2006

A yeast nuclear extract supported transcription from the CYC1 and PYK1 promoters. Transcription was initiated in vitro at or near sites used in vivo. Deletion of "TATA" sequences abolished the reaction. alpha-Amanitin (10 micrograms/ml) and chloride (100 mM) were highly inhibitory.
Heat-labile regulatory factor is required for 3' processing of histone precursor mRNAs.
Authors: Gick O , Krämer A , Vasserot A , Birnstiel M L
Published: May 2006

In addition to Sm antigen-type small nuclear ribonucleoprotein particle(s) [snRNP(s)], at least one more factor is involved in the in vitro 3' processing of histone precursor mRNAs (pre-mRNAs) in a HeLa cell nuclear extract. This factor can be completely inactivated by mild heat treatment but is resistant to digestion by micrococcal nuclease and is not immunoprecipitated by antisera of the Sm serotype. Both snRNP (the presumed human homologue of the U7 snRNP of the sea urchin) and the heat-labile factor described above show closely similar properties when fractionated on DEAE, heparin, and Mono Q columns. Fractions, after extensive purification, still contain both heat-labile factor and snRNP activity. When analyzed by gel filtration, the heat-labile component distributes bimodally, the smaller component possessing an apparent molecular weight on the order of 40,000, and the larger, of ca. 300,000.
High-level expression of sperm whale myoglobin in Escherichia coli.
Authors: Springer B A , Sligar S G
Published: May 2006

Sperm whale myoglobin was expressed in Escherichia coli from a totally synthetic gene inserted in the expression vector pUC19. The gene was constructed as 23 overlapping oligonucleotides encoding both strands of the DNA. Gene synthesis provides several advantages over traditional eukaryotic gene-cloning techniques, allowing the incorporation of an efficient ribosome binding site, appropriate initiation and termination sequences, restriction enzyme sites for convenient subcloning and future mutagenesis, and frequently used codons for highly expressed E. coli genes. The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coli. We credit the use of frequently used E. coli codons for the observed high-level expression. The sperm whale myoglobin produced is stable, easily purified to homogeneity, and indistinguishable from commercially available sperm whale myoglobin by optical and magnetic spectroscopic methods.
Human immunoglobulin C lambda 6 gene encodes the Kern+Oz-lambda chain and C lambda 4 and C lambda 5 are pseudogenes.
Authors: Dariavach P , Lefranc G , Lefranc M P
Published: May 2006

Six nonallelic immunoglobulin lambda constant region genes have been previously characterized on a 40-kilobase stretch of DNA. The nucleotide sequences of the three upstream genes of this cluster (C lambda 1, C lambda 2, C lambda 3) have been determined by other workers and shown to encode, respectively, the isotypic Mcg, Kern-Oz-, and Kern-Oz+ constant region of the lambda chains. In this paper, we report the sequence of the three downstream genes of this cluster and show that two of them (C lambda 4 and C lambda 5) are pseudogenes. However, C lambda 6 encodes a Kern+Oz- chain and corresponds to the fourth isotype described among the lambda proteins sequenced so far. A potentially active J lambda (joining) segment, with the canonical heptamer and nonamer sequences for rearrangement, is located 1.5 kilobases upstream of C lambda 6. The amino acid sequence encoded by the C lambda 6 gene is compared with the constant region sequences of various monoclonal Bence Jones lambda proteins. Allotypic and isotypic differences confirm the polymorphism and complexity of the human C lambda locus.
Induction of protective immunity against rabies by immunization with rabies virus ribonucleoprotein.
Authors: Dietzschold B , Wang H H , Rupprecht C E , Celis E , Tollis M , Ertl H , Heber-Katz E , Koprowski H
Published: May 2006

We have studied the ability of rabies virus ribonucleoprotein (RNP) to induce a protective immune response in animals against lethal challenge with rabies and rabies-related lyssa viruses. Liposomes containing either RNP or the glycoprotein (G protein) of a variant virus with multiple alterations in the G antigenic structure conferred no or poor protection, respectively, against lethal intracerebral challenge with rabies virus. By contrast, liposomes containing RNP and the variant G protein induced a good protective response, comparable to that achieved with inactivated virus vaccine against intracerebral challenge. Moreover, mice or raccoons immunized with RNP alone resisted lethal peripheral challenge with homologous or heterologous virus strains. These results indicate that the RNP of rabies virus plays a crucial role in induction of protective immunity.
Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c.
Authors: Martin L H , Calabi F , Lefebvre F A , Bilsland C A , Milstein C
Published: May 2006

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have now been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure of CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the beta 2-microglobulin-binding domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.
Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines.
Authors: Yoshimura T , Matsushima K , Tanaka S , Robinson E A , Appella E , Oppenheim J J , Leonard E J
Published: May 2006

Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue injury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.
Cloning of a cDNA for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis.
Authors: Bonifas J M , Morley B J , Oakey R E , Kan Y W , Epstein E H
Published: May 2006

A human steroid sulfatase (steryl-sulfatase; steryl-sulfate sulfohydrolase, EC 3.1.6.2) cDNA 2.4 kilobases long was isolated from a human placental lambda gt11 cDNA expression library. The library was screened with monospecific rabbit antibodies elicited by injection of steroid sulfatase protein purified from human placentas. Hybridization of the cDNA with EcoRI-digested genomic DNA indicated that patients from 14 of 15 apparently unrelated families have gross deletions of the gene for steroid sulfatase. One patient had genomic DNA fragments that were identical to those from normal individuals, indicating the absence of any major deletions as the cause of his lack of steroid sulfatase enzyme activity.
Expression and rearrangement of the ROS1 gene in human glioblastoma cells.
Authors: Birchmeier C , Sharma S , Wigler M
Published: May 2006

The human ROS1 gene, which possibly encodes a growth factor receptor, was found to be expressed in human tumor cell lines. In a survey of 45 different human cell lines, we found ROS1 to be expressed in glioblastoma-derived cell lines at high levels and not to be expressed at all, or expressed at very low levels, in the remaining cell lines. The ROS1 gene was present in normal copy numbers in all cell lines that expressed the gene. However, in one particular glioblastoma line, we detected a potentially activating mutation at the ROS1 locus.
The structural characterization of endogenous factors from Vinca rosea crown gall tumors that promote cell division of tobacco cells.
Authors: Lynn D G , Chen R H , Manning K S , Wood H N
Published: May 2006

The ability of two compounds, a cytokinin and an auxin, to stimulate tobacco cell growth and differentiation has been known for greater than 30 years, but the molecular mechanism of this activation is still unknown. Previous reports of factors endogenous in crown gall tumors of Vinca rosea that could replace the cytokinin requirement in tobacco cell culture has motivated an investigation of these tissues. The optimization of a reverse-phase isolation scheme has led to the purification of sufficient material to allow for the identification of six different related compounds. The structures of two of these compounds have been assigned as a set of epimeric dehydrodiconiferyl alcohol beta-D-glycosides. The structure of these compounds suggests that they would most likely be derived from the plant cell wall.
On certain Hecke rings
Authors: Evens Sam , Bressler Paul
Published: May 2006

We examine rings that embed into the smash product of the group algebra of the Weyl group with the field of meromorphic functions on the Cartan subalgebra and are generated by elements that satisfy braid relations. We prove that every such ring is isomorphic to either the Hecke algebra, the nil Hecke ring, or the group algebra of the Weyl group.
Nucleotide sequence of Bacillus subtilis dnaB: a gene essential for DNA replication initiation and membrane attachment.
Authors: Hoshino T , McKenzie T , Schmidt S , Tanaka T , Sueoka N
Published: May 2006

The complete nucleotide sequence of the Bacillus subtilis dnaB gene and its flanking regions was determined. The dnaB gene is essential for both replication initiation and membrane attachment of the origin region of the chromosome and plasmid pUB110. It has been known that there are two different classes (dnaBI and dnaBII) in the dnaB mutants; dnaBI is essential for both chromosome and pUB110 replication, whereas dnaBII is necessary only for chromosome replication. The nucleotide sequence revealed that dnaBI and dnaBII are two functional domains in the single dnaB gene. The mutation sites of two mutants, belonging to dnaBI and dnaBII, respectively, were also determined as substitutions of amino acids. The putative DnaB protein deduced from nucleotide sequence consists of 472 amino acids (55 kDa) with no cysteine residue. A 55-kDa polypeptide produced in an in vitro transcription-translation system was labeled with [35S]methionine but not with [35S]cysteine. The DnaB protein has a highly hydrophobic sequence of 20 amino acids in its N-terminal region, a possible DNA binding site, and two possible ATP binding sites. The dnaBI domain is between the DNA binding site and one of the ATP binding sites; the dnaBII domain is close to the other ATP binding site. Comparison of the amino acid sequence between the "dnaB protein" and those of other dna genes of Escherichia coli showed no homology, suggesting that the dnaB gene of B. subtilis may be analogous to a hitherto undiscovered gene in E. coli.
Rat gene encoding the 78-kDa glucose-regulated protein GRP78: its regulatory sequences and the effect of protein glycosylation on its expression.
Authors: Chang S C , Wooden S K , Nakaki T , Kim Y K , Lin A Y , Kung L , Attenello J W , Lee A S
Published: May 2006

The 78-kDa glucose-regulated protein GRP78 is a stress-inducible protein ubiquitously expressed in animal cells. In this paper we show that the first exon of this endoplasmic reticulum-localized protein consists of an 18 amino acid leader sequence rich in hydrophobic residues, followed by a highly acidic mature N-terminus and an 11 amino acid domain that is shared by members of the 70-kDa heat shock protein family. The end of this shared domain also marks the beginning of the first intron of this gene. A DNA region upstream of the promoter element important for induction by calcium ionophore and by a temperature-sensitive mutation was identified by deletion analysis. Our results indicate that a region spanning from 85 to 480 nucleotides upstream of the major transcription initiation site is important for both induction conditions. With evidence suggesting that perturbations in protein glycosylation may be one of the common stimuli involved in transcription activation of the GRPs, we measured the rate of glycosylation during A23187, glucose starvation, and temperature-shift induced conditions. The inverse correlation observed between the rate of glycosylation and the steady-state level of the GRP78 transcripts lends support to this hypothesis.
Epstein-Barr virus-containing B-cell line produces an interleukin 1 that it uses as a growth factor.
Authors: Wakasugi H , Rimsky L , Mahe Y , Kamel A M , Fradelizi D , Tursz T , Bertoglio J
Published: May 2006

We report the establishment of a spontaneous interleukin 1 (IL-1)-producing subclone derived from the human Epstein-Barr virus (EBV)-containing B-lymphoblastoid cell line (721 LCL) and show that the IL-1 produced by this B-cell subclone is distinct from other types of IL-1. The parental cell line 84.5, a deletion mutant of the 721 LCL cell line, can be induced to produce IL-1 activity when stimulated by certain inducers such as phorbol 12-myristate 13-acetate in the presence of fetal calf serum. From this parental 721/84.5 clone, a subclone, termed 3B6, has been developed. This 3B6 subclone has an immature B-cell phenotype, expresses only HLA class II DP subregion antigens, and spontaneously releases IL-1 in the culture supernatant with relatively few inhibitory molecules under serum-free culture conditions. The 3B6-derived IL-1 was purified from 3B6 conditioned medium with a three-step procedure. The molecular weight of this IL-1 is 13,500, and the isoelectric point values are pH 4.9 and 5.1 without any component focusing near pH 7. The N-terminal amino acid sequence differs markedly from those reported for the two IL-1 species produced by monocytes. The purified material shares several biological properties with monocyte IL-1, since it could induce the proliferation of murine thymocytes, the production of interleukin 2 by phytohemagglutinin-stimulated cloned HSB2 T cells, and the proliferation of human fibroblasts. However, this IL-1 activity could not be blocked by polyclonal anti-monocytic IL-1 antibodies, and, more importantly, it was not pyrogenic in rabbits. Finally, it promotes the growth of B-cell clones derived from the parental 721/84.5 lines in the absence of fetal calf serum, which suggests that it could act as an autocrine growth factor in this Epstein-Barr virus-transformed B-cell line.
Incorporation of a circulating protein into megakaryocyte and platelet granules.
Authors: Handagama P J , George J N , Shuman M A , McEver R P , Bainton D F
Published: May 2006

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.
Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent.
Authors: Manuelidis E E , Fritch W W , Kim J H , Manuelidis L
Published: May 2006

Isolates from six patients with Creutzfeldt-Jakob disease (CJD) were injected into various strains of hamsters and mice, and the infective agent was propagated. Serially passaged cultures were established from these CJD agent-infected brains and from uninfected control brains. All healthy cultures (21 out of 21) from CJD agent-infected brains became immortal and/or transformed. In contrast only 3 out of 13 normal brain cultures became immortal, and the rest died out with serial propagation in vitro. The fact that permanent cell lines were readily derived from multiple rodent strains and all CJD isolates tested suggests that a transforming capability is an intrinsic property of CJD agents. This conclusion is supported by demonstrations of in vitro cell transformation by CJD infectious brain fractions. Although the molecular mechanism of transformation events associated with the CJD agent is not presently known, a provocative possibility is that the CJD agent has a direct effect on the host genome by mechanisms analogous to those known for slowly oncogenic retroviruses.
Treatment of nitrosamine-induced pancreatic tumors in hamsters with analogs of somatostatin and luteinizing hormone-releasing hormone.
Authors: Paz-Bouza J I , Redding T W , Schally A V
Published: May 2006

Pancreatic ductal adenocarcinoma was induced in female Syrian golden hamsters by injecting N-nitrosobis(2-oxopropyl)amine (BOP) once a week at a dose of 10 mg per kg of body weight for 18 weeks. Hamsters were then treated with somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) or with [6-D-tryptophan]luteinizing hormone-releasing hormone [( D-Trp6]LH-RH) delayed delivery systems. Microcapsules of somatostatin analog RC-160, designed to release a dose of 5 micrograms/day, were injected twice a month and microcapsules of [D-Trp6]LH-RH, calculated to liberate 25 micrograms per day, once a month. After 18 weeks of BOP administration, the hamsters were divided into three groups of 10-20 animals each. Group I consisted of untreated controls, group II was injected with RC-160, and group III was injected with [D-Trp6]LH-RH. A striking decrease in tumor weight and volume was obtained in animals treated with [D-Trp6]LH-RH or with the somatostatin analog RC-160. After 45 days of treatment with either analog, the survival rate was significantly higher in groups II and III (70%), as compared with the control group (35%). The studies, done by light microscopy, high-resolution microscopy, and electron microscopy, showed a decrease in the total number of cancer cells and changes in the epithelium, connective tissue, and cellular organelles in groups II and III treated with the hypothalamic analogs as compared to controls. These results in female hamsters with induced ductal pancreatic tumors confirm and extend our findings, obtained in male animals with transplanted tumors, that [D-Trp6]LH-RH and somatostatin analogs inhibit the growth of pancreatic cancers.
Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons.
Authors: Kincaid R L , Balaban C D , Billingsley M L
Published: May 2006

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.
Gene encoding cytoskeletal proteins in Drosophila rhabdomeres.
Authors: Matsumoto H , Isono K , Pye Q , Pak W L
Published: May 2006

The ninaC gene is one of eight nina (neither inactivation nor afterpotential) genes identified from mutations that drastically reduce the amount of rhodopsin in the compound eye of Drosophila melanogaster. The gene has been cytogenetically localized to the 27E-28B region of the second chromosome. NaDodSO4/PAGE analysis of eye proteins of flies carrying one, two, or three copies of the ninaC region shows that two eye-specific proteins of molecular weight 170,000 and 130,000 display a strong dependence on the dosage of the ninaC gene, although the dependence is evident only when the dosage is decreased and not when it is increased. All mutations in the ninaC gene studied to date have pronounced effects on these two polypeptides. These results suggest that the ninaC locus encodes these two polypeptides. Ultrastructural studies show that the polypeptides encoded by ninaC are very likely to be important components of the cytoskeletal structure of rhabdomeral microvilli.
Effect of elastin peptides on ion fluxes in mononuclear cells, fibroblasts, and smooth muscle cells.
Authors: Jacob M P , Fülöp T , Foris G , Robert L
Published: May 2006

Elastin peptides prepared by alcoholic potassium hydroxide degradation of highly purified fibrous elastin from bovine ligamentum nuchae (kappa-elastin) were shown to act on the ion channels of human monocytes, aorta smooth muscle cells, and skin fibroblasts. In small amounts (between 0.1 and 1 microgram/ml), elastin peptides strongly increased calcium influx and inhibited calcium efflux by an apparently calmodulin-dependent mechanism. They also were shown to increase sodium influx and to decrease rubidium influx in monocyte preparations obtained from human blood. Only the ouabain-sensitive portion of rubidium influx was inhibited. The action of elastin peptides is strongly concentration-dependent; the maximal activity observed in the above reactions was less than 1 microgram/ml. These results suggest that elastin peptides may play a role in the regulation of the biological activity of mesenchymal cells, in the proximity of which they are released by the action of elastase-type enzymes. Such enzymes were demonstrated in aorta smooth muscle cells (membrane-bound serine protease) and in fibroblasts (metalloprotease). Monocytes and polymorphonuclear leukocytes were also shown to carry elastase-type enzymes. The release of peptides from elastin by elastase-type enzymes and the action of such peptides on the ion fluxes through the cell membrane may well be involved in mechanisms of the modulation of the phenotype of mesenchymal cells during aging as well as in the development of age-dependent pathologies such as arterioclerosis.
Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.
Authors: Rajavashisth T B , Eng R , Shadduck R K , Waheed A , Ben-Avram C M , Shively J E , Lusis A J
Published: May 2006

Macrophage colony-stimulating factor (CSF-1) stimulates the production of macrophages from bone marrow progenitor cells. We have identified a cDNA clone for murine CSF-1 by antibody screening of a mouse L-cell cDNA library in the expression vector lambda gt11. A screen of about 150,000 recombinant plaques yielded 6 clones that reacted well with an antibody raised against denatured and reduced mouse L-cell CSF-1. These clones were further screened with synthetic oligonucleotides based on the amino-terminal amino acid sequence of CSF-1. One clone, which hybridized to the oligonucleotides, was sequenced and found to contain a single open reading frame. This encompassed 68 amino acids of the mature protein, including the entire amino-terminal sequence we previously reported. This is preceded by what appears to be a 31 amino acid signal peptide. Blot analysis showed that this cDNA hybridizes to a major mRNA species of about 4.5 kilobases (kb) as well as several smaller, less abundant mRNA species (3.8, 2.3, and 1.4 kb) present in mouse L cells. A similar pattern of hybridization was observed with mRNA from a human pancreatic carcinoma cell line that produces CSF-1. Striking differences in the qualitative and quantitative expression of mRNA species for CSF-1 were observed in various mouse tissues. Liver expressed primarily a 1.4-kb species, heart and lung expressed primarily a 4.5-kb species, brain expressed high levels of both the 4.5-kb and 1.4-kb species, and intestine lacked detectable CSF-1 transcripts. Southern blot analysis suggests that the CSF-1 gene is present as a single copy in the mouse haploid genome and that it is not rearranged or amplified in L cells.
Amino acid sequence of rabbit fast-twitch skeletal muscle calsequestrin deduced from cDNA and peptide sequencing.
Authors: Fliegel L , Ohnishi M , Carpenter M R , Khanna V K , Reithmeier R A , MacLennan D H
Published: May 2006

Partial amino acid sequence analysis of rabbit fast-twitch skeletal muscle calsequestrin permitted the construction of synthetic oligonucleotides that were used as both primers and probes for the synthesis and isolation of cDNAs encoding calsequestrin from neonatal rabbit skeletal muscle libraries. The cDNA sequence encodes a processed protein of 367 residues with a Mr of 42,435 and a 28-residue amino-terminal signal sequence. The deduced amino acid sequence agreed closely with the portions of the mature protein that were sequenced using standard protein sequencing. The neonatal protein, however, contains an acidic carboxyl-terminal extension not present in the adult protein, suggesting that the cDNA sequence may have arisen from an alternatively spliced neonatal transcript. A single transcript of 1.9-2.0 kilobases was seen in neonatal skeletal muscle mRNA. A glycosylation site and two potential phosphorylation sites were detected. Although the protein contains about two acidic residues for each Ca2+ bound, there is no repeating distribution of acidic residues and no evidence of EF hand structures. Hydropathy plots show no transmembrane sequences, and structural analyses suggest that less than half of the protein is likely to be highly structured. This sequence defines the characteristics of a class of high-capacity, moderate-affinity, Ca2+ binding proteins.