bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo induction of antigen-specific transplantation tolerance to Qa1a by exposure to alloantigen in the absence of T-cell help.
Authors: Rees M A , Rosenberg A S , Munitz T I , Singer A
Published: May 2006

A goal of transplantation immunology is to be able to induce antigen-specific tolerance in transplant recipients. In the present study we describe an in vivo model of antigen-specific transplantation tolerance to skin allografts using mice congenic at Qa1, a ubiquitously expressed class I-like molecule encoded to the right of H-2D. B6 mice are deficient in Qa1a-specific T-helper cells and only reject Qa1a disparate tail skin grafts when a second graft expressing additional helper determinants is also present. We report that animals initially engrafted with Qa1a disparate skin, in the absence of any source of additional help, are rendered tolerant to Qa1a disparate skin allografts despite the subsequent presence of inducer skin grafts expressing additional helper allodeterminants. The nonresponsive state is Qa1a-specific, because HY-bearing inducer grafts are rejected normally. In vitro, Qa1a-tolerant animals are specifically unable to generate anti-Qa1a T-killer cells, which provides the cellular basis for their failure in vivo to reject Qa1a skin allografts. Thus, initial exposure to Qa1a allodeterminants, in the absence of T-cell help, leads to a state of Qa1a-specific transplantation tolerance. This study suggests that antigen-specific transplantation tolerance may be induced by exposing naive T-killer cells to tissue alloantigens under conditions in which T-cell help is not generated.
Paraquat toxicity is increased in Escherichia coli defective in the synthesis of polyamines.
Authors: Minton K W , Tabor H , Tabor C W
Published: May 2006

We have shown that toxicity of paraquat for Escherichia coli is increased over 10-fold in strains defective in the biosynthesis of spermidine compared to isogenic strains containing spermidine. The increased sensitivity of these spermidine-deficient mutants to paraquat is eliminated by growth in medium containing spermidine or by endogenous supplementation of spermidine by the use of a speE+D+ plasmid. No paraquat toxicity is seen in the absence of oxygen, even in amine-deficient strains, indicating that superoxide is the agent responsible for the increased toxicity. However, the specific mechanisms responsible for the increased paraquat toxicity in the spermidine-deficient mutants remain to be determined. The marked sensitivity to paraquat of E. coli deficient in spermidine is of particular interest, since such mutants have no other phenotypic properties that can be easily assayed. This increased sensitivity has been used as the basis of a convenient method for scoring for mutants in polyamine biosynthesis and for the detection of plasmids containing the biosynthetic genes.
Human cytotoxic T-cell responses against Epstein-Barr virus nuclear antigens demonstrated by using recombinant vaccinia viruses.
Authors: Murray R J , Kurilla M G , Griffin H M , Brooks J M , Mackett M , Arrand J R , Rowe M , Burrows S R , Moss D J , Kieff E
Published: May 2006

The potentially pathogenic effects of infection with Epstein-Barr virus (EBV), a B-lymphotropic agent with cell growth-transforming potential, are contained in healthy virus carriers by virus-specific cytotoxic T-lymphocyte (CTL) surveillance. The target antigens against which such CTL responses are directed are yet undefined, but the antigens probably derived from one or more of the EBV "latent" proteins constitutively expressed in virus-transformed B cells. We have analyzed target specificity of CTL responses from two EBV-immune donors that are preferentially reactive against autologous cells transformed with type A but not with type B virus isolates. Coding sequences for four EBV latent proteins with allelic polymorphism between A and B virus types--namely, the EBV nuclear antigens (EBNAs) EBNA 2, EBNA 3a, EBNA 3c, and EBNA leader protein--have been introduced into vaccinia virus vectors under control of vaccinia promoter P7.5 and used to express relevant EBNA proteins in appropriate target cells. Thus the CTL response from one donor has been mapped to type A EBNA 2 protein and from a second donor to type A EBNA 3a protein. Thereafter, a series of recombinant vaccinia viruses were constructed that carried specific internal deletions within the EBNA 2 type A coding sequence; by using these vectors, the above EBNA 2 type A-specific CTL response was shown to be directed against an epitope within a 100-amino acid fragment near the N terminus of the protein. This work clearly shows human CTL recognition of virus-coded nuclear antigens in the EBV system; moreover, it establishes an experimental approach that can be extended to all EBV latent proteins and to the more common CTL responses that cross-react against type A and type B virus isolates.
Glyoxal oxidase of Phanerochaete chrysosporium: its characterization and activation by lignin peroxidase.
Authors: Kersten P J
Published: May 2006

Glyoxal oxidase (GLOX) is an extracellular H2O2-generating enzyme produced by ligninolytic cultures of Phanerochaete chrysosporium. The production, purification, and partial characterization of GLOX from agitated cultures are described here. High-oxygen levels are critical for GLOX production as for lignin peroxidase. GLOX purified by anion-exchange chromatography appears homogeneous by NaDod-SO4/PAGE (molecular mass = 68 kDa). However, analysis by isoelectric focusing indicates two major bands (pI 4.7 and 4.9) that stain as glycoproteins as well as for H2O2-producing activity in the presence of methylglyoxal. Purified GLOX shows a marked stimulation in activity when incubated with Cu2+; full activation takes more than 1 hr with 1 mM CuSO4 at pH 6. The steady-state kinetic parameters for the GLOX oxidation of methylglyoxal, glyceraldehyde, dihydroxyacetone, glycolaldehyde, acetaldehyde, glyoxal, glyoxylic acid, and formaldehyde, were determined by using a lignin peroxidase coupled-assay at pH 4.5. Of these substrates, the best is the extracellular metabolite methylglyoxal with a Km of 0.64 mM an apparent rate of catalysis, kcat, of 198 s1 under air-saturated conditions. The Km for oxygen is greater than the concentration of oxygen possible at ambient pressure--i.e., >1.3 mM at 25 degrees C. Importantly, oxygen-uptake experiments show that purified GLOX is inactive unless coupled to the peroxidase reaction. With this coupled reaction, for each mol of methylglyoxal, veratryl alcohol (a lignin peroxidase substrate), and oxygen consumed, 1 mol each of pyruvate and veratraldehyde is produced. The importance of these results is discussed in relation to the physiology of lignin biodegradation and possible extracellular regulatory mechanisms for the control of oxidase and peroxidase activities.
Readily available fluorescein isothiocyanate-conjugated antibodies can be easily converted into targeted phototoxic agents for antibacterial, antiviral, and anticancer therapy.
Authors: Devanathan S , Dahl T A , Midden W R , Neckers D C
Published: May 2006

Fluorescein-labeled antibodies have little, if any, photodynamic effect because energy acquired by light absorption is rapidly dissipated in fluorescence. However, they can be easily and efficiently converted to selective photodynamic sensitizers by iodination under mild conditions. We have outlined general experimental procedures that can be used to turn a fluorescein-labeled anti-Escherichia coli antibody into a photodynamic sensitizer that selectively kills E. coli while sparing closely related Salmonella typhimurium. These results demonstrate that iodination did not destroy the specificity or activity of the antibody. This technique should be applicable to the large number of fluoresceinated antibodies that are commercially available. Thus, this strategy provides a simple way to rapidly prepare a large number of targeted phototoxic agents that can be used for the selective destruction with light of nearly any type of tissue or organism.
Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.
Authors: Jackson J H , Cochrane C G , Bourne J R , Solski P A , Buss J E , Der C J
Published: May 2006

Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.
Targeted gene disruption of the endogenous c-abl locus by homologous recombination with DNA encoding a selectable fusion protein.
Authors: Schwartzberg P L , Robertson E J , Goff S P
Published: May 2006

We have introduced a substitution mutation into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene. Model constructs were initially generated that consisted of a promoterless selectable neomycin resistance marker inserted into the v-abl gene of the complete Abelson murine leukemia virus genome, designed to be expressed either as a fusion protein or by translational restart. Tests of these viral genomes for transmission of v-abl and neo markers showed more stable coexpression in a protein fusion construct. The neo fusion was subcloned from this v-abl construct into a promoterless c-abl fragment, and the resulting DNA was used to transform embryonic stem cells. Direct screening of genomic DNAs showed that a high proportion of drug-resistant clones arose from homologous recombination into the endogenous c-abl locus.
Protein model structure evaluation using the solvation free energy of folding.
Authors: Chiche L , Gregoret L M , Cohen F E , Kollman P A
Published: May 2006

A systematic study of solvation free energy of folding for proteins with known crystallographic structures is presented. There is a linear relationship between the solvation free energy of folding and the protein size. This relationship, which can be rationalized by a simple model of chain folding, allows prediction of the solvation free energy of folding for proteins for which no high resolution structures are available. All misfolded structures analyzed show solvation free energies higher than predicted; however, some of the misfolded structures have values close enough to the predicted values so that one must be very careful when using such a criterion to check the correctness of a protein model.
Method to identify genomic targets of DNA binding proteins.
Authors: Sompayrac L , Danna K J
Published: May 2006

We have devised a cyclical immunoprecipitation protocol that can be used to identify and clone a specific DNA sequence that is recognized by a DNA binding protein, even if that sequence is present in only one copy in the genome of a mammal. As an example, we have used this procedure to purify mouse genomic sequences to which the simian virus 40 tumor (T) antigen binds.
Molecular characterization and biological function of the movement protein of tobacco mosaic virus in transgenic plants.
Authors: Deom C M , Schubert K R , Wolf S , Holt C A , Lucas W J , Beachy R N
Published: May 2006

We previously demonstrated, in transgenic tobacco plants, that the role of the movement protein (MP) of tobacco mosaic virus is to facilitate the cell-to-cell spread of viral progeny during infection. An analysis of different tissues of these transgenic plants indicated that the MP accumulated in leaf, stem, and root tissue. The highest levels were detected in older leaves. The relative levels of MP in leaf tissue from transgenic plants were equivalent to, or higher than, the levels of MP in tobacco mosaic virus-infected leaf tissue. Results of subcellular fractionation of homogenates of transgenic leaf tissue showed that the MP was most abundant in the cell wall fraction of older leaves and that the protein remained at high levels in the cell wall fraction as the leaves continued to age. Significant levels of the MP were detected in a crude membrane/organelle fraction and a soluble fraction in younger leaves but decreased to low levels in older leaves. These results suggest that the MP accumulates and is stable in cell walls. We have previously shown that the MP modifies the molecular exclusion limit of plasmodesmata, which is consistent with the hypothesis that plant viruses move from cell to cell through altered plasmodesmata. We show here that the ability of the tobacco mosaic virus MP to modify the molecular exclusion limit of plasmodesmata in tobacco depends on the developmental stage of the leaf. The implications of these findings on understanding virus movement and how plasmodesmata function are discussed.
Inhibition of influenza virus replication by phosphorothioate oligodeoxynucleotides.
Authors: Leiter J M , Agrawal S , Palese P , Zamecnik P C
Published: May 2006

Oligodeoxynucleotides (ODNs) were synthesized and tested for their antiviral activity against influenza viruses. ODNs corresponded to the polymerase PB1 gene of either influenza A/WSN/33 virus or influenza C/JJ/50 virus. All compounds were 20 nucleotides long, including control ODNs containing mismatches. The phosphodiester ODNs (O-ODNs) failed to inhibit replication of influenza A and influenza C viruses at concentrations up to 80 microM, possibly due to intracellular nuclease digestion of the unmodified oligomers. By contrast, the phosphorothioate derivatives (S-ODNs) were found to inhibit replication of both influenza A and influenza C virus. The antiviral effect of S-ODNs against influenza A virus was found at concentrations as low as 1.25 microM and was present with mismatched oligomers. In the case of influenza C virus, the S-ODN complementary to the 3' end of the viral RNA of the PB1 gene revealed a sequence-specific antiviral activity at a concentration of 20 microM. (At the same concentration, S-ODNs with one or three mismatches showed little or no antiviral activity.) Reduction in plaque number reached six logarithms when this sequence-specific S-ODN was used at a concentration of 80 microM.
K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.
Authors: Schmid-Antomarchi H , Amoroso S , Fosset M , Lazdunski M
Published: May 2006

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.
Amino acid residues that affect interaction of tissue-type plasminogen activator with plasminogen activator inhibitor 1.
Authors: Madison E L , Goldsmith E J , Gerard R D , Gething M J , Sambrook J F , Bassel-Duby R S
Published: May 2006

Fibrinolysis is regulated in part by the interaction between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor of the serpin family). It is known from our earlier work that deletion of a loop of amino acids (residues 296-302) from the serine protease domain of t-PA suppresses the interaction between the two proteins without altering the reactivity of t-PA towards its substrate, plasminogen. To define more precisely the role of individual residues within this loop, we have used site-directed mutagenesis to replace Lys-296, Arg-298, and Arg-299 with negatively charged glutamic residues. Replacement of all three positively charged amino acids generates a variant of t-PA that associates inefficiently with PAI-1 and is highly resistant to inhibition by the serpin. Two t-PAs with point mutations (Arg-298----Glu and Arg-299----Glu) are partially resistant to inhibition by PAI-1 and associate with the serpin at intermediate rates. Other point mutations (Lys-296----Glu, His-297----Glu, and Pro-301----Gly) do not detectably affect the interaction of t-PA with PAI-1. None of these substitutions has a significant effect on the rate of catalysis by t-PA or on the affinity of the enzyme for its substrate, plasminogen. On the basis of these results, we propose a model in which positively charged residues located in a surface loop near the active site of t-PA form ionic bonds with complementary negatively charged residues C-terminal to the reactive center of PAI-1.
Sexual reproduction as an adaptation to resist parasites (a review).
Authors: Hamilton W D , Axelrod R , Tanese R
Published: May 2006

Darwinian theory has yet to explain adequately the fact of sex. If males provide little or no aid to offspring, a high (up to 2-fold) extra average fitness has to emerge as a property of a sexual parentage if sex is to be stable. The advantage must presumably come from recombination but has been hard to identify. It may well lie in the necessity to recombine defenses to defeat numerous parasites. A model demonstrating this works best for contesting hosts whose defense polymorphisms are constrained to low mutation rates. A review of the literature shows that the predictions of parasite coevolution fit well with the known ecology of sex. Moreover, parasite coevolution is superior to previous models of the evolution of sex by supporting the stability of sex under the following challenging conditions: very low fecundity, realistic patterns of genotype fitness and changing environment, and frequent mutation to parthenogenesis, even while sex pays the full 2-fold cost.
Transdominant inhibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors.
Authors: Treadway J L , Morrison B D , Soos M A , Siddle K , Olefsky J , Ullrich A , McClain D A , Pessin J E
Published: May 2006

Classical insulin and insulin-like growth factor I (IGF-I) receptors exist as well defined alpha 2 beta 2 heterotetrameric complexes that are assembled from two identical alpha beta heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form alpha 2 beta 2 insulin/IGF-I hybrid receptor complexes in vivo and in vitro. We have utilized hybrid receptor complexes to examine ligand-stimulated transmembrane signaling of wild-type insulin (alpha beta INS.WT) or IGF-I (alpha beta IGF.WT) half-receptors assembled with a kinase-defective insulin half-receptor mutant (alpha beta INS.A/K). In vitro assembly of either (alpha beta)IGF.WT/(alpha beta)INS.A/K or (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptors resulted in decreased substrate protein kinase activity. The degree of protein kinase inactivation directly correlated with the amount of immunologically cross-reactive hybrid receptors formed. In contrast to substrate kinase activity, insulin-stimulated autophosphorylation of the (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild-type (alpha beta)INS.WT/(alpha beta)INS.WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated beta-subunit insulin half-receptor with the kinase-defective half-receptor, (alpha beta)INS. delta CT/(alpha beta)INS.A/K, demonstrated the exclusive autophosphorylation of the (alpha beta)INS.A/K half-receptor beta subunit. These results demonstrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional beta subunits within the alpha 2 beta 2 heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.
Structural model of the nucleotide-binding conserved component of periplasmic permeases.
Authors: Mimura C S , Holbrook S R , Ames G F
Published: May 2006

The amino acid sequences of 17 bacterial membrane proteins that are components of periplasmic permeases and function in the uptake of a variety of small molecules and ions are highly homologous to each other and contain sequence motifs characteristic of nucleotide-binding proteins. These proteins are known to bind ATP and are postulated to be the energy-coupling components of the permeases. Several medically important eukaryotic proteins, including the multidrug-resistance transporters and the protein encoded by the cystic fibrosis gene, are also homologous to this family. By multiple sequence alignment of these 17 proteins, the consensus sequence, secondary structure, and surface exposure were predicted. The secondary structural motifs that are conserved among nucleotide-binding proteins were identified in adenylate kinase, p21ras, and elongation factor Tu by superposition of their known tertiary structures. The equivalent secondary structural elements in the predicted conserved component were located. These, together with sequence information, served as guides for alignment with adenylate kinase. A model for the structure of the ATP-binding domain of the permease proteins is proposed by analogy to the adenylate kinase structure. The characteristics of several permease mutations and biochemical data lend support to the model.
Ferritin mRNA: interactions of iron regulatory element with translational regulator protein P-90 and the effect on base-paired flanking regions.
Authors: Harrell C M , McKenzie A R , Patino M M , Walden W E , Theil E C
Published: May 2006

The ferritin iron regulatory element (IRE), a conserved sequence of 28 nucleotides in a hairpin loop, is a conserved mRNA-specific translational regulatory element; flanking the IRE are regions of varying sequence, which form 9-17 base pairs close to the 5' cap. P-90 is a ferritin mRNA-specific translation regulatory protein purified from animal liver and reticulocytes. To study the P-90-RNA interaction, protein nucleases (RNase S1 and T1) and chemical nucleases FeEDTA and/or 1,10-phenanthroline-Cu were used as probes of an oligonucleotide (n = 55), containing the IRE and flanking regions (FL), and natural ferritin mRNA. Footprints and "toeprints" showed that P-90 binding was confined to the stem and loop of the IRE itself. However, P-90 altered the structure of the flanking region by increasing base stacking or helicity (RNase V1 sensitivity). Comparison of the reactivity of the IRE and flanking regions in natural mRNA and the 55-mer showed that long-range interactions included protecting bulges, single-stranded, and stacked regions from protein nucleases as well as stabilizing the P-90-RNA interaction. Structural integration of the IRE with the base-paired flanking regions was indicated by common features of reactivity (periodic hypersensitivity to FeEDTA) and changes in the FL region caused by P-90. The increased secondary structure of the IRE flanking regions caused by P-90 binding to the IRE provides a likely mechanism for blocking initiation of ferritin mRNA translation, since the combined structure (IRE + FL) is so close (8-17 nucleotides) to the cap.
Brain is the major site of estrogen synthesis in a male songbird.
Authors: Schlinger B A , Arnold A P
Published: May 2006

The neural system controlling song in passerine birds can undergo striking morphological and functional changes during both development and adulthood, and many of these changes are regulated by estrogenic hormones. Estrogens circulate at high levels in blood of male songbirds and persist after castration. We measured the activity of aromatase, the enzyme that converts androgens to estrogens, in various tissues from adult male and female zebra finches. As expected, aromatase activity was present in male hypothalamus/preoptic area and pituitary and female ovary, but aromatase was unusually active in whole telencephalon of males and females. By contrast, activity was undetected in testes, adrenals, or other tissues of males. These results suggest that brain is the source of circulating estrogens in the male zebra finch and that estrogen actions on the song system result from local rather than peripheral aromatization.
Single type-2 astrocytes show multiple independent sites of Ca2+ signaling in response to histamine.
Authors: Inagaki N , Fukui H , Ito S , Yamatodani A , Wada H
Published: May 2006

Intracellular Ca2+ plays an important role in signal transduction as a second messenger. In various types of cells, inositol 1,4,5-trisphosphate-induced elevations of intracellular free Ca2+ concentration ([Ca2+]i) have been reported to be uniform in single cells or originate at discrete sites from which they then propagate throughout the cells. These observations so far imply that a single cell functions as a minimal unit for inositol 1,4,5-trisphosphate-induced Ca2+ signaling. In this study, we examined the effects of histamine on [Ca2+]i of type-2 astrocytes using fura-2-based digital imaging fluorescence microscopy and found an unusual type of Ca2+ signaling in these cells. Histamine induced [Ca2+]i elevation in type-2 astrocytes by means of histamine H1 receptors. Submaximal concentrations of histamine (10(-7)-10(-6) M) evoked multiple sites of oscillatory [Ca2+]i elevation in single type-2 astrocytes. These Ca2+ "hot spots" were localized in the processes of the astrocytes but not in the cell bodies. The time courses of [Ca2+]i oscillations in different hot spots were not synchronized, indicating that each of them formed an independent compartment of Ca2+ signaling. When higher concentrations (10(-5)-10(-4) M) of histamine were added, [Ca2+]i in the processes remained elevated at high levels and [Ca2+]i elevations propagated from the processes to the cell bodies. These results suggest that individual processes of type-2 astrocytes can form minimal units for Ca2+ signaling in response to submaximal concentrations of histamine and that single type-2 astrocytes may function as multiple units for Ca2+ signaling.
Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells.
Authors: Wagner E , Cotten M , Foisner R , Birnstiel M L
Published: May 2006

We have previously described a gene delivery system based upon the receptor-mediated endocytosis of DNA complexed with transferrin-polycation conjugates. This delivery system has been found to be very effective for both the internalization and the expression of genetic material in cells that have many transferrin receptors. Upon scrutinization of the parameters involved in this method, which we have termed transferrinfection, we note two important features of the process: the polycation in polycation-transferrin conjugates, as expected, serves to attach the transferrin moiety to the DNA and, in addition, the polycation functions to condense the DNA into a doughnut structure. Electron microscopic analysis of a range of poorly active to highly active transferrinfection samples reveals a strong correlation between DNA condensation and cellular DNA uptake. Furthermore, we demonstrate that the transfection activity of the DNA complex can be increased by addition of free polycation as long as a sufficient quantity of polycation-transferrin conjugates remains in the complex to ensure its binding to the cellular receptor.
Immunoglobulin variable-region-like domains of diverse sequence within the major histocompatibility complex of the chicken.
Authors: Miller M M , Goto R , Young S , Chirivella J , Hawke D , Miyada C G
Published: May 2006

The highly polymorphic B-G antigens are considered to be part of the major histocompatibility complex (MHC) of the chicken, the B system of histocompatibility, because they are encoded in a family of genes tightly linked with the genes encoding MHC class I and class II antigens. To better understand these unusual MHC antigens, full-length B-G cDNA clones were isolated from B21 embryonic erythroid cell cDNA library, restriction-mapped, and sequenced. Five transcript types were identified. Analysis of the deduced amino acid sequences suggests that the B-G polypeptides are composed of single extracellular domains that resemble immunoglobulin domains of the variable-region (V) type, single membrane-spanning domains typical of integral membrane proteins, and long cytoplasmic tails. Sequence diversity among the five transcript types was found in all domains, notably including the B-G immunoglobulin V-like domains. The cytoplasmic tails of the B-G antigens are made up entirely of units of seven amino acid residues (heptads) that are typical of an alpha-helical coiled-coil conformation. The heptads vary in number and sequence between the different transcripts. The presence within B-G polypeptides of polymorphic immunoglobulin V-like domains warrants further investigations to determine the degree and nature of variability within this domain in these unusual MHC antigens.
Fast oscillations (20-40 Hz) in thalamocortical systems and their potentiation by mesopontine cholinergic nuclei in the cat.
Authors: Steriade M , Dossi R C , Paré D , Oakson G
Published: May 2006

Previous investigations in various motor and sensory cortical areas have shown that fast oscillations (20-80 Hz) of focal electroencephalogram and multiunit activities occur spontaneously during increased alertness or are dependent upon optimal sensory stimuli. We now report the presence of 20- to 40-Hz rhythmic activities in intracellularly recorded thalamocortical cells of the cat. In some neurons, subthreshold oscillations were triggered by depolarizing pulses and eventually gave rise to action potentials. In other neurons, the oscillations consisted of fast prepotentials, occasionally generating full spikes that arose from the resting or even from hyperpolarized membrane potential levels, and leading to trains of spikes at more depolarized levels. The rhythmic nature of these fast prepotentials was confirmed by means of an autocorrelation study, which demonstrated clear peaks at 25-ms intervals (40 Hz). In view of the recent evidence that mesopontine cholinergic nuclei trigger and maintain activation processes in thalamocortical systems, we tested the possibility that stimulation of these brainstem nuclei potentiates the 40-Hz waves on the background of the cortical electroencephalogram. This was indeed the case. The potentiation outlasted the stimulation by 10-20 s. The brainstem-induced facilitation of cortical 40-Hz oscillations was blocked by scopolamine, a muscarinic antagonist. That this facilitation was transmitted by brainstem-thalamic cholinergic projections was confirmed by persistence of the phenomenon after large excitotoxic lesions of the nucleus basalis of Meynert.
Full-length CD4 electroinserted in the erythrocyte membrane as a long-lived inhibitor of infection by human immunodeficiency virus.
Authors: Zeira M , Tosi P F , Mouneimne Y , Lazarte J , Sneed L , Volsky D J , Nicolau C
Published: May 2006

Recombinant full-length CD4 expressed in Spodoptera frugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was "correctly" oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments (125I-CD4 and 51Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, we showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance of HIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cells. RBC-CD4, but not RBCs, aggregated with chronically HIV-1-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.
Free-flow electrophoresis with trapping by a transverse inhomogeneous field.
Authors: Ajdari A , Prost J
Published: May 2006

We propose an electrophoretic technique combining the use of a series of dielectric traps controlled by an ac electric field and conventional continuous field free-flow electrophoresis. From the theoretical model that we describe, one can expect, for DNA electrophoresis, an improvement of one to two orders of magnitude in the selectivities or experimental durations.
Distinguishing African and European honeybee matrilines using amplified mitochondrial DNA.
Authors: Hall H G , Smith D R
Published: May 2006

Previous DNA studies have revealed that feral neotropical African bees have largely retained an African genetic integrity. Additional DNA testing is needed to confirm these findings, to understand the processes responsible, and to follow African bee spread into the temperate United States. To facilitate surveys, the polymerase chain reaction was utilized. African and European honeybee mitochondrial DNA (mtDNA) was identified through amplified segments that carry informative restriction site and length polymorphisms. The ability to discriminate among honeybee subspecies was established by testing a total of 129 colonies from Africa and Europe. Matriline identities could thus be determined for imported New World bees. Among 41 managed and feral colonies in the United States and north Mexico, two European lineages (west and east) were distinguished. From neotropical regions, 72 feral colonies had African mtDNA and 4 had European mtDNA. The results support earlier conclusions that neotropical African bees have spread as unbroken African maternal lineages. Old and New World African honeybee populations exhibit different frequencies of a mtDNA length polymorphism. Through standard analyses, a north African mtDNA type that may have been imported previously from Spain or Portugal was not detected among neotropical African bees.
Kinetics of antigenic peptide binding to the class II major histocompatibility molecule I-Ad.
Authors: Tampé R , McConnell H M
Published: May 2006

Using high-performance size-exclusion chromatography and fluorescence spectroscopy, we investigated the kinetics of fluorescent peptide reactions with detergent-solubilized I-Ad, a class II molecule of the mouse major histocompatibility complex. At pH 7.0 and 37 degrees C the half-time for the binding of a fluorescein-labeled synthetic peptide representing ovalbumin amino acids 323-339 [FOva-(323-339)Y] to I-Ad was 32 hr, independent of added fluorescent peptide concentration in the range 5-200 microM. Peptide exchange experiments were also carried out, where it was found that the half-time of FOva-(323-339)Y binding was equal to the half-time of dissociation of the Texas Red-labeled peptide TROva-(323-339)Y. These experiments show that slow peptide binding to class II major histocompatibility molecules may be limited by the slow dissociation of prebound peptides. Paradoxically, however, this kinetic behavior--a peptide concentration-insensitive on-reaction with a half-time for peptide binding approximately equal to the half-time for dissociation--can be modeled in more than one way. Models involving a kinetic intermediate are particularly attractive. The kinetics were significantly different at pH 5.0. The half-times for peptide binding and dissociation were approximately 7 times shorter than at pH 7.0. In addition the complex of the I-Ad alpha/beta heterodimer with FOva-(323-339)Y was unstable and dissociated into separate alpha and beta chains with a half-time of approximately 7 hr.
Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier.
Authors: Friden P M , Walus L R , Musso G F , Taylor M A , Malfroy B , Starzyk R M
Published: May 2006

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. We have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration (Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London) 312, 162-163), labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate approximately 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.
Localized cytosolic domains of the erythropoietin receptor regulate growth signaling and down-modulate responsiveness to granulocyte-macrophage colony-stimulating factor.
Authors: Quelle D E , Wojchowski D M
Published: May 2006

Erythrocyte development in mammals depends in part upon the interaction of the glycopeptide hormone erythropoietin (EPO) with cell surface receptors on committed erythroid progenitor cells. Both this factor and an EPO receptor polypeptide previously have been cloned, yet little is presently understood concerning molecular mechanisms of receptor activation and signal transduction. To identify cytosolic receptor domains necessary for signaling, we have compared the activities of a series of deletionally mutated EPO receptor constructs by their expression in interleukin 3-dependent, myeloid FDC-P1 cells. EPO-induced growth was transduced efficiently in these cells by the full-length receptor (507 amino acids), and no measurable loss in activity resulted from the deletion of up to 111 carboxyl-terminal residues. In contrast, the deletion of 44 additional residues led to a dramatic loss (86.3% +/- 7.8%; mean +/- SD) in the ability of this receptor to mediate EPO-induced growth, thus indicating that residues between Gly-352 and Met-396 constitute a functionally critical cytosolic subdomain. Interestingly, the expression of full-length EPO receptors in FDC-P1 cells also led to a selective inhibition of normal proliferative responsiveness to the alternative hematopoietic factor granulocyte-macrophage colony-stimulating factor. Moreover, this inhibition was progressively reversed in forms of the EPO receptor in which distal cytosolic residues were sequentially deleted. These results suggest that EPO receptors normally may trans-modulate components in the pathway of granulocyte-macrophage colony-stimulating factor-induced proliferation and that this down-modulation, as exerted by intact EPO receptors, may play a role in promoting erythroid commitment during myeloid blood cell development.
cAMP response element-binding protein is activated by Ca2+/calmodulin- as well as cAMP-dependent protein kinase.
Authors: Dash P K , Karl K A , Colicos M A , Prywes R , Kandel E R
Published: May 2006

In a variety of nerve cells of the brain, action potentials activate gene expression by means of Ca2+ influx. To determine how Ca2+ influx alters gene expression, we have examined the pattern of phosphorylation of a protein that binds to the cAMP response element (CRE). We have found that purified bovine brain CRE-binding protein is a substrate for the Ca2+/calmodulin-dependent kinase II (Cam kinase) as it is for the cAMP-dependent protein kinase (kinase A). Tryptic peptide maps show that the same peptide is phosphorylated in vitro both by kinase A and by Cam kinase. Moreover, in vitro transcription assays using a CRE-containing c-fos promoter indicate that phosphorylation of CRE-binding protein by Cam kinase increases gene transcription. Thus, action potentials in nerve cells and the consequent influx of Ca2+ can activate CRE-binding proteins by means of Cam kinase. This kinase therefore provides a direct second-messenger pathway by which impulse activity at the membrane can influence gene transcription. This has been shown independently by Sheng et al. (Sheng, M., Thomson, M. A. & Greenberg, M. E. (1991) Science, in press), who found that depolarization and Ca2+ influx mediate induction of c-fos in PC12 rat pheochromocytoma cells through phosphorylation of CRE-binding protein. These several findings indicate that CRE-binding protein(s) is a convergence point for synaptic activity acting through kinase A and impulse activity acting through Cam kinase. Together the two kinases could activate transcription in a synergistic manner, which could allow CRE-binding protein to couple short-term to long-term associative forms of synaptic plasticity.
Alloreactive T cells discriminate among a diverse set of endogenous peptides.
Authors: Heath W R , Kane K P , Mescher M F , Sherman L A
Published: May 2006

Previous studies have demonstrated that class I major histocompatibility complex (MHC) molecules are occupied by peptides of endogenously synthesized self proteins. Since graft rejection appears to be mediated by the normal occurrence of high frequencies of cytolytic T lymphocytes (CTLs) specific for allogeneic MHC molecules, it is important to know if such CTLs recognize specific MHC-peptide complexes (as opposed to the MHC molecule per se) and, if so, whether allorecognition is the result of the recognition of a limited spectrum of antigenic determinants or, alternatively, the recognition of a diverse array of MHC-self peptide complexes. This issue has been examined using a mutant cell line, T2Kb, that expresses class I molecules devoid of endogenously derived peptides. This cell line was not recognized by Kb-specific alloreactive CTLs. However, upon exposure to peptides derived by cyanogen bromide cleavage of cytoplasmic proteins these cells became sensitized for recognition and lysis by a majority of the CTL clones examined. Reverse-phase HPLC fractionation of the heterogeneous cell-derived peptides revealed that individual CTL clones were specific for different peptide antigen(s). Thus, the high frequency of alloreactive T cells that is responsible for graft rejection appears to represent the sum of numerous T-cell clones specific for a diverse array of endogenous peptide antigens presented in the context of allogeneic class I molecules.
Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus.
Authors: Lai C J , Zhao B T , Hori H , Bray M
Published: May 2006

Dengue virus is an enveloped positive-strand RNA virus with a genome approximately 11 kilobases in length. The four serotypes of dengue virus are currently the most important members of the flavivirus family in terms of geographical distribution and the incidence of infection in humans. In this communication we describe successful cloning of a stable full-length cDNA copy of dengue type 4 virus that can be used as the template for in vitro transcription of infectious RNA. Evidence is presented that dengue virus recovered from permissive cells transfected with the in vitro RNA transcripts retained a mutation that was engineered into full-length cDNA. The properties of the virus produced by cells transfected with infectious RNA transcripts of dengue cDNA resembled those of the virus from which the cDNA clone was derived. The dengue virus recombinant DNA system should prove helpful in gaining a better understanding of the molecular biology of dengue viruses and should facilitate the development of a safe and effective live vaccine for use in humans.
Developmental expression of the 25-kDa synaptosomal-associated protein (SNAP-25) in rat brain.
Authors: Oyler G A , Polli J W , Wilson M C , Billingsley M L
Published: May 2006

The developmental expression and subcellular distribution of the neuron-specific 25-kDa synaptosomal protein (SNAP-25) were investigated by using Northern (RNA) blots, immunoblots, and immunocytochemistry. Both SNAP-25 protein and mRNA were present at low levels in embryonic day 15 rat brain, and levels of both increased during early postnatal maturation. Developmental immunoblots with antipeptide antisera demonstrated that a 25-kDa peptide was the major isoform in brain, and this form increased steadily from embryonic day 15 through adulthood. A second 27-kDa immunoreactive isoform was present in brain only during early development. Immunoblots of two-dimensional SDS/polyacrylamide gels revealed the presence of a predominant 25-kDa isoform of SNAP-25 in adult brain. Immunocytochemical studies indicated that as immunoreactivity for SNAP-25 increased during development, the cellular localization of SNAP-25 immunoreactivity concomitantly shifted from axons and cell bodies to presynaptic terminals. These data suggest that the SNAP-25 protein shifts in subcellular localization during development and may play a role in the establishment and stabilization of specific presynaptic terminals in brain.