bluuebunny/crossref_metadata_2025 huggingface dataset:
Agonist-independent activation of acetylcholine receptor channels by protein kinase A phosphorylation.
Authors: Ferrer-Montiel A V , Montal M S , Díaz-Muñoz M , Montal M
Published: May 2006

Protein phosphorylation is a ubiquitous and one of the most effective means of regulating protein activity. Receptor phosphorylation is a key event in signal transduction. The question, therefore, that arises is whether this modulatory mechanism might produce functional changes in a membrane receptor in the absence of its naturally occurring ligand. To examine this issue, single-channel properties of purified acetylcholine receptors (AChRs) from Torpedo californica reconstituted in lipid bilayers were studied in the absence of ACh in both unphosphorylated preparations and after in vitro phosphorylation by a purified catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A). Notably, the spontaneous open-channel probability of phosphorylated AChRs is significantly higher than that of unphosphorylated AChRs. Channel activation by protein kinase A is correlated with AChR phosphorylation and is abolished by alpha-bungarotoxin. Analysis of probability distributions of the open dwell times indicates that, similar to unphosphorylated AChR has two distinct open states, short- and long-lived. The frequency of occurrence of the long openings over the short and the magnitude of both time constants increase after phosphorylation, as they do with agonist concentration. Thus, phosphorylation of AChR gamma and delta subunits activates AChR channel opening in the absence of ligand binding. This result is compatible with the notion that protein phosphorylation may effectively act as an intracellular ligand with the phosphorylation sites envisioned as cytoplasmic ligand binding sites.
Synchronized overproduction of neurotransmitter receptors in diverse regions of the primate cerebral cortex.
Authors: Lidow M S , Goldman-Rakic P S , Rakic P
Published: May 2006

A remarkable diversity of neurotransmitter receptors develops concurrently in disparate areas of the primate cerebral cortex. The density of dopaminergic, adrenergic, serotonergic, cholinergic, and GABAergic receptors (where GABA is gamma-aminobutyric acid) in rhesus monkey reaches a maximum level between 2 and 4 months of age and then declines gradually to adult levels in all layers of sensory, motor, and association regions. The synchronized development of neurotransmitter receptors in diverse layers and regions of the neocortex occurs pari passu with synaptogenesis, demonstrating unusual coordination of biochemical and structural maturation and supporting the hypothesis that the entire cerebral cortex matures as an integrated network, rather than as a system-by-system cascade.
Nuclear matrins: identification of the major nuclear matrix proteins.
Authors: Nakayasu H , Berezney R
Published: May 2006

A preparative two-dimensional polyacrylamide gel system was used to separate and purify the major Coomassie blue-stained proteins from the isolated rat liver nuclear matrix. Approximately 12 major proteins were consistently found. Of these, 5 proteins represented identified proteins, including nuclear lamins A, B, and C, the nucleolar protein B-23, and residual components of core heterogeneous nuclear ribonucleoproteins. The remaining eight major proteins termed the nuclear matrins consisted of matrin 3 (125 kDa, slightly acidic), matrin 4 (105 kDa, basic), matrins D-G (60-75 kDa, basic), and matrins 12 and 13 (42-48 kDa, acidic). Peptide mapping and two-dimensional immunoblot studies indicate that matrins D-G compose two pairs of related proteins (matrins D/E and F/G) and that none of the matrins resemble the nuclear lamins or any of the other major proteins detected on our two-dimensional gels. Subfractionation immunoblot experiments demonstrated the nearly exclusive localization of matrins F/G and other matrins to the nuclear matrix fraction of the cell. These results were further supported by indirect immunofluorescence microscopy that showed a strictly interior nuclear localization of the matrins in intact cells in contrast to the peripherally located nuclear lamins. We conclude that the nuclear matrins are a major class of proteins of the nuclear matrix interior and are distinct from the nuclear lamins.
Insulin-like growth factor I shifts from promoting cell division to potentiating maturation during neuronal differentiation.
Authors: Påhlman S , Meyerson G , Lindgren E , Schalling M , Johansson I
Published: May 2006

SH-SY5Y neuroblastoma cells undergo neuronal differentiation and their proliferation is inhibited when they are treated with phorbol 12-myristate 13-acetate (PMA). Insulin and insulin-like growth factor I (IGF-I) are mitogens for the nontreated SH-SY5Y cells, whereas the proliferative response to such factor stimulation is lost upon differentiation, in spite of the fact that the receptors for insulin and IGF-I remain expressed and functional in the differentiated cells. Here we show that the PMA-induced differentiation of SH-SY5Y cells grown in a serum-free medium is strongly potentiated by nanomolar concentrations of IGF-I, as judged by morphology and markers for neuronal differentiation--e.g., neuropeptide tyrosine and growth-associated protein 43. Also, insulin and IGF-II potentiated the phorbol ester-induced differentiation, although less efficiently than IGF-I. Using blocking anti-receptor antibodies, it could be shown that the differentiation induced by these factors, in combination with PMA, was primarily mediated through the IGF-I receptor.
Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli.
Authors: Nagai H , Yuzawa H , Yura T
Published: May 2006

When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion. Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.
Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease.
Authors: Smith C D , Carney J M , Starke-Reed P E , Oliver C N , Stadtman E R , Floyd R A , Markesbery W R
Published: May 2006

The relationship between Alzheimer disease (AD) and aging is not currently known. In this study, postmortem frontal- and occipital-pole brain samples were obtained from 16 subjects with AD, 8 age-matched controls, and 5 young controls. These samples were analyzed both for protein oxidation products (carbonyl) and the activities of two enzymes vulnerable to mixed-function oxidation, glutamine synthetase and creatine kinase. Glutamine synthetase is more sensitive to mixed-function oxidation than creatine kinase. Carbonyl content rises exponentially with age, at double the rate in the frontal pole compared with the occipital pole. Compared with young controls, both aged groups (AD and age-matched controls) have increased carbonyl content and decreased glutamine synthetase and creatine kinase activities, which are more marked in the frontal than occipital pole in all instances. We conclude that protein oxidation products accumulate in the brain and that oxidation-vulnerable enzyme activities decrease with aging in the same regional pattern (frontal more affected than occipital). However, only glutamine synthetase activity distinguishes AD from age-matched controls: Because glutamine synthetase activity is differentially reduced in the frontal pole in AD, we suggest that AD may represent a specific brain vulnerability to age-related oxidation.
Nucleosome positioning is determined by the (H3-H4)2 tetramer.
Authors: Dong F , van Holde K E
Published: May 2006

It is demonstrated that the histone (H3-H4)2 tetramer can find specific positions on DNA, even in the absence of other histones. Purified histone (H3-H4)2 tetramers were reconstituted onto 208-base-pair (bp) DNA molecules containing a nucleosome-positioning sequence by using salt-gradient dialysis. The stoichiometry of histone tetramer to DNA was shown to be 1:1. Digestion with micrococcal nuclease led to formation of protected DNA fragments of approximately 73 bp. Cleavage of the 73-bp DNA with restriction enzymes produced a small set of defined bands, demonstrating positioning of the (H3-H4)2 tetramer on DNA. Analysis of the restriction digests shows that the 73-bp DNA corresponds mainly to two fragments, one lying on either side of the pseudo-dyad axis of the major position adopted by complete histone octamers on this DNA. This result means that a single (H3-H4)2 histone tetramer can fold approximately 146 bp of DNA with the same positioning as the complete octamer but that a region near the pseudo-dyad is only weakly protected against micrococcal nuclease attack in the absence of histones H2A and H2B.
Detailed physical map of human chromosomal region 11q12-13 shows high meiotic recombination rate around the MEN1 locus.
Authors: Janson M , Larsson C , Werelius B , Jones C , Glaser T , Nakamura Y , Jones C P , Nordenskjöld M
Published: May 2006

We have constructed a physical map of the region q12-13 on chromosome 11 by combining data generated from a panel of radiation-reduced somatic cell hybrids and pulsed-field gel electrophoresis (PFGE). Twenty different genetic markers have been sublocalized and ordered within this region and a total of 8.0 megabases has been mapped in detail using rare-cutting restriction endonucleases and PFGE. In two instances, the long-range restriction PFGE map spans the total distance between pairs of loci that have been previously mapped by genetic linkage in reference families. Comparison of this physical map with the available linkage map indicates a great variation in the recombination frequency over the region. The recombination rate is higher than expected, particularly for markers flanking the MEN1 region. Thus, for the closest pair of linked markers on the centromeric side, one centimorgan corresponds to approximately 300 kilobases, and for markers on the telomeric side, one centimorgan corresponds to approximately 350-600 kilobases.
Mutations in p53 as potential molecular markers for human breast cancer.
Authors: Runnebaum I B , Nagarajan M , Bowman M , Soto D , Sukumar S
Published: May 2006

Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors, we investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. We examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell lines tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studies by single-stranded conformation polymorphism analysis. We conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.
Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.
Authors: Wightman R M , Jankowski J A , Kennedy R T , Kawagoe K T , Schroeder T J , Leszczyszyn D J , Near J A , Diliberto E J , Viveros O H
Published: May 2006

Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.
Epstein-Barr nuclear antigen 1 mediates a DNA loop within the latent replication origin of Epstein-Barr virus.
Authors: Frappier L , O'Donnell M
Published: May 2006

Epstein-Barr virus-encoded nuclear antigen 1 (EBNA-1) binds and activates the viral latent origin of DNA replication, oriP. We have used electron microscopy to examine the assembly of EBNA-1 onto oriP. The oriP region consists of two essential elements separated by approximately 1 kilobase pair of DNA. One element contains 20 tandom EBNA-1 binding sites [called the family of repeats (FR)] and serves to activate initiation of replication at the dyad symmetry (DS) element, which contains 4 EBNA-1 binding sites. Titration of homogeneous EBNA-1 produced in baculovirus (bEBNA-1) onto oriP DNA showed an order to the assembly of bEBNA-1 onto oriP. At low concentrations, bEBNA-1 was located exclusively on the FR element. As the level of bEBNA-1 was raised, a loop between the FR and DS elements became the most prevalent DNA-protein complex. These data suggest protein-mediated DNA looping may play a role in activating latent-phase replication of the Epstein-Barr virus.
Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation.
Authors: Stein G H , Drullinger L F , Robetorye R S , Pereira-Smith O M , Smith J R
Published: May 2006

Senescent human diploid fibroblasts (HDF) contain no detectable cdc2 mRNA or p34cdc2 protein. Similarly, young quiescent HDF have only low levels of cdc2 mRNA and protein. After serum stimulation, quiescent HDF accumulate increasing amounts of cdc2 mRNA and protein and go through DNA synthesis and mitosis. In contrast, serum-stimulated senescent HDF fail to accumulate detectable amounts of cdc2 mRNA and protein and fail to enter S phase. Mitosis is likewise deficient in senescent cells even when they have been induced to synthesize DNA by simian virus 40 large tumor antigen. Since p34cdc2 or its homologues appear to be required for DNA synthesis and mitosis in eukaryotes, a lack of these molecules in serum-stimulated senescent HDF could be an important reason for their inability to enter S phase or mitosis. Nuclear microinjection of cdc2 DNA into senescent HDF causes rounding up of the cells but no induction of DNA synthesis. Since cyclins A and B are important cofactors of the protein kinase activity of p34cdc2 or its homologues, we analyzed expression of these genes in serum-stimulated senescent HDF and determined that they contain little or no cycA or cycB mRNA. These deficiencies may be relevant to the lack of DNA synthesis and mitosis in senescent HDF.
Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family.
Authors: Suemori S , Lynch-Devaney K , Podolsky D K
Published: May 2006

The trefoil peptide family encompasses a group of small proteins that appear to assume a distinctive secondary structure that leads to intrinsic resistance to protease digestion. Induction of these peptides has been associated with response to injury in the gastrointestinal tract and related organs. Using an oligonucleotide derived from N-terminal amino acid sequencing of a transformed growth-inhibiting protein, a cDNA was cloned from rat intestinal villus epithelial cells that encodes a protein 81 amino acids in length with the characteristic trefoil peptide cysteine residue motif. Northern blot analysis demonstrates specific expression of a single transcript of 0.43 kilobase in small and large intestinal epithelium in rat and man. Indirect immunofluorescent staining with antiserum raised using a synthetic peptide based on the predicted C-terminal sequence of this protein, designated intestinal trefoil factor, demonstrated that it is primarily expressed and secreted onto the intestinal surface by goblet cells, suggesting that it may be an important component of intrinsic mechanisms for defending mucosal integrity.
Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase.
Authors: Kinouchi H , Epstein C J , Mizui T , Carlson E , Chen S F , Chan P H
Published: May 2006

Oxygen-derived free radicals have been implicated in the pathogenesis of vasogenic edema and infarction caused by ischemia and reperfusion injury. In earlier studies, exogenously supplied liposome-entrapped CuZn superoxide dismutase (CuZn-SOD) ameliorated ischemic brain edema and infarction in rats following focal cerebral ischemia. To ascertain directly the role of SOD in the protection against superoxide radical-induced injury, we measured infarct size and water content 24 hr following focal cerebral ischemia in nontransgenic mice and in transgenic mice bearing the human SOD1 gene. These transgenic mice have 3.1-fold higher cellular CuZn-SOD activity in the brain than do their nontransgenic littermates. We also measured antioxidant levels (reduced glutathione and reduced ascorbate) of contralateral cortex, infarct cortex, surrounding cortex, and striatum. Infarct size and brain edema were significantly decreased in transgenic mice compared with nontransgenic mice. Reduced glutathione and reduced ascorbate levels decreased in the ischemic hemisphere, but levels in surrounding cortex and striatum were significantly higher in transgenic mice than in nontransgenic mice. These results indicate that increased endogenous SOD activity in brain reduces the level of ischemic damage and support the concept that superoxide radicals play an important role in the pathogenesis of infarction and edema following focal cerebral ischemia.
A nondeletional mechanism of peripheral tolerance in T-cell receptor transgenic mice.
Authors: Morahan G , Hoffmann M W , Miller J F
Published: May 2006

To investigate tolerance to extrathymic self molecules, we produced two groups of transgenic mice: one expressed the major histocompatibility complex molecule H-2Kb in pancreatic beta cells, and the other expressed rearranged T-cell receptor genes encoding an anti-H-2Kb receptor. The transgenic T-cell receptor genes were shown to confer the correct specificity and to be expressed appropriately. T cells bearing this receptor were activated by H-2Kb in vitro and in vivo, and they underwent negative selection in mice expressing H-2Kb in the thymus. To determine the fate and function of these anti-H-2Kb T cells in mice expressing H-2Kb exclusively in the periphery, the two groups of transgenic mice were mated to produce double transgenic offspring. In these, transgene-expressing T cells were present in both thymus and periphery. Persisting T cells had not down-regulated either their antigen-specific receptors or their CD8 molecules. Despite the persistence of large numbers of potentially reactive T cells, the mice were tolerant of H-2Kb in that they could not reject H-2Kb-bearing skin grafts, although they did reject third-party grafts. The results show that peripheral T-cell tolerance, unlike that imposed in the thymus, does not involve deletion of T cells. The existence of T cells bearing receptors specific for self components raises the possibility that aberrant activation of such cells may lead to the development of autoimmune disease.
Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy.
Authors: Papahadjopoulos D , Allen T M , Gabizon A , Mayhew E , Matthay K , Huang S K , Lee K D , Woodle M C , Lasic D D , Redemann C
Published: May 2006

The results obtained in this study establish that liposome formulations incorporating a synthetic polyethylene glycol-derivatized phospholipid have a pronounced effect on liposome tissue distribution and can produce a large increase in the pharmacological efficacy of encapsulated antitumor drugs. This effect is substantially greater than that observed previously with conventional liposomes and is associated with a more than 5-fold prolongation of liposome circulation time in blood, a marked decrease in uptake by tissues such as liver and spleen, and a corresponding increased accumulation in implanted tumors. These and other properties described here have expanded considerably the prospects of liposomes as an effective carrier system for a variety of pharmacologically active macromolecules.
Human interferon gamma potently induces the synthesis of a 55-kDa protein (gamma 2) highly homologous to rabbit peptide chain release factor and bovine tryptophanyl-tRNA synthetase.
Authors: Fleckner J , Rasmussen H H , Justesen J
Published: May 2006

An interferon gamma (IFN-gamma)-inducible protein, gamma 2, was identified by two-dimensional gel electrophoresis of transformed human amnion (AMA) cell proteins. cDNA clones coding for this protein have been isolated and characterized as encoding a polypeptide with a predicted molecular weight of 53,165 and a pI of 6.16. Both values are in good agreement with those observed in two-dimensional gel electrophoresis. The gamma 2 protein is found to be highly induced by IFN-gamma, whereas no induction was seen after addition of IFN-alpha to AMA cells. A gamma 2-specific 2.7-kilobase mRNA was likewise seen to accumulate selectively in response to IFN-gamma in these cells. Comparison of the predicted amino acid sequence of gamma 2 to proteins in GenBank data bases revealed that gamma 2 is highly homologous to rabbit peptide chain release factor [Lee, C. C., Craigen, W. J., Muzny, D. M., Harlow, E. & Caskey, C. T. (1990) Proc. Natl. Acad. Sci. USA 87, 3508-3512] and bovine tryptophanyl-tRNA synthetase [M. Garret, V. Trezeguet, B. Pajot, J. C. Gandar, M. Merle, M. Guegiev, J. P. Benedetto, C. Sarger, J. Alteriot, J. La Bouessec, J. Labouesse, and J. Bonnet (1990), GenBank accession no. X52113]. Amino acid sequence similarities of 94% and 97%, respectively, are found, which in general would indicate that gamma 2 represents the human equivalent to either of these two mammalian genes. Based on these sequence similarities, the current data raise the possibility that tryptophanyl-tRNA charging and peptide chain release are carried out by the same enzyme. The gamma 2 protein is shown to possess tryptophan-dependent aminoacyl-tRNA synthetase activity and thus constitutes an enzymatic activity involved in the biological activity of IFN-gamma.
Recombinant fusion protein identified by lepromatous sera mimics native Mycobacterium leprae in T-cell responses across the leprosy spectrum.
Authors: Laal S , Sharma Y D , Prasad H K , Murtaza A , Singh S , Tangri S , Misra R S , Nath I
Published: May 2006

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a lambda gt11 recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of approximately 10 kDa.
In vivo T-cell activation by staphylococcal enterotoxin B prevents outgrowth of a malignant tumor.
Authors: Newell K A , Ellenhorn J D , Bruce D S , Bluestone J A
Published: May 2006

Treatment of T cells with staphylococcal enterotoxins in vitro is known to activate T cells in a subset restricted manner based on beta-chain variable region (V beta) gene expression. In particular, staphylococcal enterotoxin B (SEB) activates T cells bearing V beta 7 or V beta 8. We examined the ability of SEB to activate T cells in vivo. Treatment of C3H mice with doses of SEB ranging from 5 to 250 micrograms resulted in a dose-dependent activation of V beta 8+ T cells as reflected by increased interleukin 2 receptor (IL-2R) expression, proliferation to exogenous IL-2 and allogeneic cells, and production of gamma interferon. SEB also caused proliferation of the CD8+ subset of V beta 8+ cells in vivo. Thus, T-cell activation by SEB in vivo appears to be specific since V beta 2+ cells (non-SEB reactive) did not show increases in IL-2R expression similar to those seen with V beta 8+ cells nor did they proliferate. We then studied the ability of these activated cells to potentiate the immune response to a malignant progressor tumor. Treatment of C3H mice with 50 micrograms of SEB at the time of inoculation with tumor fragments resulted in a statistically significant decrease in the frequency of tumor outgrowth. These data demonstrate that treatment of C3H mice with SEB results in specific activation of V beta 8+ cells in vivo and that these activated cells are capable of preventing the outgrowth of a malignant tumor.
Herpes simplex virus latency-associated transcript is a stable intron.
Authors: Farrell M J , Dobson A T , Feldman L T
Published: May 2006

The latency-associated transcript (LAT) is the major viral transcript detected by in situ hybridization of mouse and human sensory ganglia latently infected with herpes simplex virus type 1. The last 750 bases of LAT are complementary to infected-cell polypeptide 0, a herpes simplex virus type 1 immediate-early gene that encodes a transactivating protein that may facilitate re-activation of the virus from the latent state. Several laboratories have shown that LAT accumulates in the nucleus and is not polyadenylylated. Recently, we showed that the promoter for LAT lies 688 bases upstream from its 5' end. We report here that LAT is actually a uniquely stable intron. Furthermore, LAT effectively inhibits transactivation of gene expression by infected-cell polypeptide 0 in transient transfection assays.
Kinetics of self-assembly of alpha alpha-tropomyosin coiled coils from unfolded chains.
Authors: Mo J M , Holtzer M E , Holtzer A
Published: May 2006

The two-chain coiled-coil structural motif is found in fibrous muscle proteins and leucine zippers. Unfolding/refolding studies abound, but none establishes the time scale or mechanism of structural assembly from separated, unfolded chains. Stopped-flow circular dichroism studies of such refolding of alpha-tropomyosin chains are reported here. The backbone spectral region (222 nm) reveals a fast phase (less than 0.04 s), yielding an intermediate possessing approximately 70% of the equilibrium helix content. A subsequent slow phase is first order [k-1 (20 degrees C) = 1.67 s, Ea = 12.7 kcal.mol-1 (1 kcal = 4.18 kJ)], so dimerization is fast. The same rate constant characterizes folding in the Cys-190 crosslinked chains, so the intermediate has parallel and nearly registered chains. The tyrosine spectral region (280 nm) reveals only a fast phase, so these six chain sites are native in the intermediate.
Cloning of the DNA-binding subunit of human nuclear factor kappa B: the level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor alpha.
Authors: Meyer R , Hatada E N , Hohmann H P , Haiker M , Bartsch C , Röthlisberger U , Lahm H W , Schlaeger E J , van Loon A P , Scheidereit C
Published: May 2006

The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-kappa B binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNF alpha or phorbol ester. Thus, both factors not only activate NF-kappa B protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-kappa B.
Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex.
Authors: Rollins T E , Siciliano S , Kobayashi S , Cianciarulo D N , Bonilla-Argudo V , Collier K , Springer M S
Published: May 2006

We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the alpha and beta subunits of Gi, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm our earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.
Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine.
Authors: Kalebic T , Kinter A , Poli G , Anderson M E , Meister A , Fauci A S
Published: May 2006

The effects of glutathione (GSH), glutathione ester (GSE), and N-acetyl-L-cysteine (NAC) on the induction of human immunodeficiency virus (HIV) expression were investigated in the chronically infected monocytic U1 cell line, a previously described cellular model for HIV latency. U1 cells constitutively express low levels of virus, which can be increased by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and other inducers. GSH, GSE, and NAC suppressed in a dose-dependent fashion the induction of HIV expression mediated by PMA, TNF-alpha, and IL-6, in the absence of cytotoxic or cytostatic effects. Reverse transcriptase activity, inducible by PMA, TNF-alpha, or IL-6, was decreased by 80-90% after pretreatment with GSH, GSE, or NAC. The induction of total HIV protein synthesis was also decreased appreciably after pretreatment with GSH, GSE, or NAC. The accumulation of HIV mRNA was substantially suppressed after pretreatment with NAC but to a lesser extent after pretreatment with GSH or GSE. Although PMA induces the expression of TNF-alpha in U1 cells, the suppressive effect of GSH, GSE, and NAC on PMA-induced HIV expression in U1 cells was not associated with the inhibition of TNF-alpha expression. The present findings, which elucidate relationships between cellular GSH and HIV expression, suggest that therapy with thiols may be of value in the treatment of HIV infection.
Activated protooncogenes in human lung tumors from smokers.
Authors: Reynolds S H , Anna C K , Brown K C , Wiest J S , Beattie E J , Pero R W , Iglehart J D , Anderson M W
Published: May 2006

Fourteen primary human lung tumor DNAs from smokers were analyzed for transforming activity by two DNA transfection assays. Activated protooncogenes were detected in 3 of 11 tumor DNAs by the NIH 3T3 focus assay, whereas activated protooncogenes were detected in 11 of 13 tumor DNAs by the NIH 3T3 cotransfection-nude mouse tumorigenicity assay. K- or NRAS genes activated by point mutation at codons 12 or 61 were detected in a large cell carcinoma, a squamous cell carcinoma, and 5 adenocarcinomas. An HRAS oncogene activated by a different mechanism was detected in an epidermoid carcinoma. One adenocarcinoma was found to contain an activated RAF gene. Two unidentified transforming genes were detected in a squamous cell carcinoma DNA and two adenocarcinoma DNAs. Eight of 10 lung adenocarcinomas that had formed metastases at the time of surgery were found to contain RAS oncogenes. No significant increase in metastasis was observed in the lung adenocarcinomas that contained one or more 6-kilobase EcoRI alleles of the LMYC gene. Overall, 12 of 14 (86%) of the lung tumor DNAs from smokers were found to contain activated protooncogenes. RAS oncogenes appear to play a role in the development of metastases in lung adenocarcinomas.
Evolution of life history variation among female mammals.
Authors: Charnov E L
Published: May 2006

A unified approach is developed for the evolutionary structure of mammalian life histories; it blends together three basic components (individual growth or production rate as a function of body size, natural selection on age of maturity, and stable demography) to predict both the powers and the intercepts of the scaling allometry of life history variables to adult size. The theory also predicts the signs (+, -) of the correlations between life history variables when body size is held constant. Finally, the approach allows us to eliminate body size to predict the dimensionless relationships between the life history variables themselves.
An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae.
Authors: Edwards H , Trézéguet V , Schimmel P
Published: May 2006

While the Escherichia coli Su-3 (tyrT) tyrosine tRNA suppressor inserts only tyrosine at amber codons in E. coli, we show here that in Saccharomyces cerevisiae this tRNA inserts leucine and no significant amounts of any other amino acid. Thus, the E. coli tyrosine tRNA is functionally a leucine tRNA in yeast cytoplasm. This functional identity may correlate with a structural relationship between the E. coli tyrosine and yeast leucine tRNAs, which are both members of the uncommon type II class of tRNA structures. The results raise the possibility that in evolution a tRNA may be more closely related to a tRNA of different acceptor specificity, but of the same type class, than to one with the same amino acid specificity, but of a different type class.
Genetic diversity and adaptedness in tetraploid Avena barbata and its diploid ancestors Avena hirtula and Avena wiestii.
Authors: García P , Morris M I , Sáenz-de-Miera L E , Allard R W , Pérez de la Vega M , Ladizinsky G
Published: May 2006

Avena barbata, a tetraploid grass, is much more widely adapted and successful in forming dense stands than its diploid ancestors. The success of such polyploids has often been attributed to heterosis associated with ability to breed true for a highly heterozygous state in which allelic differences between the parents are fixed in the polyploid by chromosome doubling. We have examined the relationship between genetic diversity and adaptedness for 14 allozyme loci in A. barbata and its diploid ancestors in samples collected from diverse habitats in Israel and Spain. The relationship varied from locus to locus: superior adaptedness was associated with genetic uniformity for five loci, in part with genetic uniformity and in part with genetic diversity (monomorphism for a single heteroallelic quadriplex) for one locus, and with allelic diversity in the form of heteroallelic quadriplexes combined with genotypic diversity in the form of complex polymorphisms among different homoallelic and/or heteroallelic quadriplexes for the eight remaining loci. These results indicate that allelic diversity fixed in nonsegregating form through chromosome doubling was an important factor in the evolution of adaptedness in A. barbata. However, it is unlikely that heterosis associated with heterozygosity contributed significantly to superior adaptedness in either the diploids or the tetraploid because virtually all loci (approximately 99%) were homozygous in the Avena diploids and tetraploid.
Characterization of lipid domains in erythrocyte membranes.
Authors: Rodgers W , Glaser M
Published: May 2006

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.
Mononuclear phagocytes: a major population of effector cells responsible for rejection of allografted tumor cells in mice.
Authors: Yoshida R , Takikawa O , Oku T , Habara-Ohkubo A
Published: May 2006

To understand the in situ mechanism of immunological response of recipient animals to allografted tumor cells, the types of cells that infiltrated into the rejection site were examined. When Meth A cells (H-2d) were given i.p. to an allogeneic [C57BL/6 (H-2b)] strain of mouse, the tumor cells ceased to grow on the 6th day, accompanied by an i.p. infiltration of leukocytes. The tumor cells were totally eliminated from the peritoneal cavity around the 12th day. The highest cytotoxic activity against Meth A cells was obtained with the peritoneal exudate cells harvested on day 8. On this day, the exudate cells consisted of three populations when examined by flow cytometry, and each was isolated by sorting. Each of them appeared to be homogeneous, and they were morphologically identified as lymphocytes; granulocytes; and medium-sized, mononuclear, less-granular cells. The cytotoxic activity was confined exclusively to the last population. The effector cells (H-2b) were cytotoxic against not only Meth A cells (H-2d) but also concanavalin A-stimulated allogeneic spleen cells [C3H/He (H-2k), CBA/N (H-2k), A/J (H-2a), BALB/c (H-2d), and DBA/2 (H-2d) strains of mouse]. The effector cells were totally inert against concanavalin A-activated syngeneic spleen cells [C57BL/6 (H-2b) and C57BL/10 (H-2b) strains of mouse]. The effector cells were phenotypically (Thy-1.2- CD3- Lyt-1- Lyt-2- L3T4- immunoglobulin- asialo GM1-), morphologically, and functionally distinct from cytotoxic T cells, natural killer cells, and lymphokine-activated killer cells but were adherent mononuclear phagocytes.
Gene silencing in a polyploid homosporous fern: paleopolyploidy revisited.
Authors: Gastony G J
Published: May 2006

Because of their high chromosome numbers, homosporous vascular plants were considered paleopolyploids until recent enzyme electrophoretic studies rejected this hypothesis by showing that they express only diploid numbers of isozymes. In polyploid sporophytes of the homosporous fern pelleae rufa, however, progressive diminution of phosphoglucoisomerase activities encoded by one ancestral genome culminates in tetraploid plants exhibiting a completely diploidized electrophoretic phenotype for this enzyme. The demonstration that such gene silencing can make a polyploid fern look isozymically like a diploid questions the validity of isozyme evidence for testing the paleopolyploid hypothesis and supports the proposed role of polyploidization followed by genetic diploidizaton in the evolutionary history of homosporous pteridohytes.
Monomeric IgG2a promotes maturation of bone-marrow macrophages and expression of the mannose receptor.
Authors: Schreiber S , Blum J S , Stenson W F , MacDermott R P , Stahl P D , Teitelbaum S L , Perkins S L
Published: May 2006

The macrophage mannose receptor, a 172-kDa lineage-specific glycoprotein, partakes in nonopsonin-mediated phagocytosis by recognition of terminal mannose residues on targeted particles. Because appearance of the receptor progresses with monocyte/macrophage differentiation, its expression is indicative of the maturational state of the cell. Monomeric IgG2a and IgG2b up-regulate mannose-receptor surface expression and biosynthesis by murine bone-marrow macrophage precursors as much as 7- to 12-fold in a dose-dependent manner. IgG2a accelerates macrophage mannose-receptor expression by several days during in vitro bone-marrow differentiation; however, treated and control cells ultimately express equivalent levels of receptor. Moreover, the effect is independent of cell cycle or ambient levels of colony-stimulating factor 1. The coinduction of another maturation-dependent lineage-specific antigen, F4/80, and the fact that macrophage precursors respond to IgG2a only within the first day of culture, indicate that the targeted cell is an early myelomonocytic precursor, responsive only during a short, early developmental window. The effect is specific for immunoglobulin molecules of the IgG2a and IgG2b subclasses and probably involves an Fc gamma-receptor signal-transduction pathway but not macrophage priming or activation. Most importantly, a paracrine mechanism of immunoglobulin-mediated bone-marrow macrophage differentiation is suggested by experiments in which basal levels of mannose-receptor expression are reduced by continual removal of B-cell-generated IgG from marrow cultures. Thus, IgG2a and IgG2b prompt mannose-receptor synthesis and bone-marrow macrophage differentiation and may, therefore, play a role in the regulation of macrophage differentiation in host defense.
Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli.
Authors: Claverie-Martin F , Magasanik B
Published: May 2006

The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system. One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogen-limiting conditions. The general nitrogen regulatory protein (NRI) binds to two overlapping sites centered at -109 and -122 from the transcription start site and, when phosphorylated, activates transcription of glnHp2 by catalyzing isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. The DNA-bending protein integration host factor (IHF) binds to a site immediately upstream of glnHp2 and enhances the activation of open complex formation by NRI phosphate. The NRI-binding sites can be moved several hundred base pairs further upstream without altering the ability of NRI phosphate to activate open complex formation. We propose that the IHF-induced bend can facilitate or obstruct the interaction between NRI phosphate and the closed complex depending on the relative positions of NRI phosphate and sigma 54-RNA polymerase on the DNA.
Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii.
Authors: Kindle K L , Richards K L , Stern D B
Published: May 2006

Chloroplast transformation of Chlamydomonas reinhardtii has been accomplished by agitating cell wall-deficient cells in the presence of glass beads and DNA. By using the atpB gene as the selected marker and cells grown in 0.5 mM 5-fluorodeoxyuridine, we have recovered up to 50 transformants per microgram of DNA. This method is easy and does not require specialized equipment, although it is not as efficient as the tungsten particle bombardment method [Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. (1988) Science 240, 1534-1537]. By using particle bombardment, we have developed a cotransformation approach in which spectinomycin-resistant 16S rRNA-encoding DNA is the selected marker, and we have demonstrated that cotransformation of an unselected marker on an independent replicon is very efficient. We have used this strategy (i) to recover transformants with partially deleted atpB genes that could not otherwise have been selected since they did not restore photosynthetic capability to a recipient carrying a more extensive atpB deletion and (ii) to generate specific deletion mutations in a wild-type recipient. This methodology should allow the introduction of any desired change into the chloroplast genome, even in the absence of phenotypic selection, and thus a detailed functional analysis of any chloroplast DNA sequence should be possible.