bluuebunny/crossref_metadata_2025 huggingface dataset:
Two adjacent promoter elements mediate nerve growth factor activation of the c-fos gene and bind distinct nuclear complexes.
Authors: Visvader J , Sassone-Corsi P , Verma I M
Published: May 2006

Protooncogene fos is rapidly and transiently induced by nerve growth factor (NGF) in rat pheochromocytoma PC12 cells. Two adjacent promoter elements have been identified to mediate the NGF response. One element colocalizes with the serum response element (SRE) centered at position -308, previously shown to confer inducibility by serum, phorbol 12-myristate 13-acetate, and epidermal growth factor, whereas the other element, termed SRE-2, maps approximately 20 base pairs downstream of the SRE and contains several sequence repeats. This element also confers serum responsiveness. Gel mobility shift assays have demonstrated that there are specific nucleoprotein complexes associated with each element and that these exist in the cell prior to NGF induction. The NGF response is independent of the cAMP-regulatory element(s) and does not require cAMP-dependent protein kinase II, as induction by NGF is retained in the mutant PC12 cell line A126-1B2. Finally, the human heat shock HSP70 promoter is also transcriptionally activated by NGF and appears to bind the same nuclear complex as the SRE-2 element of the c-fos promoter.
Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression.
Authors: Glukhova M A , Kabakov A E , Frid M G , Ornatsky O I , Belkin A M , Mukhin D N , Orekhov A N , Koteliansky V E , Smirnov V N
Published: May 2006

Vinculin- and caldesmon-immunoreactive forms and actin isoform patterns were studied in samples of normal and atherosclerotic human aorta. After removal of adventitia and endothelium, the remaining tissue was divided into three layers: media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. In media of normal aorta, meta-vinculin accounted for 41.0 +/- 0.9% (mean +/- SEM) of total immunoreactive vinculin (meta-vinculin + vinculin); 150-kDa caldesmon accounted for 78.2 +/- 5.1% of immunoreactive caldesmon (150-kDa + 70-kDa); the fractional contents of alpha-smooth muscle actin, beta-nonmuscle, and gamma-isoactins were 49.0 +/- 0.6%, 30.4 +/- 0.6%, and 20.8 +/- 0.8%, respectively. Muscular-elastic intima was very similar to media by these criteria. In subendothelial intima, the fractional content of meta-vinculin and 150-kDa caldesmon was significantly lower (6.9 +/- 1.5% and 32.7 +/- 7.0%, respectively) than in muscular-elastic intima and media, whereas the isoactin pattern was identical to that in adjacent layers, demonstrating the smooth muscle origin of subendothelial intima cells. In atherosclerotic fibrous plaque, the fractional content of alpha-actin was decreased in subendothelial intima, rather than in media and muscular-elastic intima. Additionally, the proportion of subendothelial intima cells [i.e., the cells that express low amounts of smooth muscle phenotype markers (meta-vinculin, 150-kDa caldesmon, and alpha-actin)] in the total intima cell population increased dramatically in atherosclerotic fibrous plaque. The results suggest that changes in the relative content of meta-vinculin and 150-kDa caldesmon as well as alpha-actin in human aortic intima are associated with atherosclerosis although, in subendothelial intima of normal aorta, a certain smooth muscle cell population exists that expresses reduced amounts of "contractile" phenotype markers, even in the absence of the disease.
Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160.
Authors: Willey R L , Bonifacino J S , Potts B J , Martin M A , Klausner R D
Published: May 2006

The synthesis and processing of the human immunodeficiency virus 1 (HIV-1) envelope precursor glycoprotein gp 160 was studied in an infected CD4+ lymphocytic cell line. Surprisingly, only a small percentage (5-15%) of gp160 is cleaved to produce the mature gp120 component. Intracellular sorting results in the transfer of most uncleaved gp160 to lysosomes, where it is degraded, while gp120 is transported to the cell surface and subsequently secreted. Cleavage of gp160 to generate gp120 occurs intracellularly and can be inhibited by NH4Cl. Taken together, these results indicate that intracellular cleavage of gp160 determines the intracellular transport and survival of the envelope glycoproteins necessary to produce infectious virus.
Sequences of the gypsy transposon of Drosophila necessary for its effects on adjacent genes.
Authors: Peifer M , Bender W
Published: May 2006

The Drosophila melanogaster transposon gypsy is the cause of numerous spontaneous mutations, most of which are suppressible by mutations in the suppressor of Hairy wing [su(Hw)] locus. We have examined the phenotype of four revertants of the gypsy element-induced mutation bithoraxoid1 (bxd1) and determined the molecular basis of these reversions. All four revertants have undergone deletions within the gypsy element. The altered gypsy element from one of the partial revertants has been cloned. It has a deletion of only 109 base pairs near the 5' end of the gypsy transcription unit. Similar deletion gypsy elements exist elsewhere in the Drosophila genome. We discuss a mechanism by which the 109-base segment might affect the bxd phenotype.
Molecular cloning and chromosomal localization of the human T-cell receptor zeta chain: distinction from the molecular CD3 complex.
Authors: Weissman A M , Hou D , Orloff D G , Modi W S , Seuanez H , O'Brien S J , Klausner R D
Published: May 2006

The T-cell antigen receptor (TCR) is a multisubunit receptor complex specific to T cells subserving both antigen recognition and signal transduction functions. The zeta chain of the TCR is a component of all surface receptor complexes. This chain was first identified in murine T cells by virtue of the fact that it coimmunoprecipitates with the TCR complex using antibodies directed against either the clone-specific subunits or invariant CD3 subunits of the receptor. Recently, we have isolated a cDNA encoding the murine zeta. Using this as a probe, we have now isolated cDNAs encoding the human zeta. Sequence analysis of cDNAs encoding human and murine zeta reveals that it is a highly conserved protein. In addition to amino acid homology, there is remarkable interspecies conservation in the nucleotide sequence of the 5' and 3' untranslated regions of the zeta mRNA. The previously characterized invariant delta, epsilon, and gamma chains of the TCR, referred to as the CD3 complex, share significant sequence and structural homology with each other and are all located within 300 kilobases of each other on human chromosome 11 (11q23). zeta has no sequence similarity to the CD3 chains and the localization of the human zeta gene to the centromeric region of chromosome 1 underscores the fact that it is a distinct genetic component of the TCR.
Prevention of diabetes in nonobese diabetic mice by anti-I-A monoclonal antibodies: transfer of protection by splenic T cells.
Authors: Boitard C , Bendelac A , Richard M F , Carnaud C , Bach J F
Published: May 2006

The nonobese diabetic (NOD) mouse has been developed as a model for insulin-dependent diabetes. One gene required for the development of diabetes is associated with the major histocompatibility complex. This gene possibly could be linked to class II genes, which show a unique pattern in NOD mice. To evaluate the role of the I-A class II antigen expressed in NOD mice, we studied the effect of anti-I-A monoclonal antibodies on disease onset in vivo. Long-term treatment with anti-class II IgG2a antibodies specific for NOD I-A antigen prevented the spontaneous development of diabetes, as opposed to control antibodies shown not to react with NOD I-A antigen. Anti-class II antibodies apparently elicited active immune suppression, requiring a fully immunocompetent host, rather than passive blockade of class II antigen. Treatment with anti-class II antibody effectively prevented the adoptive transfer of diabetes produced by splenocytes from diabetic NOD mice into newborn mice but failed to prevent adoptive transfer into irradiated adult NOD recipients. Direct evidence for the induction of suppressor cells was obtained from the passive transfer of spleen cells from anti-class II antibody-treated NOD donors. The injection of anti-class II antibody-treated spleen cells collected from NOD donors prevented the development of diabetes, which normally follows transfer of diabetogenic spleen cells into irradiated 8-week-old male NOD recipients. Depletion experiments indicate that CD4+ cells are responsible for anti-class II-induced protection transferred by spleen cells.
Brain-specific src oncogene mRNA mapped in rat brain by in situ hybridization.
Authors: Ross C A , Wright G E , Resh M D , Pearson R C , Snyder S H
Published: May 2006

Brain src protooncogene is expressed in two forms, one identical to message in other tissues, and one containing an 18-nucleotide insert specific to brain. We have mapped mRNA for the two forms of src in rat brain with selective antisense oligonucleotide probes to the brain (src+) and peripheral (src-) forms. Fetal rat src mRNA levels were much higher in the central nervous system than any peripheral organ. In adult brain, src+ mRNA level was highest in the internal granular layer of the olfactory bulb, pyramidal cells of the hippocampus, granule cells of the dentate gyrus, and cerebellar granule cells. src+ and src- levels were similar in hindbrain, but src+ levels were higher than those of src- in forebrain. These distributions suggest that src+ may play roles in a number of neural processes, possibly including neuronal plasticity.
Use of a foreign epitope as a "tag" for the localization of minor proteins within a cell: the case of the immunity protein to colicin A.
Authors: Geli V , Baty D , Lazdunski C
Published: May 2006

The immunity protein to colicin A (Cai), which is constitutively expressed at a very low level in Escherichia coli strains, has been studied in recombinant plasmid constructs allowing expression of various immunity fusion proteins under the control of inducible promoters. The 13-amino acid NH2-terminal region of Cai was substituted by polypeptides from beta-galactosidase or from colicin A. Upon induction of the chimeric proteins, the rate of expression of the immunity protein could be correlated to the level of resistance to colicin A. The immunity protein has been "tagged" with an epitope from the colicin A protein for which a monoclonal antibody is available. Using this technique, we have directly demonstrated that the immunity protein is located in the cytoplasmic membrane. The results indicate that the NH2-terminal region of Cai is directed toward the cytoplasm and is probably not required for Cai insertion into the membrane or for its function.
Cauliflower mosaic virus gene VI produces a symptomatic phenotype in transgenic tobacco plants
Authors: Baughman Gail A. , Jacobs Jerry D. , Howell Stephen H.
Published: May 2006

Gene VI of the cauliflower mosaic virus (CaMV) genome encodes a protein (P
66
) in virus-infected plants that accumulates in cytoplasmic inclusion bodies. When a segment of the CaMV genome bearing gene VI is transferred to tobacco plants by the
Agrobacterium tumefaciens
Ti plasmid, the resulting transgenic plants display viral-like symptoms. Symptoms produced by the DNA from two different viral isolates (CaMV Cabb B-JI and CM1841) were distinct—symptoms from the first were mosaic-like, whereas the other caused uniform bleaching of leaves. That gene VI was responsible for the symptomatic phenotype was demonstrated by showing that symptom production was blocked by deletions and by a frame-shifting linker mutation in gene VI. Furthermore, in primary transformants, there was a strict correlation between the appearance of symptoms and the presence of gene VI product, P
66
, detected by immunoblots. Hence, a protein encoded by the CaMV genome produces viral-like symptoms in transgenic tobacco plants.
A "housekeeping" gene on the X chromosome encodes a protein similar to ubiquitin.
Authors: Toniolo D , Persico M , Alcalay M
Published: May 2006

An X chromosome gene located 40 kilobases downstream from the G6PD gene, at Xq28, was isolated and sequenced. This gene, which we named GdX, spans about 3.5 kilobases of genomic DNA. GdX is a single-copy gene, is conserved in evolution, and has the features of a "housekeeping" gene. At its 5' end, a cluster of CpG dinucleotides is methylated on the inactive X chromosome and unmethylated on the active X chromosome. The GdX gene can code for a 157 amino acid protein, GdX. Residues 1-74 of GdX show 43% identity to ubiquitin, a highly conserved 76 amino acid protein. The COOH-terminal moiety of GdX is characterized in its central part (residues 110-128) by a sequence homologous to the COOH-terminal hormonogenic site of thyroglobulin. The structural organization of the GdX protein suggests the existence of a family of genes, in addition to the ubiquitin gene, that could play specific roles in key cellular processes, possibly through protein-protein recognition.
High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.
Authors: Miller J F , Dower W J , Tompkins L S
Published: May 2006

Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.
Precursor B cells transformed by Epstein-Barr virus undergo sterile plasma-cell differentiation: J-chain expression without immunoglobulin.
Authors: Kubagawa H , Burrows P D , Grossi C E , Mestecky J , Cooper M D
Published: May 2006

Human bone marrow cells were depleted of B lymphocytes to enrich for precursor B cells that could be transformed with Epstein-Barr virus. Transformed immunoglobulin-negative precursors either maintained their immunoglobulin genes in the germ-line configuration or had undergone DJ or abortive VDJ rearrangements (V, D, and J represent variable, diversity, and joining gene segments). All cell lines and their derivative clones, even those with no detectable immunoglobulin gene rearrangements, generated subpopulations of cells that produced high levels of joining (J) chain when analyzed by immunoprecipitation after biosynthetic labeling and by blot hybridization of cytoplasmic RNA. Morphologic and immunofluorescence analyses revealed that J-chain production was confined to clonal progeny that had exited the cell cycle to undergo plasma-cell differentiation. Analysis of cell surface antigens revealed expression of several B-cell maturational markers, including complement receptor type 2 (CR2) and plasma cell antigen 1 (PCA-1). Epstein-Barr virus can thus transform B-cell progenitors, allowing them to proliferate and undergo terminal B-cell differentiation coupled with J-chain expression. These events appear to occur independently of the immunoglobulin gene status of the transformed cells.
Lipid composition and fluidity of the human immunodeficiency virus.
Authors: Aloia R C , Jensen F C , Curtain C C , Mobley P W , Gordon L M
Published: May 2006

Lipid analyses of the human immunodeficiency virus (HIV) propagated in Hut 78 cells indicated a low total lipid/protein ratio, a high cholesterol/phospholipid molar ratio, and major phospholipids consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine; comparable lipid profiles were noted for human erythrocytes and other RNA viruses. Electron spin resonance (ESR) studies of HIV labeled with 5-nitroxide stearate (N-oxy-4',4'-dimethyloxazolidine derivative of ketostearate) showed a low "fluidity" at 37 degrees C, similar to other enveloped RNA viruses and erythrocytes and probably due to the high cholesterol/phospholipid ratio. Ethanol (50%) completely disrupts the envelope, contributing to the rapid inactivation of HIV by ethanol. Contrarily, heating to 57 degrees C causes much less fluidization, and this heating may play a role in the slower viral inactivation at high temperatures. Should a critical minimum ordering in the HIV envelope be necessary for viral stability and infectivity, manipulating the lipid composition or fluidizing the HIV membrane, or both, may provide an untried therapeutic approach.
Old mice recover the ability to produce IgG and high-avidity antibody following irradiation with partial bone marrow shielding.
Authors: Tsuda T , Kim Y T , Siskind G W , Weksler M E
Published: May 2006

The splenic plaque-forming-cell (PFC) response to trinitrophenylated bovine gamma globulin of 18- to 20-month-old mice is markedly depressed, with a preferential loss of indirect (IgG) PFC and high-avidity-antibody-secreting cells compared to 6- to 8-week-old animals. The anti-trinitrophenyl response of old mice, whose peripheral lymphoid system has been reconstituted from their own bone marrow after irradiation while their bone marrow was partially shielded, includes high-avidity and IgG PFCs relatively comparable to those of normal young mice. If young mice are irradiated while their bone marrow is partially shielded and given purified splenic T cells from either old or young donors during recovery from irradiation, then the avidity distribution and the ratio of IgG/IgM PFCs they produce in response to trinitrophenylated bovine gamma globulin reflects the characteristic immune response of the T-cell donor. These results are consistent with the hypothesis that the bone marrows of old and young mice are similar with regard to the spectrum of B-cell clones that they can generate and that it is peripheral regulatory effectors that are responsible for much of the age-related change in the immune response. In addition, if one calculates the PFC avidity distribution taking into account those cells whose secretion of antibody was inhibited by anti-idiotype autoantibodies, then it is clear that there are more high-avidity B cells present in old mice than are detected by the conventional plaque-inhibition assay. Thus, the reduced avidity of the PFC response of old mice appears to be, at least in part, due to down regulation by anti-idiotype autoantibodies.
An asymmetric form of muscle acetylcholinesterase contains three subunit types and two enzymic activities in one molecule.
Authors: Tsim K W , Randall W R , Barnard E A
Published: May 2006

We have purified completely the principal asymmetric ("heavy") form of acetylcholinesterase (Ac-ChoEase; EC 3.1.1.7) from chick muscle (i.e., the synaptic form in the twitch muscle fibers) by using a monoclonal antibody that recognizes AcChoEase but not pseudocholinesterase (ChoEase; cholinesterase, EC 3.1.1.8). The purified protein exhibits catalytic and inhibition properties characteristic of AcChoEase and ChoEase and contains three distinct subunits of apparent sizes 110 kDa, 72 kDa, and 58 kDa in the ratio 2:2:1. The discovery of an AcChoEase/ChoEase hybrid asymmetric form has been further supported by (i) the identification of active site properties of AcChoEase in the 110-kDa subunit and of ChoEase in the 72-kDa subunit, (ii) the purification or precipitation of both activities together by, also, a ChoEase-specific monoclonal antibody, and (iii) evidence that all subunits are bound in the asymmetric forms by disulfide bonds. The 58-kDa subunit is the only one that is sensitive to digestion with purified collagenase; it carries the collagenous "tail" of the asymmetric form. A model is proposed for this form of AcChoEase.
Nerve growth factor induces the genes for two proteins related to a family of calcium-binding proteins in PC12 cells.
Authors: Masiakowski P , Shooter E M
Published: May 2006

Differential hybridization of a cDNA library from rat pheochromocytoma PC12 cells with cDNA probes from naive PC12 cells and from PC12 cells exposed to nerve growth factor for 7 days identified cDNA sequences of two genes induced by NGF. The mRNA species detected by these cDNA sequences, designated 42A and 42C, reached maximal levels after 24 hr of treatment with NGF and were still significantly higher than control levels after 7 days. Epidermal growth factor transiently induced both mRNAs but at much lower levels. The mRNAs code for 95- (42C) and 101- (42A) amino acid residue peptides whose sequences are homologous to those of a family of calcium-binding proteins including the S-100 protein. The conservation of primary and secondary structure between 42A, 42C, and the other proteins suggests a possible role for them in the regulation of cell growth and differentiation.
Norepinephrine inhibits gamma-interferon-induced major histocompatibility class II (Ia) antigen expression on cultured astrocytes via beta-2-adrenergic signal transduction mechanisms.
Authors: Frohman E M , Vayuvegula B , Gupta S , van den Noort S
Published: May 2006

The astrocyte is now recognized as a facultative immunocompetent antigen-presenting cell that can initiate intracerebral immune responses. However, despite the presence of activated T lymphocytes and their associated lymphokines within the central nervous system, there is a paucity in the expression of the major histocompatibility (MHC) antigens on normal neural tissue. These membrane-localized glycoproteins are required for the process of antigen presentation and, therefore, for the initiation of immune responses. To date, little is understood regarding the nature of inhibitory mechanisms that might be responsible for maintaining the brain as an immunoprivileged site. In this study we found that norepinephrine, a major brain transmitter, significantly inhibited gamma interferon-induced MHC class II antigen expression on astrocytes derived from neonatal Lewis rats. We show that this inhibition can be attenuated by the addition of a beta-adrenergic antagonist, propranolol, but not by the addition of a beta 1-selective antagonist, atenolol, or by an alpha-adrenergic antagonist, phentolamine. Furthermore, it was found that a similar inhibition could be achieved by the addition of either dibutyryl-cAMP or dipyridimole, a phosphodiesterase inhibitor. Therefore, it seems that norepinephrine-mediated inhibition of MHC class II antigen expression on astrocytes works through beta 2-adrenergic signal transduction pathways. Taken together, these in vitro results suggest that the brain contains inhibitory factors that may play a pivotal role in the regulation of intracerebral immune responses by modulating the expression of MHC antigens on astrocytes.
Size of bacterial ice-nucleation sites measured
in situ
by radiation inactivation analysis
Authors: Govindarajan Arepura G. , Lindow Steven E.
Published: May 2006

Four bacterial species are known to catalyze ice formation at temperatures just below 0°C. To better understand the relationship between the molecular structure of bacterial ice-nucleation site(s) and the quantitative and qualitative features of the ice-nucleation-active phenotype, we determined by γ-radiation analysis the
in situ
size of ice-nucleation sites in strains of
Pseudomonas syringae
and
Erwinia herbicola
and in
Escherichia coli
HB101 carrying the plasmid pICE1.1 (containing a 4-kilobase DNA insert from
P. syringae
that confers ice-nucleation activity). Lyophilized cells of each bacterial strain were irradiated with a flux of γ radiation from 0 to 10.2 Mrad (1 Mrad = 10
6
J/kg). Differential concentrations of active ice nuclei decreased as a first-order function of radiation dose in all strains as temperature was decreased from -2°C to -14°C in 1°C intervals. Sizes of ice nuclei were calculated from the γ-radiation flux at which 37% of initial ice nuclei active within each 1°C temperature interval remained. The minimum mass of a functional ice nucleus, active only between -12°C and -13°C, was about 150 kDa for all strains. The size of ice nuclei increased logarithmically with increasing temperature from -12°C to -2°C, where the estimated nucleant mass was 19,000 kDa. The ice nucleant in these three bacterial species may represent an oligomeric structure, composed at least in part of an
ice
gene product that can self-associate to assume many possible sizes.
Cloning and sequencing of the cytoplasmic precursor to the alpha subunit of rat liver mitochondrial succinyl-CoA synthetase.
Authors: Henning W D , Upton C , McFadden G , Majumdar R , Bridger W A
Published: May 2006

Succinyl-CoA synthetase [succinate-CoA ligase (GDP-forming); EC 6.2.1.4] of rat liver, an alpha beta dimer, is a component of the enzymology of the tricarboxylic acid cycle and functions within the mitochondrial matrix. We have isolated and determined the sequence of a cDNA clone containing the coding sequence of the cytoplasmic precursor to the alpha subunit of this enzyme together with stretches of nontranslated sequence at the 5' and 3' ends. The translated amino acid sequence indicates the presence of a 27-residue N-terminal signal sequence for mitochondrial targeting. The amino acid sequence of the mature alpha subunit shows an extraordinary degree of homology to the alpha subunit of Escherichia coli succinyl-CoA synthetase, with greater than 70% of the residues identical. This suggests that the fundamental differences in the quaternary structures and catalytic functions of the mammalian and bacterial enzymes must be attributable to differences in the beta subunits. mRNA that hybridizes to the cloned DNA is approximately equal to 1800 nucleotide residues in length, confirming that each of the two subunits is encoded separately and does not arise by proteolysis of a primary gene product containing both subunits of the mature protein.
Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.
Authors: Bretaudiere J P , Tapon-Bretaudiere J , Stoops J K
Published: May 2006

Well-preserved structures of native and alpha-chymotrypsin-bound alpha 2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native alpha 2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the alpha 2-macroglobulin-chymotrypsin conjugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of alpha 2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex.
Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.
Authors: Mulligan R M , Houtz R L , Tolbert N E
Published: May 2006

Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], but the stoichiometry of binding of the reaction-intermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was resistant. Three tryptic peptides were isolated and characterized after digestion of the activated enzyme; the tryptic-sensitive sites were identified at Lys-8, Lys-14, and Lys-466 of the large subunit. Tryptic digestion of the enzyme complexed with the reaction-intermediate analogue released only two peptides by hydrolysis at Lys-8 and at Lys-14. The loss of susceptibility of Lys-466 to trypsin may be the result of a conformational change that limits the accessibility of the carboxyl-terminal region after binding of the reaction-intermediate analogue. Analysis of the amino-terminal tryptic peptide by composition and fast atom bombardment mass spectrometry demonstrated that the actual amino-terminal residue is proline at position 3 of the DNA-deduced sequence and that this proline is blocked with an N-acetyl moiety. Thus, posttranslational processing of the chloroplast-encoded large subunit of the enzyme must occur to remove Met-1 and Ser-2 and to acetylate the amino terminus.
Evidence that insect embryogenesis is regulated by ecdysteroids released from yolk proteins.
Authors: Bownes M , Shirras A , Blair M , Collins J , Coulson A
Published: May 2006

That the yolk proteins (YPs), or vitellins, stored in the oocytes of insects are a nutritional store for subsequent embryogenesis has long been assumed. Exhaustive data base searching programs revealed highly significant sequence similarity between the three YPs of Drosophila melanogaster and part of the triacylglycerol lipase of the domestic pig. Based upon time of degradation of YPs during embryogenesis, existence of maternally stored ecdysteroid conjugates in embryos, location of these conjugates in locust embryos, and the fact that free active ecdysteroid hormones are released at a specific time in embryogenesis to trigger cuticle deposition, we postulate that the similarity reflects a common property of Drosophila YPs--the ability to bind the fatty acid ecdysteroid conjugates. Our finding of conjugated ecdysteroids tightly bound to purified Drosophila YP supports this prediction.
Looping out and deletion mechanism for the immunoglobulin heavy-chain class switch.
Authors: Jäck H M , McDowell M , Steinberg C M , Wabl M
Published: May 2006

In the mouse pre-B-cell line 18-81, cells can switch production in vitro from immunoglobulin mu chain to gamma 2b chain. The gene encoding the gamma 2b chain is created by a rearrangement of the mu gene. This rearrangement always takes place within a homolog. In cells with a gamma 2b gene, most of the time the gene segment encoding the constant region of the mu chain is deleted, but often the rearrangement leads to cells that produce no immunoglobulin, and all DNA sequences are retained. The latter result is due to an inversion. Inversions exclude the unequal sister chromatid exchange model of the heavy-chain class switch. Looping out is an intermediate step in the process of generating an inversion. Our findings demonstrate that the switch rearrangement occurs by looping out and deletion.
Immunologic modeling of a 75-kDa malarial protein with carrier-free synthetic peptides.
Authors: Richman S J , Reese R T
Published: May 2006

A protein of 75 kDa is produced in large quantities by the human malarial parasite Plasmodium falciparum and is present on the surface of the merozoite, whose function is to infect erythrocytes. Based on nucleotide sequence coding for 40% of this protein, two nonoverlapping model peptides 13 and 19 residues long were synthesized, coupled to a keyhole limpet hemocyanin carrier, and used to immunize rabbits. Although both antisera had high titers of anti-peptide antibodies, only that raised against the 13-residue peptide showed good reactivity against the original protein. Although the 19-mer adopted the helical secondary structure predicted for the corresponding protein region, antisera against this peptide reacted with the native protein weakly or not at all. Concluding that the poor anti-protein reactivity was due to modification of lysine-containing epitopes by glutaraldehyde conjugation, we used a carrier-free 28-residue peptide presented as a 56-residue disulfide-bonded dimer to model the same region. This peptide, in contrast to the conjugated 19-mer, stimulated the production of IgG antibodies that reacted at high dilution with the authentic protein in immunoblots, ELISA, and radioimmunoprecipitation assays. These data indicate that large carrier-free peptides may be successfully used as immunogens. In addition, our results show that this strategy may greatly improve the ability of conjugation-sensitive peptides to stimulate antibodies reactive with the original protein and therefore has substantial practical application.
Induced and natural break sites in the chromosomes of Hawaiian Drosophila.
Authors: Tonzetich J , Lyttle T W , Carson H L
Published: May 2006

gamma-Irradiation of a laboratory strain of the Hawaiian species Drosophila heteroneura yielded 310 breaks in the five major acrocentric polytene chromosomes. Their map positions conform to the Poisson distribution, unlike most of the 436 natural breaks mapped in 105 closely related species endemic to Hawaii. Genome element E is longer and has more induced breaks than the others. Both in Hawaiian and related species groups, this element shows increased polymorphism and fixation of naturally occurring inversions. The X chromosome (element A) also accumulates many natural breaks; the majority of the resulting aberrations become fixed rather than remain as polymorphisms. Although size may play a small role in initial break distribution, the major effects relative to the establishment of a rearrangement in natural populations are ascribed to the interaction of selection and drift. Nonconformance of the natural breaks to the Poisson distribution appears to be due to the tendency for breaks to accumulate both in the proximal euchromatic portion of each arm and in heterochromatic regions that are not replicated in the polytene chromosomes.
On a notion of simplicial depth
Authors: Liu Regina Y.
Published: May 2006

For a distribution
F
on R

p

and a point
x
in R

p

the simplicial depth
D
(
x
), which is the probability that
x
be inside a random simplex whose vertices are
p
+ 1 independent observations from
F
, is introduced.
D
(
x
) can be viewed as a measure of depth of the point
x
relative to
F
, and its empirical version gives rise to a natural ordering of the data points from the center outward. This ordering provides an approach for detecting outliers in a multivariate data cloud and leads to the introduction of affine equivariant multivariate generalizations of the univariate sample median and L-statistics. This sample median is shown to be consistent for the center of any angularly symmetric distribution.
Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities.
Authors: Tanese N , Goff S P
Published: May 2006

The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity.
Two genetic loci participate in the regulation by iron of the gene for the human transferrin receptor.
Authors: Casey J L , Di Jeso B , Rao K , Klausner R D , Harford J B
Published: May 2006

Iron regulation of the human transferrin receptor gene was examined in murine cells transformed with chimeric constructs containing the human transferrin receptor gene's promoter and either the structural gene for bacterial chloramphenicol acetyltransferase or the human transferrin receptor cDNA. The activity of the transferrin receptor gene's promoter with the heterologous indicator gene was found to be approximately equal to 3-fold higher in cells treated with the iron chelator desferrioxamine than in cells treated with the iron source, hemin. A higher degree of iron regulation was seen in the expression of the human transferrin receptor cDNA driven by its own promoter. The receptor cDNA under the control of the simian virus 40 early promoter was also iron-regulated. Several human transferrin receptor transcripts differing in their 3' end were produced in the murine cells regardless of the promoter used, with the shorter transcripts being relatively unregulated by iron. Deletion of cDNA corresponding to most of the 3' untranslated portion of the mRNA for the receptor ablated the iron regulation. We conclude that at least two genetic elements exist for the regulation of the transferrin receptor gene by iron. One has its locus in the DNA upstream of the transferrin receptor gene's transcription start site, and the other is dependent upon the integrity of the sequences in the 3' end of the gene.
Antigen-independent regulation of cytoplasmic calcium in B cells with a 12-kDa B-cell growth factor and anti-CD19.
Authors: Ledbetter J A , Rabinovitch P S , June C H , Song C W , Clark E A , Uckun F M
Published: May 2006

Increases in cytoplasmic free calcium ([Ca2+]i) can be induced in resting B cells either by a low molecular weight (12-kDa) B-cell growth factor (LMW-BCGF) or by crosslinking the B-cell antigen CD19 with monoclonal antibody (mAb). LMW-BCGF causes a slow [Ca2+]i increase in peripheral blood and tonsillar B cells but has no effect on [Ca2+]i in resting T cells. B-cell [Ca2+]i responses mediated by anti-surface immunoglobulin (sIg) or anti-CD19 are potentiated by LMW-BCGF, but anti-sIg and anti-CD19 do not show additive [Ca2+]i responses. LMW-BCGF- and anti-CD19-induced [Ca2+]i signals are similar to the sIgM or sIgD-mediated signals in that they are inhibited by prior treatment with phorbol 12-myristate 13-acetate. However, LMW-BCGF- and CD19-mediated signals do not depend on the expression of sIg, since they were also observed on sIg-B-cell precursor acute lymphoblastic leukemia (ALL) cells. Both anti-CD19 and LMW-BCGF stimulated in vitro colony formation by ALL cells and showed additive effects when used together. [Ca2+]i responses to LMW-BCGF or CD19 cross-linking were also evident on certain pre-B-cell and lymphoma B-cell lines.
Sigma-phase packing of icosahedral clusters in 780-atom tetragonal crystals of Cr
5
Ni
3
Si
2
and V
15
Ni
10
Si that by twinning achieve 8-fold rotational point-group symmetry
Authors: Pauling Linus
Published: May 2006

A 780-atom primitive tetragonal unit with edges 27.3, 27.3, and 12.6 Å is assigned to rapidly solidified Cu
5
Ni
3
Si
2
and V
15
Ni
10
Si by analysis of electron diffraction photographs with the assumption that the crystals contain icosahedral clusters. There are thirty 26-atom clusters at the sigma-phase positions. Apparent 8-fold symmetry results from 45° twinning on the basal plane.
Benzo[alpha]pyrene diol epoxide I binds to DNA at replication forks.
Authors: Paules R S , Cordeiro-Stone M , Mass M J , Poirier M C , Yuspa S H , Kaufman D G
Published: May 2006

The distribution of lesions in DNA caused by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [alpha]pyrene (B[alpha]P diol epoxide-I) was studied in synchronized C3H/10T1/2 cells treated in S phase. Sites of carcinogen modification of DNA were identified by polyclonal rabbit antibodies elicited against DNA modified with B[alpha]P diol epoxide-I in vitro. This antigenic DNA contained trans-(7R)-N2-[10-(7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene)-yl]- deoxyguanosine; other adducts were not detected by liquid chromatography. In this study, DNA replication forks with antibodies bound to B[alpha]P diol epoxide-I adducts were detected by electron microscopy. The frequency of replication forks containing carcinogen adducts associated with the fork junction was found to be 8-fold higher than expected for an average distribution. The proportion of replication forks that were apparently blocked at the site of the DNA damage increased when replication was allowed to occur after carcinogen exposure. These results support the conclusions that the fork junction is particularly vulnerable to adduction by B[alpha]P diol epoxide-I and that B[alpha]P diol epoxide-I adducts block the displacement of replication forks during DNA synthesis in intact cells.
Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants.
Authors: Bookstein R , Lee E Y , To H , Young L J , Sery T W , Hayes R C , Friedmann T , Lee W H
Published: May 2006

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a restriction and exon map of the RB gene was constructed by aligning overlapping genomic clones, yielding three contiguous regions ("contigs") of 150 kilobases total length separated by two gaps. At least 20 exons were identified in genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origins of their shortened RB mRNA transcripts. The same probe detected genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastoma. Third, this probe also detected a polymorphic site for BamHI with allele frequencies near 0.5/0.5. Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families.
Amino acid sequence of heavy chain from Xenopus laevis IgM deduced from cDNA sequence: implications for evolution of immunoglobulin domains.
Authors: Schwager J , Mikoryak C A , Steiner L A
Published: May 2006

Present understanding of the evolution of immunoglobulins is derived almost entirely from studies of a few mammalian species. To obtain information about immunoglobulin genes in Xenopus laevis, a cDNA library was prepared in the expression vector lambda gt11 from mitogen-stimulated splenocytes of this species. Of approximately equal to 50,000 clones screened, 18 were found to express IgM epitopes. One of these, lambda XIg14, hybridized with RNA of RNA of approximately equal to 2 kilobases from splenocytes. The insert of this clone appears to encode a variable region and part of a mu constant region; that of another clone, lambda XIg8, appears to encode a variable region and a complete mu constant region. Both inserts contain sequence corresponding to the three gene segments (VH, DH, and JH) that encode heavy-chain variable regions. The heavy-chain constant region (CH) encoded by lambda XIg8 has the characteristic features of C mu, including a four-domain structure and a carboxyl-terminal tail. The amino acid sequences of two mu-chain peptides agree with the cDNA sequence. The identity in amino acid sequence between the corresponding Xenopus and mouse C mu domains ranges from 31 to 47%. The C mu domains vary in the extent to which their sequences resemble the sequences of other immunoglobulins, consistent with previous suggestions that the immunoglobulin domains have an independent evolutionary history.
cDNA cloning of the immunoglobulin heavy chain binding protein.
Authors: Haas I G , Meo T
Published: May 2006

A cDNA library was constructed from size-fractionated poly(A)+ RNA prepared from a murine pre-B-cell hybridoma expressing high levels of immunoglobulin heavy chain binding protein (BiP) and mu heavy chains. Transformed bacterial colonies were screened for recombinant plasmids containing cDNA coding for BiP by hybrid-selected mRNA translation. A clone, pMBiP, containing a 736-base-pair insert was shown to encode the protein. Translation in vitro of hybridoma mRNA selected by hybridization to the pMBiP cDNA yielded a single polypeptide of BiP-like size. The authenticity of this mRNA was verified by comparing the peptides obtained by the limited proteolysis of its in vitro translation product with those obtained from the in vivo produced BiP. Likewise, the authenticity of the cDNA insert was verified by an RNase A protection assay of heteroduplex molecules obtained by annealing a uniformly labeled single-strand copy of the cDNA clone with the same mRNA selected by hybridization and tested by translation. The nucleotide sequence of this clone enabled us to deduce the carboxyl-terminal 142 amino acids of BiP and to establish its kinship with the 70-kDa heat shock protein family. The finding of a single copy of the BiP gene in DNA blots of mouse and rat implies that the BiP-related RNA transcripts constitutively expressed in various murine tissues and cell lines are indeed products of the same gene. These findings imply that BiP plays a more general role than previously anticipated on the basis of the discovery of its association with immunoglobulin heavy chains.
Cloning and expression of a cDNA for the T-cell-activating protein TAP.
Authors: Reiser H , Coligan J , Palmer E , Benacerraf B , Rock K L
Published: May 2006

The T-cell-activating protein TAP is a murine phosphatidylinositol-anchored glycoprotein whose expression is controlled by the Ly-6 locus. Previous studies have suggested an important role for this protein in physiological T-cell activation. Using oligonucleotide probes, we have now isolated a cDNA clone whose predicted sequence would encode a protein with an NH2-terminal sequence identical to that of the TAP molecule. Further analysis of the predicted protein sequence revealed a cysteine-rich protein with a hydrophobic domain at the COOH terminus and without N-linked glycosylation sites--all features consistent with our previous analysis of the TAP protein. In Southern blot analysis, the Ly-6.2 cDNA clone detects a multigene family and a restriction fragment length polymorphism that maps precisely to the Ly-6 locus. Expression of the cDNA clone in COS cells demonstrates that it codes for TAP and clarifies the relationship between the epitopes recognized by various alpha Ly-6 monoclonal antibodies. Finally, we have studied the expression of Ly-6 mRNA in a variety of cell lineages. Ly-6 transcripts were detected in all organs examined, including spleen, kidney, lung, brain, and heart. This demonstrates that the Ly-6 locus is transcriptionally active in a wide range of organs and suggests that the role of TAP or TAP-like proteins might extend to other tissues.