bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase.
Authors: Banerjee P , Ahmad M F , Grove J R , Kozlosky C , Price D J , Avruch J
Published: May 2006

The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase alpha; both kinases were cloned from H4 hepatoma cDNA libraries. The 70-kDa S6 kinase (calculated molecular mass of 59,186 Da) exhibits a single catalytic domain that is most closely related in amino acid sequence (56% identity) to the amino-terminal, kinase C-like domain of the rat p85 S6 kinase (calculated molecular mass of 82,695 Da); strong similarity extends through a further 67 residues carboxyl-terminal to the catalytic domain (40% identity), corresponding to a region also conserved among the kinase C family. Outside of this segment of approximately 330 amino acids, the structures of the p70 and p85 S6 kinases diverge substantially. The p70 S6 kinase is known to be activated through serine/threonine phosphorylation by unidentified insulin/mitogen-activated protein kinases. A model for the regulation of p70 S6 protein kinase activity is proposed wherein the low activity of the unphosphorylated enzyme results from the binding of a basic, inhibitory pseudosubstrate site (located carboxyl-terminal to the extended catalytic domain) to an acidic substrate binding region (located amino-terminal to the catalytic domain); substrate binding is thereby prevented. S6 kinase activation requires displacement of this inhibitory segment, which is proposed to occur consequent to its multiple phosphorylation. The putative autoinhibitory segment contains several serine and threonine residues, each followed directly by a proline residue. This motif may prevent autophosphorylation but permit transphosphorylation; two of these serine residues reside in a maturation promoting factor (MPF)/cdc-2 consensus motif. Thus, hormonal regulation of S6 kinase may involve the action of MPF/cdc-2 or protein kinases with related substrate specificity.
Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas.
Authors: Sugawa N , Ekstrand A J , James C D , Collins V P
Published: May 2006

The epidermal growth factor receptor gene has been found to be amplified and rearranged in human glioblastomas in vivo. Here we present the sequence across a splice junction of aberrant epidermal growth factor receptor transcripts derived from corresponding and uniquely rearranged genes that are coamplified and coexpressed with non-rearranged epidermal growth factor receptor genes in six primary human glioblastomas. Each of these six tumors contains aberrant transcripts derived from identical splicing of exon 1 to exon 8 as a consequence of a deletion-rearrangement of the amplified gene, the extent of which is variable among these tumors. In spite of this intertumoral variability, each intragenic rearrangement results in loss of the same 801 coding bases (exons 2-7) and creation of a new codon at the novel splice site in their corresponding transcripts. These rearrangements do not, however, affect the mRNA sequence for the signal peptide, the first five codons, or the reading frame downstream of the rearrangement.
Mutations in the
DET1
gene affect cell-type-specific expression of light-regulated genes and chloroplast development in
Arabidopsis
Authors: Chory Joanne , Peto Charles A.
Published: May 2006

When grown in the absence of light, the
det1
mutant of
Arabidopsis thaliana
develops characteristics of a light-grown plant by morphological, cellular, and molecular criteria. Here, we show that recessive mutations at the
DET1
locus also result in cell-type inappropriate accumulation of RNAs for light-regulated nuclear and chloroplast genes.
det1
root plastids are differentiated into chloroplasts and are present in very high numbers in root cortex cells in contrast to the few starch-containing amyloplasts normally found in
Arabidopsis
roots. To assay the effect of the
det1
mutation on the expression of photoregulated promoters, we used fusion constructs to stably transform wild-type and
det1
mutants. We show that the three red-light-regulated chlorophyll a/b binding protein promoters are inappropriately expressed in the roots of
det1
seedlings and the blue-light-controlled anthocyanin biosynthetic gene, chalcone synthase, is expressed ectopically in leaf mesophyll cells. These results, together with out previous findings, suggest that the
DET1
gene product is a negatively acting regulatory molecule that is used in common by the light stimulus transduction pathway and by temporal or spatial regulatory signals in plants.
Nerve growth factor potentiates the neurotoxicity of beta amyloid.
Authors: Yankner B A , Caceres A , Duffy L K
Published: May 2006

The role of growth factors in the pathogenesis of Alzheimer disease is unknown. The beta-amyloid protein accumulates abnormally in the brain in Alzheimer disease and is neurotoxic to differentiated hippocampal neurons in culture. Nerve growth factor (NGF) increased the neurotoxic potency of a beta-amyloid polypeptide by a factor of approximately 100,000, which resulted in a reduction of the beta-amyloid neurotoxic EC50 from 0.1 microM to 1 pM. This potentiating effect of NGF was reversed by a monoclonal antibody against NGF and was not observed for a variety of other neurotrophic growth factors. Exposure of hippocampal neurons to very low concentrations of beta amyloid alone resulted in a marked induction of immunoreactive NGF receptors. Addition of NGF with beta amyloid resulted in the appearance of neurodegenerative changes in NGF receptor-positive neurons. The early and profound degeneration of hippocampal and basal forebrain cholinergic neurons that occurs in Alzheimer disease may result from a neurotoxic interaction of beta amyloid with NGF.
Aluminum access to the brain: a role for transferrin and its receptor.
Authors: Roskams A J , Connor J R
Published: May 2006

The toxicity of aluminum in plant and animal cell biology is well established, although poorly understood. Several recent studies have identified aluminum as a potential, although highly controversial, contributory factor in the pathology of Alzheimer disease, amyotrophic lateral sclerosis, and dialysis dementia. For example, aluminum has been found in high concentrations in senile plaques and neurofibrillary tangles, which occur in the brains of subjects with Alzheimer disease. However, a mechanism for the entry of aluminum (Al3+) into the cells of the central nervous system (CNS) has yet to be found. Here we describe a possible route of entry for aluminum into the cells of the CNS via the same high-affinity receptor-ligand system that has been postulated for iron (Fe3+) delivery to neurons and glial cells. These results suggest that aluminum is able to gain access to the central nervous system under normal physiological conditions. Furthermore, these data suggest that the interaction between transferrin and its receptor may function as a general metal ion regulatory system in the CNS, extending beyond its postulated role in iron regulation.
Xenopus Y-box transcription factors: molecular cloning, functional analysis and developmental regulation.
Authors: Tafuri S R , Wolffe A P
Published: May 2006

We describe the cloning and characterization of two cDNAs from Xenopus laevis that encode sequence-specific DNA binding proteins called FRG Y1 and FRG Y2 (frog Y-box proteins 1 and 2). During oogenesis and embryogenesis, the genes encoding these proteins are differentially expressed. FRG Y1 mRNA is present in oocytes, embryos, and all adult tissues examined, whereas FRG Y2 mRNA is found only in testis and immature oocytes. The FRG Y1 and FRG Y2 proteins are shown to stimulate transcription from a promoter containing a Y box (CTGATTGGCCAA). This promoter element is found in both mammalian major histocompatibility complex class II and Xenopus germ-cell-specific genes. FRG Y1, FRG Y2, and a human Y-box binding protein are homologous and represent a distinct family of sequence-specific DNA binding proteins. We identify protamine-like regions that are present within this family of transcription factors, suggesting that they use unusual means of binding to DNA.
Evidence against a requisite role for defective virus in the establishment of persistent hepadnavirus infections.
Authors: Miller R H , Girones R , Cote P J , Hornbuckle W E , Chestnut T , Baldwin B H , Korba B E , Tennant B C , Gerin J L , Purcell R H
Published: May 2006

The factors involved in the establishment of persistent hepadnavirus infection are poorly understood. Recent findings demonstrate that the sequence of the genome of hepatitis B virus (HBV) is variable in infected individuals and that, in some cases, virus mutants predominate. Our objectives in the present study were to analyze the variability of woodchuck hepatitis virus (WHV) genomes in an infected animal and to determine whether sequence heterogeneity played a critical role in the ability of WHV to induce chronic infection. We cloned and determined the complete nucleotide sequence of three supercoiled genomes from an animal that became infected after inoculation with a standardized WHV serum pool (i.e., the WHV7 virus pool). We found that there were four nucleotide substitutions among the three genome sequences as well as a 73-nucleotide deletion in one of the recombinants. DNA transfection experiments revealed that only one of the three recombinants was capable of independent replication. These data suggest that a significant proportion of replicative templates in woodchucks that are infected with WHV are defective virus genomes. Next, we compared the outcome of acute infection after inoculation with a serum pool containing a uniform population of replication competent virus (i.e., the WHV7R pool) with a serum pool composed of WHV genomes of variable sequence. The WHV7R serum pool originated from a woodchuck that became a chronic carrier after in vivo transfection of the liver with the infectious WHV7 recombinant. Neonatal woodchucks were inoculated with 5 x 10(6) WHV genome equivalents of either the WHV7 pool or the WHV7R pool. All animals in the study became acutely infected with WHV. Of the animals infected with the WHV7 serum pool, 65% became chronic carriers, while 80% of the animals infected with the WHV7R serum pool developed chronic infection. Thus, infection of woodchucks with a serum pool containing defective virus resulted in a rate of chronic WHV infection that was similar to, or even lower than, a rate from a pool containing only wild-type virus. This suggests that the presence of defective virus in the inoculum is not a prerequisite for the establishment of persistent hepadnavirus infections.
Effects of auxin and abscisic acid on cytosolic calcium and pH in plant cells.
Authors: Gehring C A , Irving H R , Parish R W
Published: May 2006

Dark-grown corn coleoptiles and parsley hypocotyls and their roots were loaded with acetoxymethyl esterified forms of the Ca2+ indicator fluo-3, and the pH indicator 2',7'-bis (2-carboxyethyl)-5(and-6)-carboxyfluorescein. These tissues were treated with the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin analogue, or abscisic acid (ABA), and the cytosolic pH (pHcyt) and cytosolic Ca2+ ([Ca2+]cyt) changes were monitored by confocal scanning optical microscopy. Over a period of 4 min pHcyt decreased 0.1-0.2 pH unit and [Ca2+]cyt increased from 280 to 380 nM in response to 2,4-D. ABS, on the other hand, induced cytosolic alkalinization of 0.05-0.1 pH unit with a concomitant increase in [Ca2+]cyt from 240 to 320 nM over a 4-min period. Responses similar to these were observed in all the tissues tested. We suggest that pHcyt profoundly influences signaling by[Ca2+]cyt, possibly by regulating Ca2+-protein binding, and that the divergent effects of auxin and ABA on pHcyt underlie their mutual antagonism.
Genetic basis of the difference in alcohol dehydrogenase expression between Drosophila melanogaster and Drosophila simulans.
Authors: Laurie C C , Heath E M , Jacobson J W , Thomson M S
Published: May 2006

Drosophila melanogaster and its sibling species, Drosophila simulans, differ in expression of the enzyme alcohol dehydrogenase (ADH). Adult melanogaster flies that are homozygous for the Slow allozyme have approximately twice the level of ADH activity and crossreacting material as simulans adults. There is no corresponding difference in ADH mRNA, however, so this difference in ADH protein level is evidently due to a difference in the rate of translation of the two RNAs and/or to a difference in protein stability. Here we report an interspecific gene-transfer experiment, using P-element transformation, to determine whether this expression difference is due to genetic background differences between the species (trans-acting modifiers) or to cis-acting factors within the Adh gene. When the Adh genes from D. melanogaster and D. simulans are put into the same genetic background, there is no detectable difference in their level of expression. The level is relatively high in the melanogaster background and relatively low in the simulans background. Therefore, the interspecific difference in Adh expression is due entirely to trans-acting modifiers, in spite of the many sequence differences between the Adh genes of the two species, which include two amino acid substitutions.
Cell mutants defective in synthesizing a heparan sulfate proteoglycan with regions of defined monosaccharide sequence.
Authors: De Agostini A L , Lau H K , Leone C , Youssoufian H , Rosenberg R D
Published: May 2006

We have demonstrated that mouse LTA cells synthesize cell-surface heparan sulfate proteoglycans (HSPGs) with regions of defined monosaccharide sequence that specifically interact with antithrombin (HSPGact). It remains unclear how HSPGact can be generated by a biosynthetic pathway with no simple template for directing the ordered assembly of monosaccharide units. To examine this issue, we treated LTA cells with ethyl methanesulfonate and then isolated seven stable mutants that synthesize only 8-27% of the wild-type HSPGact but produce normal amounts of other HSPGs. These mutants are recessive in nature and fall into at least two different complementation groups. The delineation of the molecular basis of these defects should help to elucidate the manner by which cells synthesize HSPGs with regions of defined monosaccharide sequence.
Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone.
Authors: Celeste A J , Iannazzi J A , Taylor R C , Hewick R M , Rosen V , Wang E A , Wozney J M
Published: May 2006

Characterization of the polypeptides present in bone-inductive protein extracts from bovine bone has led to the cloning of seven regulatory molecules, six of which are distantly related to transforming growth factor beta. The three human bone morphogenetic proteins (BMPs) we describe herein, BMP-5, BMP-6, and BMP-7, show extensive sequence similarity to BMP-2, a molecule that by itself is sufficient to induce de novo bone formation in vivo. The additive or synergistic contribution of these BMP-2-related molecules to the osteogenic activity associated with demineralized bone is strongly implicated by the presence of these growth factors in the most active fractions of highly purified bone extract.
Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.
Authors: Thomale J , Huh N H , Nehls P , Eberle G , Rajewsky M F
Published: May 2006

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.
Antagonistic effects of a covalently dimerized insulin derivative on insulin receptors in 3T3-L1 adipocytes.
Authors: Weiland M , Brandenburg C , Brandenburg D , Joost H G
Published: May 2006

In the present study we describe the antagonistic effects of the covalently dimerized insulin derivative B29,B29'-suberoyl-insulin on insulin receptors in 3T3-L1 mouse cells. In differentiated 3T3-L1 adipocytes, the derivative fully inhibits binding of 125I-labeled insulin to its receptor with about the same affinity as unlabeled insulin. In contrast, the dimerized derivative only partially (approximately 20%) mimics insulin's effects on glucose transport and DNA synthesis in the absence of insulin. In the presence of insulin, the agent competitively inhibits insulin-stimulated DNA synthesis ([3H]thymidine incorporation into total DNA), glucose transport activity (2-deoxyglucose uptake rate), and insulin receptor tyrosine kinase activity. In rat adipocytes, in contrast, the dimerized derivative stimulates glucose transport (initial 3-O-methylglucose as well as 2-deoxyglucose uptake rates) to the same extent as insulin does, and it fails to inhibit the effect of insulin. The data indicate that the dimerized insulin derivative B29,B29'-suberoyl-insulin is an insulin receptor antagonist (partial agonist) which retains a moderate intrinsic activity. The effects of this agent reveal a striking difference in insulin receptor-mediated stimulation of glucose transport between 3T3-L1 fatty fibroblasts and the mature rat adipocyte.
Observation of a water-depletion region surrounding loblolly pine roots by magnetic resonance imaging.
Authors: MacFall J S , Johnson G A , Kramer P J
Published: May 2006

Magnetic resonance imaging was used to study sand containing various amounts of water and roots of loblolly pine planted into similar sand. Spin-lattice (T1) relaxation times of sand with water contents ranging from 0 to 25% (wt/wt) ranged from 472 to 1265 ms and increased with water content. Spin-spin (T2) relaxation times ranged from 54 to 76 ms and did not change in a discernible pattern with water content. Based on water content and measured T1 and T2 values, the signal intensity of sand/water images was predicted to increase with water content in a linear fashion, with the slope of the lines increasing with the time of acquisition repetition (TR). Measured signal intensity from images of sand with various water contents was found to follow a similar pattern. This allows interpretation of dark images of sand/water to be regions of low water content, and bright images to have comparatively greater water content. Images of loblolly pine seedling roots planted in identical sand showed the formation of a distinct water-depletion region first around the woody taproot and later showed the region extended and expanded around the lateral roots and clusters of mycorrhizal short roots. This observation strongly suggests that water uptake is occurring through the suberized region of the woody taproot.
EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages.
Authors: Lancaster J R , Hibbs J B
Published: May 2006

Activated macrophage cytotoxicity is characterized by loss of intracellular iron and inhibition of certain enzymes that have catalytically active nonheme-iron coordinated to sulfur. This phenomenon involves the oxidation of one of the terminal guanidino nitrogen atoms of L-arginine, which results in the production of citrulline and inorganic nitrogen oxides (NO2-, NO3-, and NO). We report here the results of an electron paramagnetic resonance spectroscopic study performed on cytotoxic activated macrophage (CAM) effector cells, which develop the same pattern of metabolic inhibition as their targets. Examination of activated macrophages from mice infected with Mycobacterium bovis (strain bacillus Calmette-Guérin) that were cultured in medium with lipopolysaccharide and L-arginine showed the presence of an axial signal at g = 2.039, which is similar to previously described iron-nitrosyl complexes formed from the destruction of iron-sulfur centers by nitric oxide (NO). Inhibition of the L-arginine-dependent pathway by addition of NG-monomethyl-L-arginine (methyl group on a terminal guanidino nitrogen) inhibits the production of nitrite, nitrate, citrulline, and the g = 2.039 signal. Comparison of the hyperfine structure of the signal from cells treated with L-arginine with terminal guanidino nitrogen atoms of natural abundance N14 atoms or labeled with N15 atoms showed that the nitrosyl group in this paramagnetic species arises from one of these two atoms. These results show that loss of iron-containing enzyme function in CAM is a result of the formation of iron-nitrosyl complexes induced by the synthesis of nitric oxide from the oxidation of a terminal guanidino nitrogen atom of L-arginine.
Isolation of a functional antigen-Ia complex.
Authors: Srinivasan M , Pierce S K
Published: May 2006

The helper T-cell recognition of globular protein antigens requires that the antigen be processed and presented by an I-region associated (Ia)-expressing antigen-presenting cell (APC). Processing involves the uptake of antigen into an intracellular, proteolytic, acidic compartment; release of peptide fragments containing the T-cell antigenic determinant; association of these peptides with Ia; and presentation of these complexes on the cell surface for recognition by the specific T cells. The molecular mechanisms by which processed antigenic peptides associate with Ia within the APC are poorly understood. To date, functional antigen-Ia complexes have not been isolated from cells that have processed native antigens, although the resolution of the structure of a major histocompatibility complex (MHC) class I protein indicates that peptide is bound in a groove between two alpha-helical regions of the molecule and synthetic peptides have been demonstrated to bind purified MHC both in detergent solution and incorporated into planar membranes, where the MHC-peptide complexes function to activate specific T cells. Here we demonstrate that Ia purified from APCs that have processed the native globular protein antigen cytochrome c, when incorporated into lipid membranes, stimulates cytochrome c-specific T cells in the absence of exogenous antigenic peptide. The T-cell response to Ia purified from cytochrome c-pulsed APCs shows the same MHC restriction and antigen fine specificity as the response to antigen-pulsed APCs. Indeed, T-cell recognition of pigeon cytochrome c (Pc) shows a well documented high-affinity heteroclitic cross-reaction to insect cytochromes c-namely, those of Drosophila melanogaster (DMc) and tobacco hornworm moth (THMc). The same heteroclitic response is observed when purified Ia from unpulsed cells, incorporated into lipid membranes, is used to present antigenic peptides of Pc and of THMc. Significantly, Ia purified from APCs that have processed DMc is approximately 50-fold more active in stimulating specific T cells compared to Ia purified from APCs that have processed Pc. The peptide-Ia complex isolated here may provide the necessary material for analysis of the physiochemical properties of the processed form of the antigen that is produced by the APC and associates with Ia.
Host regulation of the cauliflower mosaic virus multiplication cycle.
Authors: Covey S N , Turner D S , Lucy A P , Saunders K
Published: May 2006

The DNA genome of cauliflower mosaic virus (CaMV) replicates in the cytoplasm of infected plant cells by reverse transcription of an RNA template. Viral RNA is generated in the nucleus by transcription of an episomal minichromosome containing supercoiled DNA. We have assessed the relative activities of the nuclear and cytoplasmic phases of the CaMV multiplication cycle by monitoring unencapsidated viral DNA forms and polyadenylylated RNAs in different organs of one host plant and in different host species. Systemically infected leaves of a highly susceptible host, turnip (Brassica rapa), contained abundant 35S RNA and 19S RNA transcripts and unencapsidated reverse transcription products but relatively little supercoiled DNA. In contrast, supercoiled DNA accumulated in roots and other tissues of turnip plants but without significant amounts of steady-state viral RNA. Infected but asymptomatic leaves of a less susceptible CaMV host, kohlrabi (Brassica oleracea), contained supercoiled DNA almost exclusively but negligible viral RNA and DNA products of reverse transcription. An allotetraploid species, rape (Brassica napus), exhibited infection characteristics and minichromosome expression levels intermediate between the other two species from which it was derived. We conclude that expression of the CaMV minichromosome is a key phase of the virus multiplication cycle, which is regulated differentially in organs of a highly susceptible host species. Furthermore, this regulation exhibits genetic variation among different Brassica species and controls host susceptibility to CaMV infection.
1H NMR visibility of mammalian glycogen in solution.
Authors: Zang L H , Rothman D L , Shulman R G
Published: May 2006

High-resolution 1H NMR spectra of rabbit liver glycogen in 2H2O were obtained at 500 MHz, and several resonances were assigned by comparison with the chemical shifts of alpha-linked diglucose molecules. The NMR relaxation times T1 and T2 of glycogen in 2H2O were determined to be 1.1 and 0.029 s, respectively. The measured natural linewidth of the carbon-1 proton (12 +/- 2 Hz) is in excellent agreement with that calculated from T2. The visibility measurements made by digesting glycogen and comparing glucose and glycogen signal intensities demonstrate that in spite of the very high molecular weight, all of the proton nuclei in glycogen contribute to the NMR spectrum. The result is not unexpected, since 100% NMR visibility was previously observed from the carbon nuclei of glycogen, due to the rapid intramolecular motions.
Phospholipids enhance the binding of peptides to class II major histocompatibility molecules.
Authors: Roof R W , Luescher I F , Unanue E R
Published: May 2006

The binding of a lysozyme and ovalbumin peptide to purified class II major histocompatibility molecules in detergents was increased by the addition of certain lipids. Natural lipids from B lymphoma cells enhanced the binding and so did phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylethanolamine, sphingomyelin, and cholesterol had no effect. There was no major difference between the effects of a phospholipid and its lyso derivative. As studied with phosphatidylcholine, the increase in peptide binding was also dependent on the fatty acid composition of the lipid. The binding affinity was increased 10- to 50-fold in the presence of lipid as a result of an increase in the association rate while the off-rate remained essentially unchanged. Our results suggest that lipids, directly or indirectly, induce conformational changes in class II molecules that favor their peptide-binding property.
Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.
Authors: Lowy R J , Sarkar D P , Chen Y , Blumenthal R
Published: May 2006

We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.
Biochemical identification of glycosylation inhibiting factor.
Authors: Katamura K , Iwata M , Mori A , Ishizaka K
Published: May 2006

Rat monoclonal antibody against mouse glycosylation inhibiting factors was obtained, and radiolabeled glycosylation inhibiting factors from the mouse T-cell hybridoma, 231F1 cells, were purified by using the monoclonal antibody and antilipomodulin antibody. Analysis of the affinity-purified lymphokine by PAGE demonstrated two proteins of 14.4 kDa and 41 kDa. Both proteins migrated similarly under the reduced and unreduced conditions, indicating that each of the two species consist of a single polypeptide chain. Biologic activity of the lymphokine was recovered by extraction of the proteins from gel slices followed by renaturation. Evidence was obtained that suggested the 14.4-kDa peptide was a degradation product of the 41-kDa molecules. The 14.4-kDa peptide was also recovered by extraction of the surface-labeled 231F1 cells with Ca2+ chelator, indicating that the glycosylation inhibiting factor is associated with the cell surface.
Nuclear myxovirus-resistance protein Mx is a minor histocompatibility antigen.
Authors: Speiser D E , Zürcher T , Ramseier H , Hengartner H , Staeheli P , Haller O , Zinkernagel R M
Published: May 2006

Minor histocompatibility antigens (MiHAgs) cause slow-to-rapid organ transplant rejection by immunocompetent hosts and mild-to-severe graft-versus-host reactions in immunosuppressed hosts. MiHAgs are allelic forms of major histocompatibility complex (MHC) class I-restricted self-antigens recognized by cytotoxic T cells and usually are defined immunogenetically. Although structurally not identified as yet, it is assumed that MiHAgs are internal cell antigens that are processed and then presented by MHC class I proteins similar to viral antigens. To define a MiHAg both molecularly and functionally, we took advantage of the allelic difference of the structurally characterized intracellular myxovirus-resistance protein (Mx) and investigated its antigenicity. Skin grafts from congenic Mx+ mice carrying the functional Mx1 gene were rejected by mice lacking a functional Mx1 gene (Mx- mice). In parallel, cytotoxic MHC class I-restricted effector T cells specific for Mx protein and the H-2Kk antigen (but not for several other allelic H-2 antigens) were strongly induced in Mx- mice immunized with spleen cells from interferon-treated Mx+ mice. These data show that allelic forms of cell internal proteins presented by MHC class I may act as MiHAgs.
Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.
Authors: Casadei J , Powell M J , Kenten J H
Published: May 2006

A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli. The gene was expressed as apoaequorin and, by using luciferin isolated from Renilla reniformis, its activity was found essentially identical to native aequorin. The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion of apoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody. The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene. Transfection of the chimeric gene into a cell line expressing the complementary lambda 1 light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten. The Ca2(+)-dependent bioluminescent activity of aequorin was also recovered.
Neoplastic transformation and tumorigenesis by the human protooncogene MYC.
Authors: Ramsay G M , Moscovici G , Moscovici C , Bishop J M
Published: May 2006

Damage to the protooncogene MYC has been implicated in the genesis of diverse human tumors, but the tumorigenic potential of the isolated gene has been disputed. Here we report the use of a retroviral vector to test the potency of human MYC for neoplastic transformation in avian cells. We found that sustained and abundant expression of MYC can transform both embryonic fibroblasts and hematopoietic cells and elicit granulocytic leukemias in chickens. Transformation by MYC is accompanied by changes in diverse aspects of cellular phenotype, including morphology, ability to grow in suspension, rate of proliferation, the structure of the cytoskeleton, and the composition of the extracellular matrix. Nevertheless, the biological potency of MYC is inherently constrained when compared to that of the retroviral oncogene v-myc. Our findings enlarge on previous descriptions of neoplastic transformation by MYC and sustain the view that ungoverned expression of the gene can contribute to the genesis of human tumors.
K(+)-evoked Müller cell depolarization generates b-wave of electroretinogram in toad retina.
Authors: Wen R , Oakley B
Published: May 2006

We tested the hypothesis that a light-evoked increase in [K+]o produces a depolarization of the Müller cell membrane, which in turn generates the electroretinogram b-wave current. Using Bufo marinus isolated retinas and K(+)-selective microelectrodes, we recorded two distinct light-evoked increases in extracellular K+ concentration: one in the inner plexiform layer and the other near the outer plexiform layer; the "distal" K+ increase was found over only 10-microns depth and had a maximum amplitude of 0.3 mM. We also recorded the electroretinogram and the light-evoked responses of rods and Müller cells. After correction for the response time of the K(+)-selective microelectrode, the waveforms of all three of these responses were almost exactly as predicted by an earlier computer simulation of the K+/Müller cell hypothesis of the b-wave by Newman and Odette [Newman, E.A. & Odette, L.L. (1984) J. Neurophysiol. 51, 164-182]. The distal K+ increase and the b-wave varied in a similar manner as a function of stimulus irradiance. Superfusion with 0.2 mM Ba2+ attenuated both the Müller cell depolarization and the b-wave by approximately 65% but had no significant effect upon the distal K+ increase. Because Ba2+ reduces K+ conductance of Müller cells, these results are very strong support of the K+/Müller cell hypothesis of the origin of the electroretinogram b-wave; the light-evoked increase in extracellular potassium concentration still is present during superfusion with Ba2+, but the K(+)-evoked Müller cell depolarization and the b-wave are decreased in amplitude because Müller cell K+ conductance is reduced.
Rhabdomyosarcoma-associated locus and MYOD1 are syntenic but separate loci on the short arm of human chromosome 11.
Authors: Scrable H J , Johnson D K , Rinchik E M , Cavenee W K
Published: May 2006

The MYOD1 locus is preferentially expressed in skeletal muscle and at higher levels in its related neoplasm, rhabdomyosarcoma. We have combined physical mapping of the human locus with meiotic and physical mapping in the mouse, together with synteny homologies between the two species, to compare the physical relationship between MYOD1 and the genetically ascertained human rhabdomyosarcoma-associated locus. We have determined that the myogenic differentiation gene is tightly linked to the structural gene for the M (muscle) subunit of lactate dehydrogenase in band p15.4 on human chromosome 11 and close to the p and Ldh-1 loci in the homologous region of mouse chromosome 7. Because the rhabdomyosarcoma locus maps to 11p15.5, MYOD1 is very unlikely to be the primary site of alteration in these tumors. Further, these analyses identify two syntenic clusters of muscle-associated genes on the short arm of human chromosome 11, one in the region of rhabdomyosarcoma locus that includes IGF2 and TH and the second the tightly linked MYOD1 and LDHA loci, which have been evolutionarily conserved in homologous regions of both the mouse and the rat genomes.
Recombinant human bone morphogenetic protein induces bone formation.
Authors: Wang E A , Rosen V , D'Alessandro J S , Bauduy M , Cordes P , Harada T , Israel D I , Hewick R M , Kerns K M , LaPan P
Published: May 2006

We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans.
Antibody-targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA selectively inhibit viral replication.
Authors: Leonetti J P , Machy P , Degols G , Lebleu B , Leserman L
Published: May 2006

Mouse L929 cells were incubated with antibody-targeted liposomes containing oligodeoxyribonucleotides (oligomers). When the oligomer was a 15-mer complementary to the 5'-end region of the mRNA encoding the N protein of vesicular stomatitis virus, the cells became less permissive for multiplication of that virus; greater than 95% reduction of viral multiplication was achieved. Protection was not seen for "empty" liposomes, liposomes containing a random oligomer sequence, or liposomes containing a sequence complementary to the 5' end of c-myc protooncogene mRNA targeted by the same antibody, nor was it seen when the liposomes containing the N-protein antisense oligomer were targeted by an antibody that does not bind to L929 cells. Antibody-bearing liposomes containing antisense oligomers thus have a double specificity: a particular cell selected by the targeting antibody on the liposome and a particular mRNA in the cell selected by sequence complementarity with the liposome-encapsulated oligomer. Nonencapsulated oligomers are sensitive to nucleases and usually must be administered to cells at high concentrations. Oligomers encapsulated in liposomes resist DNase and are active in amounts 1-2 orders of magnitude lower than for those reported for unencapsulated oligomer sequences.
Synaptic currents in cerebellar Purkinje cells.
Authors: Konnerth A , Llano I , Armstrong C M
Published: May 2006

Cerebellar Purkinje cells are known to receive strong excitatory input from two major pathways originating outside the cerebellum and inhibitory input from two types of neurons in the cerebellar cortex. The functions and synaptic strengths of these pathways are only partially known. We have used the patch-clamp technique applied to Purkinje cells in thin slices of rat cerebellum to measure directly the postsynaptic currents arising from the two major excitatory pathways and one of the inhibitory inputs. Inhibitory synaptic currents occur spontaneously with high frequency and are variable in amplitude, ranging, in our recording conditions with high internal Cl-, from less than 100 pA to more than 1 nA. These currents are blocked by the gamma-aminobutyrate type A antagonist bicuculline. One of the excitatory inputs is all or none. For threshold stimulation, the synaptic current is either full amplitude, when the presynaptic fiber is successfully stimulated, or completely absent. This synaptic current is often larger than 1 nA and is virtually eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione, a blocking agent thought to be specific for glutamate receptors that are not of the N-methyl-D-aspartate type. Its all-or-none character identifies it as arising from a climbing-fiber synapse. The other excitatory input produces a synaptic current that is smoothly graded as a function of stimulus intensity. This response we believe arises from the stimulation of mossy fibers or granule cells. The synaptic current associated with this input is also largely eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione.
Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.
Authors: Medin J A , Hunt L , Gathy K , Evans R K , Coleman M S
Published: May 2006

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.
The t(10;14)(q24;q11) of T-cell acute lymphoblastic leukemia juxtaposes the delta T-cell receptor with TCL3, a conserved and activated locus at 10q24.
Authors: Zutter M , Hockett R D , Roberts C W , McGuire E A , Bloomstone J , Morton C C , Deaven L L , Crist W M , Carroll A J , Korsmeyer S J
Published: May 2006

We cloned the t(10;14) recurrent translocation from CD3-negative T-cell acute lymphoblastic leukemia cells. The breakpoint at 14q11 involved an intermediate rearrangement of the delta T-cell receptor locus, suggesting that the translocation arose at the time of antigen receptor assemblage. Translocation introduced chromosome segment 10q24 as proven by hybridization of a breakpoint-derived probe to flow-sorted chromosomes and metaphase chromosomes. Two t(10;14) breakpoints were clustered within a 600-base-pair region of 10q24 but no heptamer-spacer-nonamer motifs resembling T-cell receptor/immunoglobulin rearrangement signals were noted at the breakpoint. A locus distinct from terminal deoxynucleotidyltransferase was found at 10q24. Evolutionarily conserved regions surrounding the 10q24 breakpoint were examined for transcriptional activity. A region telomeric to the 10q24 breakpoint, expected to translocate to the der(14) chromosome, recognized an abundant 2.9-kilobase RNA in a t(10;14) T-cell leukemia. This locus was not active in a variety of other normal and neoplastic T cells, arguing that it was deregulated by the introduction of the T-cell receptor. This locus is a candidate for a putative protooncogene, TCL3, involved in T-cell neoplasia.
Constructing deletions with defined endpoints in Drosophila.
Authors: Cooley L , Thompson D , Spradling A C
Published: May 2006

Chromosomes bearing small deletions are valuable tools in Drosophila genetics. We have investigated a method for efficiently constructing precise chromosomal deficiencies. Two P transposable elements were positioned within a progenitor strain at the sites of the desired deletion endpoints. Deletions spanning the two transposons were recovered at high frequency when P element transposase was expressed in these flies, but only if the flanking P elements were in a cis rather than a trans configuration. Appropriate progenitor strains can now be constructed to delete virtually any chromosomal region by utilizing an extensive collection of lines containing single P element insertions throughout the Drosophila genome.
Immunocytochemical localization of the alpha subspecies of protein kinase C in rat brain.
Authors: Ito A , Saito N , Hirata M , Kose A , Tsujino T , Yoshihara C , Ogita K , Kishimoto A , Nishizuka Y , Tanaka C
Published: May 2006

The distribution of the alpha subspecies of protein kinase C (PKC) in rat brain was demonstrated immunocytochemically by using polyclonal antibodies raised against a synthetic oligopeptide corresponding to the carboxyl-terminal sequence of alpha-PKC. The alpha-PKC-specific immunoreactivity was widely but discretely distributed in both gray and white matter. The immunoreactivity was associated predominantly with neurons, particularly with perikaryon, dendrite, or axon, but little was seen in the nucleus. Glial cells expressed this PKC subspecies poorly, if at all. The highest density of immunoreactivity was seen in the olfactory bulb, septohippocampal nucleus, indusium griseum, islands of Calleja, intermediate part of the lateral septal nucleus, and Ammon's horn. A moderately high density of the immunoreactivity was seen in the anterior olfactory nucleus, anterior commissure, cingulate cortex, dentate gyrus, compact part of the substantia nigra, interpeduncular nucleus, inferior olive, and olivocerebellar tract. This distribution pattern was consistent with that obtained by in situ hybridization histochemistry. The distribution of alpha-PKC immunoreactivity was different from that of beta I-, beta II-, and gamma-PKC immunoreactivity. These findings suggest that alpha-PKC is involved heavily in the control of specific functions of some restricted neurons.