bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonal evolution of a follicular lymphoma: evidence for antigen selection.
Authors: Bahler D W , Levy R
Published: May 2006

The potential role antigens play in growth stimulation or in clonal selection of follicular lymphomas is unknown. To study this issue, we sequenced the immunoglobulin heavy chain variable region genes expressed by a follicular lymphoma from multiple biopsy specimens and also cloned and sequenced the corresponding germ-line variable gene from this patient. Comparison to the germ-line gene revealed numerous nucleotide substitutions in all of the lymphoma variable gene sequences. Some of the substitutions may have occurred in the nonmalignant precursor B cell that gave rise to this lymphoma because they were shared among all of the variable genes, but many of the mutations accumulated as the malignant clone expanded. The mutations were distributed in such a way that strongly suggested the majority of tumor cells had been positively selected through their antigen receptor. This was especially evident for the mutations that developed late in the clonal evolution of this lymphoma. These findings indicate that antigen stimulation may be involved in the growth of follicular lymphoma tumors.
Song presentation induces gene expression in the songbird forebrain.
Authors: Mello C V , Vicario D S , Clayton D F
Published: May 2006

We investigated the participation of genomic regulatory events in the response of the songbird brain to a natural auditory stimulus of known physiological and behavioral relevance, birdsong. Using in situ hybridization, we detected a rapid increase in forebrain mRNA levels of an immediate-early gene encoding a transcriptional regulator (ZENK; also known as zif-268, egr-1, NGFI-A, or Krox-24) following presentation of tape-recorded songs to canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). ZENK induction is most marked in a forebrain region believed to participate in auditory processing and is greatest when birds hear the song of their own species. A significantly lower level of induction occurs when birds hear the song of a different species and no induction is seen after exposure to tone bursts. Cellular analysis indicates that the level of induction reflects the proportion of neurons recruited to express the gene. These results suggest a role for genomic responses in neural processes linked to song pattern recognition, discrimination, or the formation of auditory associations.
Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis.
Authors: Fuchs E , Esteves R A , Coulombe P A
Published: May 2006

Epidermolytic hyperkeratosis (EH; previously called bullous congenital ichthyosiform erythroderma) is an autosomal dominant skin disease of unknown etiology, affecting approximately 1 out of 300,000 people. It is typified by hyperkeratotic scaliness, blistering due to cytolysis within suprabasal epidermal cells, and hyperproliferation in basal cells. Histologically, EH epidermis exhibits a thickened stratum corneum and granular layer, with enlarged and irregular-shaped cells. Ultrastructurally, only suprabasal layers are affected, with three major aberrancies: (i) tonofilament clumping, (ii) nuclei and keratohyalin granules of irregular shape and size, and (iii) cell degeneration. We have discovered that transgenic mice expressing a mutant keratin 10 gene have the EH phenotype, thereby suggesting that a genetic basis for human EH residues in mutations in genes encoding suprabasal keratins K1 and K10. In addition, we show that (i) stimulation of basal cell proliferation can arise from a defect in suprabasal cells, and (ii) distortion of nuclear shape or aberrations in cytokinesis can occur when an intermediate filament network is perturbed.
Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity.
Authors: Venis M A , Napier R M , Barbier-Brygoo H , Maurel C , Perrot-Rechenmann C , Guern J
Published: May 2006

The major auxin-binding protein in maize membranes is thought to function as a physiological receptor. From earlier information, including the use of site-directed irreversible inhibitors, several of the amino acids likely to form part of the active auxin-binding site were provisionally assigned. Inspection of the amino acid sequence of the auxin-binding protein showed a short region containing all but one of these amino acids. We find that antisera raised against a synthetic peptide encompassing this region recognize all isoforms of the maize auxin-binding protein together with homologous polypeptides in other species. We further find that the antibodies hyperpolarize protoplast transmembrane potential in an auxin-like manner. We conclude that these antibodies display auxin agonist activity and that we have identified an essential portion of the auxin-binding site.
Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector.
Authors: Walsh C E , Liu J M , Xiao X , Young N S , Nienhuis A W , Samulski R J
Published: May 2006

Gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. Distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. We have explored the use of a human parvovirus, the adeno-associated virus (AAV), for globin gene transfer. The human A gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was subcloned into a plasmid (psub201) containing the AAV inverted terminal repeats. This construct was cotransfected with a helper plasmid containing trans-acting AAV genes into human 293 cells that had been infected with adenovirus. The recombinant AAV vector containing hypersensitivity site 2 stably introduced on average one or two unrearranged proviral copies into human K562 erythroleukemia cells. The transferred globin gene exhibited normal regulation upon hemin induction of erythroid maturation and was expressed at a level equivalent to a native chromosomal A gamma-globin gene.
Serine tRNA complementary to the nonuniversal serine codon CUG in Candida cylindracea: evolutionary implications.
Authors: Yokogawa T , Suzuki T , Ueda T , Mori M , Ohama T , Kuchino Y , Yoshinari S , Motoki I , Nishikawa K , Osawa S
Published: May 2006

In the asporogenic yeast Candida cylindracea, the codon CUG is read as serine instead of leucine. This is an unusual instance in which the amino acid assignment of a codon deviates from the universal code. To infer the evolutionary process of this change, the tRNA with the anticodon sequence CAG, which is complementary to and thus responsible for translation of the codon CUG, has been identified. Indeed, this tRNA translates an in-frame CUG codon in a synthetic mRNA as serine in an in vitro translation system. The gene for the tRNA is interrupted by an intron in the anticodon loop. Sequence comparisons of the tRNA and its gene suggest that a single cytidine was inserted into the anticodon loop of the gene for tRNA(Ser)IGA during evolution to produce tRNA(Ser)CAG. The tRNA(Ser)CAG may be produced from its precursor molecule containing the cytidine insertion by splicing.
Association of titin and myosin heavy chain in developing skeletal muscle.
Authors: Isaacs W B , Kim I S , Struve A , Fulton A B
Published: May 2006

To understand molecular interactions that organize developing myofibrils, we examined the biosynthesis and interaction of titin and myosin heavy chain in cultures of developing muscle. Use of pulse-labeling, immunoprecipitation, and a reversible cross-linking procedure demonstrates that within minutes of synthesis, titin and myosin heavy chain can be chemically cross-linked into very large, detergent-resistant complexes retaining many features of intact myotubes. These complexes, predominantly of titin and myosin, occur very early in myofibrillogenesis as well as later. These data suggest that synthesis and assembly of titin and myosin are temporally and spatially coordinated in nascent myofibrils and support the hypothesis that titin molecules help to organize sarcomere formation.
ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae.
Authors: Blum S , Schmid S R , Pause A , Buser P , Linder P , Sonenberg N , Trachsel H
Published: May 2006

Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)] or valine [eIF-4A(A66V)] were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.
Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding.
Authors: Kitayama S , Shimada S , Xu H , Markham L , Donovan D M , Uhl G R
Published: May 2006

Polar amino acids lying within three hydrophobic regions of the dopamine transporter (DAT) are analogous to those important for ligand recognition by catecholamine receptors. Possible functional significance of these amino acids was examined by expressing DAT cDNAs mutated in these polar residues. Replacement of aspartate at position 79 with alanine, glycine, or glutamate dramatically reduced uptake of [3H]dopamine and the tritium-labeled Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and reduced the mutants' affinity for the tritium-labeled cocaine analog (-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT) without affecting Bmax. Replacement of the serine residues at positions 356 and 359 in the seventh hydrophobic region by alanine or glycine caused reductions in [3H]dopamine and [3H]MPP+ uptake, whereas [3H]CFT binding was less affected. Substitution of two serines in the eighth hydrophobic region yielded wild-type values for [3H]dopamine and [3H]MPP+ uptake and [3H]CFT binding. These results demonstrate that aspartate and serine residues lying within the first and seventh hydrophobic putative transmembrane regions are crucial for DAT function and provide identification of residues differentially important for cocaine binding and for dopamine uptake.
Inverted repeat structure of the Sry locus in mice.
Authors: Gubbay J , Vivian N , Economou A , Jackson D , Goodfellow P , Lovell-Badge R
Published: May 2006

The testis-determining gene Sry is located on the short arm of the mouse Y chromosome in a region known to have undergone duplications and rearrangements in comparison with the equivalent portion of the human Y chromosome. Detailed analysis of the Sry genomic locus reveals a further difference in that the mouse Sry open reading frame lies within 2.8 kilobases of unique sequence at the center of a large inverted repeat. This repeat, which is found in both Mus musculus musculus and Mus musculus domesticus Y chromosomes, is not present at the human SRY locus. Recombination involving the repeat region may have led to an 11-kilobase deletion, precisely excising Sry in a line of XY female mice.
Broad-spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens.
Authors: Gould F , Martinez-Ramirez A , Anderson A , Ferre J , Silva F J , Moar W J
Published: May 2006

Evolution of pest resistance to insecticidal proteins produced by Bacillus thuringiensis (Bt) would decrease our ability to control agricultural pests with genetically engineered crops designed to express genes coding for these proteins. Previous genetic and biochemical analyses of insect strains with resistance to Bt toxins indicate that (i) resistance is restricted to single groups of related Bt toxins, (ii) decreased toxin sensitivity is associated with changes in Bt-toxin binding to sites in brush-border membrane vesicles of the larval midgut, and (iii) resistance is inherited as a partially or fully recessive trait. If these three characteristics were common to all resistant insects, specific crop-variety deployment strategies could significantly diminish problems associated with resistance in field populations of pests. We present data on Bt-toxin resistance in Heliothis virescens, a major agricultural pest targeted for control with Bt-toxin-producing crops. A laboratory strain of H. virescens developed resistance in response to selection with the Bt toxin CryIA(c). In contrast to other cases of Bt-toxin resistance, this H. virescens strain exhibits cross-resistance to Bt toxins that differ significantly in structure and activity. Furthermore, the resistance in this strain is not accompanied by significant changes in toxin binding, and resistance is inherited as an additive trait when larvae are treated with high doses of CryIA(c) toxin. These findings have important implications for Bt-toxin-based pest control.
Rudimentary form of cellular "vision".
Authors: Albrecht-Buehler G
Published: May 2006

BHK cells were inoculated sparsely on one face ("sparse- or s-face") of a thin glass film whose opposite face was covered with a 2- to 3-day-old confluent layer of BHK cells ("confluent- or c-face"). After 7 hr of attaching and spreading in the absence of visible light, most of the cells on the s-face traversed with their long axes the direction of the whorls of the confluent cells on the c-face directly opposed. The effect was inhibited by a thin metal coating of the glass films. The results suggest that the cells were able to detect the orientation of others by signals that penetrated glass but not thin metallic films and, therefore, appeared to be carried by electromagnetic radiation. In contrast, the effect was not influenced by a thin coat of silicone on the glass, suggesting that the wavelength of this radiation is likely to be in the red to infrared range. The ability of cells to detect the direction of others by electromagnetic signals points to a rudimentary form of cellular "vision."
Tempo and mode of evolution revealed from molecular phylogenies.
Authors: Nee S , Mooers A O , Harvey P H
Published: May 2006

The analysis of the tempo and mode of evolution has a strong tradition in paleontology. Recent advances in molecular phylogenetic reconstruction make it possible to complement this work by using data from extant species.
Dynamic surrounds of receptive fields in primate striate cortex: a physiological basis for perceptual completion?
Authors: Fiorani Júnior M , Rosa M G , Gattass R , Rocha-Miranda C E
Published: May 2006

Visual receptive fields (RFs) were mapped inside and outside the cortical representation of the optic disk in the striate cortex (area V1) of anesthetized and paralyzed Cebus monkeys. Unexpectedly, most cells were found to be binocularly driven, and the RFs mapped with contralateral-eye stimulation progressed in a topographically appropriate fashion as the optic disk sector was crossed. Activation of these neurons by the contralateral eye was shown to depend on stimulation of the parts of the retina around the optic disk. Outside the optic disk representation, a similar effect was demonstrated by obstructing the "classical" RF with masks 5-10 times larger in size. In all cases, visual stimuli presented around the mask could be used to accurately interpolate the position of the hidden RF. These properties reflect, at a cellular level, the process of "filling in" that allows for completion of the visual image across natural and artificially induced scotomas.
Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor alpha required as a costimulatory factor for interferon gamma-induced activation.
Authors: Oswald I P , Wynn T A , Sher A , James S L
Published: May 2006

Interleukin 10 (IL-10) inhibits interferon gamma-induced macrophage activation for cytotoxicity against larvae of the human parasite Schistosoma mansoni by suppressing production of the toxic effector molecule nitric oxide (NO). In this study, the mechanism of IL-10 action was identified as inhibition of endogenous tumor necrosis factor alpha (TNF-alpha) production by interferon gamma-activated macrophages. TNF-alpha appears to serve as a cofactor for interferon gamma-mediated activation, since both schistosomulum killing and NO production were inhibited by anti-TNF-alpha antibody, whereas TNF-alpha alone was unable to stimulate these macrophage functions. IL-10 blocked TNF-alpha production by interferon gamma-treated macrophages at the levels of both protein and mRNA synthesis. Addition of exogenous TNF-alpha reversed IL-10-mediated suppression of macrophage cytotoxic activity as well as NO production. Likewise, addition of a macrophage-triggering agent (bacterial lipopolysaccharide or muramyl dipeptide), which induced the production of TNF-alpha, also reversed the suppressive effect of IL-10 on cytotoxic function. In contrast to IL-10, two other cytokines, IL-4 and transforming growth factor beta, which also inhibit macrophage activation for schistosomulum killing and NO production, did not substantially suppress endogenous TNF-alpha production. These results, therefore, describe a separate pathway by which macrophage microbicidal function is inhibited by the down-regulatory cytokine IL-10.
Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.
Authors: Kuspa A , Loomis W F
Published: May 2006

Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.
Differential response of maize catalases to abscisic acid: Vp1 transcriptional activator is not required for abscisic acid-regulated Cat1 expression.
Authors: Williamson J D , Scandalios J G
Published: May 2006

In this paper we describe the distinctive responses of the maize catalases to the plant growth regulator abscisic acid (ABA). We analyzed RNA and enzyme accumulation in excised maize embryos and found that each catalase responded differently to exogenously applied ABA. Levels of Cat1 transcript and enzyme activity rapidly increased. In contrast, levels of Cat2 transcript and protein decreased, while Cat3 transcript levels were not affected. In developing kernels of the ABA-deficient/biosynthetic viviparous mutant vp5, lower levels of Cat1 RNA correlated with lower endogenous ABA levels when compared to measured levels in comparably aged wild-type siblings from the same ear. The maize vp1 mutant line is morphologically insensitive to normal endogenous levels of ABA. Analysis of the response of Cat1 to exogenously applied ABA in mutant and wild-type vp1 sibling embryos suggests that, unlike other ABA-responsive genes analyzed to date, the Vp1 gene product is not essential for the ABA-mediated regulation of Cat1. The significance of these responses to ABA in defining the roles of the various CATs in maize is discussed.
Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth.
Authors: Carpenter J F , Hansen T N
Published: May 2006

Antifreeze proteins (AFPs) are extremely efficient at inhibiting ice recrystallization in frozen solutions. Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263] have proposed that this may be an important function of the proteins in freeze-tolerant organisms. We have tested this proposal in vitro by characterizing the influence of AFP on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during warming. Relatively low concentrations (e.g., 5-150 micrograms/ml) of winter flounder (Pseudopleuronectes americanus) AFP enhance survival of red blood cells cryopreserved in hydroxyethyl starch solutions. This effect is most apparent in samples warmed at suboptimal rates, i.e., where ice recrystallization would be exaggerated. Cryomicroscopy demonstrates that AFP inhibits ice recrystallization in the extracellular regions during the latter stages of the warming cycle. AFP concentrations that enhance survival of red cells confer partial inhibition of recrystallization. Relatively high concentrations of AFP (e.g., 1.54 mg/ml) are much more effective at inhibiting extracellular recrystallization. However, extensive growth of ice around the cell, and concomitant cell damage, is noted. The mechanism for this AFP-induced ice growth is unknown. We propose that there is a delicate balance between AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell damage and that this balance hinges on the degrees of inhibition of ice recrystallization and of preferential growth of ice around the cells. We conclude that, under appropriate conditions, one of the proposed functions of AFPs in nature can be emulated, and perhaps have application, in cryopreservation of materials of biomedical interest.
Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296.
Authors: Wetterholm A , Medina J F , Rådmark O , Shapiro R , Haeggström J Z , Vallee B L , Samuelsson B
Published: May 2006

The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be virtually inactive (less than or equal to 0.2% of unmutated enzyme). To serve as a positive control, we also replaced Ser-298 with an alanine residue, which resulted in a protein ([Ala298]LTA4 hydrolase) with catalytic properties almost indistinguishable from the wild-type enzyme. Substitution of Glu-296 by glutamine or alanine was also carried out with human LTA4 hydrolase, and the mutated human enzymes displayed specific activities similar to the corresponding mouse proteins. Zinc analyses of the purified mouse and human proteins confirmed that the mutations did not significantly influence their zinc content. In conclusion, the results of the present study indicate a direct catalytic role for Glu-296 in the peptidase reaction of LTA4 hydrolase, where it presumably acts as a base to polarize water, whereas its function, if any, is apparently not essential in the epoxide hydrolase reaction.
Uncoupling gene activity from chromatin structure: promoter mutations can inactivate transcription of the yeast HSP82 gene without eliminating nucleosome-free regions.
Authors: Lee M S , Garrard W T
Published: May 2006

DNase I-hypersensitive sites represent "nucleosome-free" regions in chromatin where the underlying DNA sequence is highly accessible to trans-acting proteins. Here we demonstrate that it is possible to uncouple gene activity from hypersensitive site formation. Point or substitution mutations were introduced into the promoter of the yeast chromosomal HSP82 gene, encoding the 83-kDa heat shock protein (HSP), via site-directed integration. Mutating either the TATA box or heat shock element 1 (HSE1) significantly reduced basal and heat-induced transcription while mutating both essentially inactivated expression. Dormant transcription units exhibited arrays of sequence-positioned nucleosomes; nevertheless, the inactivated genes still retained a hypersensitive site within their mutated promoters. In addition, all yeast strains maintained a heat-inducible hypersensitive site at -600 base pairs (bp), while several mutant strains converted a constitutive hypersensitive site at -300 bp into a heat-inducible one. Thus, mutations in cis-acting elements within a promoter can inactivate transcription without eliminating nucleosome-free regions.
Phagocytosis of liposomes by human platelets.
Authors: Male R , Vannier W E , Baldeschwieler J D
Published: May 2006

We have shown that platelets are capable of phagocytosing liposomes rather than simply sequestering particles as previously postulated. Incubation of human platelets with small neutral unilamellar liposomes (approximately 74 nm) resulted in uptake of the liposomes and retention of the lipid with rapid release of the aqueous-phase components. The lipid label [3H]-cholesterylhexadecyl ether and water-soluble [3H]inulin were used to study the fate of the liposome components. Uptake of liposomes was proportional to the number of liposomes added and to the incubation time. Approximately 250 liposomes per platelet were taken up within a 5-hr incubation period. Uptake of the liposomes occurred through the open-channel system, as evidenced by thin-section electron microscopy, and was followed by accumulation and degradation in acid- and esterase-containing vesicles, as determined by changes in fluorescence of the pH-sensitive probe, pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid), and hydrolysis of the cholesteryl [14C]oleate membrane marker. Uptake was inhibited by the addition of EDTA, cytochalasin B, or 2,4-dinitrophenol and iodoacetate to the medium. Results from the serotonin release assay, micro-aggregation assay, fluorescein diacetate membrane integrity assay, and electron microscopy indicate that neither the conditions for loading nor phagocytosis of liposomes significantly alter platelet function or morphology.
Interleukin 6: a fibroblast-derived growth inhibitor of human melanoma cells from early but not advanced stages of tumor progression.
Authors: Lu C , Vickers M F , Kerbel R S
Published: May 2006

Recently we reported that human dermal fibroblasts, or conditioned media obtained from such cells, affect the growth of human melanoma cells as a direct function of tumor progression: melanoma cells obtained from early-stage (metastatically incompetent) primary lesions were growth inhibited, whereas cells obtained from more advanced (metastatically competent) primary lesions, or metastases, were growth stimulated. Ion-exchange and gel-filtration chromatography of fibroblast conditioned medium revealed the inhibitor to be a protein of molecular mass between 20 and 30 kDa and distinct from the stimulator. This is the approximate molecular mass of interleukin 6 (IL-6), a ubiquitous multifunctional cytokine known to affect in particular many kinds of hemopoietic and lymphoid cells. Since this cytokine is known to be made by fibroblasts, we attempted to determine if the human fibroblast-derived growth inhibitor (hFDGI) was identical to IL-6. Neutralizing antibodies specific for IL-6 completely eliminated the inhibitory activity of hFDGI. Moreover, exposure to human recombinant IL-6 was found to inhibit the growth of early-stage melanoma cells obtained from radial growth phase (RGP) or early vertical growth phase (VGP) primary lesions in three of four cases. In contrast, melanoma cells from a number of more advanced VGP primary lesions, or from distant metastases, were completely resistant to this IL-6-mediated growth inhibition. Acquisition of an "IL-6-resistant" phenotype by metastatically competent melanoma cell variants may provide such cells with a proliferative advantage within the dermal mesenchyme (a hallmark of melanoma cells that are malignant), helping them eventually to dominate advanced primary lesions and to establish secondary growths elsewhere.
Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.
Authors: Takahashi S , Kawarabayasi Y , Nakai T , Sakai J , Yamamoto T
Published: May 2006

A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with striking homology to the low density lipoprotein (LDL) receptor. Like the LDL receptor, the mature protein consists of the following five domains spanning 846 amino acids: 328 N-terminal amino acids including an 8-fold repeat of 40 amino acids homologous to the ligand binding repeat of the LDL receptor; 396 amino acid residues homologous to the epidermal growth factor precursor including three cysteine-rich repeats; a region immediately outside of the plasma membrane rich in serines and threonines; 22 amino acids traversing the plasma membrane; and 54 amino acids including the NPVY sequence that is required for clustering of the LDL receptor in coated pits and that projects into the cytoplasm. LDL-receptor-deficient Chinese hamster ovary cells transfected with the cDNA bound and internalized VLDL, beta-migrating VLDL, and intermediate density lipoprotein but did not bind LDL with high affinity. The 3.6- and 9.5-kilobase mRNAs for the VLDL receptor are highly abundant in heart, muscle, and adipose tissue. Barely detectable amounts of the mRNAs were present in liver. Based on the structural features, ligand specificity, and tissue expression of the mRNAs, we suggest that this VLDL receptor may mediate uptake of apolipoprotein E-containing lipoproteins enriched with triglyceride in nonhepatic tissues that are active in fatty acid metabolism.
Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II.
Authors: Natt E , Kida K , Odievre M , Di Rocco M , Scherer G
Published: May 2006

Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame.
Cortical neurons express nerve growth factor receptors in advanced age and Alzheimer disease.
Authors: Mufson E J , Kordower J H
Published: May 2006

Using a monoclonal antibody directed against the primate nerve growth factor (NGF) receptor, we examined the expression of NGF receptors within neuronal perikarya of normal adult human cerebral cortex (27-98 years old) and individuals with Alzheimer disease (AD). This expression of cortical NGF receptors was compared with that seen in other neurological diseases and normal human development as well as in young and aged nonhuman primates. NGF receptor-containing cortical neurons were not observed in young adults (less than 50 years old) and were observed only infrequently in non-demented elderly individuals (50-80 years old). In contrast, numerous NGF receptor-containing cortical neurons were seen in AD patients of all ages and in one 98-year-old nondemented patient. In advanced age and AD, numerous NGF receptor-positive neurons were located within laminae II-VI of temporal association cortices whereas only a few were seen in the subicular complex, entorhinal cortex, parahippocampal gyrus, and amygdaloid complex. These perikarya appeared healthy, with bipolar, fusiform, or multipolar morphologies and extended varicose dendritic arbors. These neurons failed to express neurofibrillary tangle-bearing material. In contrast to AD, NGF receptor-containing cortical neurons were not observed in Parkinson disease, Pick disease, or Shy-Drager syndrome. The NGF receptor-containing cortical neurons seen in advanced age and AD were similar in morphology to those observed in human fetal cortex. No NGF receptor-containing cortical neurons were observed in young or aged nonhuman primates. These findings suggest that neurons within the human cerebral cortex exhibit plasticity in their expression of NGF receptors in AD and extreme advanced aging.
Radioprotection of mice by recombinant rat stem cell factor.
Authors: Zsebo K M , Smith K A , Hartley C A , Greenblatt M , Cooke K , Rich W , McNiece I K
Published: May 2006

Treatment with recombinant rat stem cell factor (rSCF) protects mice from the lethal effects of irradiation. Mice treated with a single dose of rSCF prior to irradiation of up to 1150 rads [given as a split dose (1 rad = 0.01 Gy)] resulted in > 80% long-term survival, whereas a single injection given after the last dose of irradiation was not radioprotective. The combination of pre- and posttreatment (-20 h, -2 h, and +4 h) with rSCF resulted in 100% survival of otherwise lethally irradiated mice. Using this optimum schedule of rSCF administration, a radioprotective factor of 1.3-1.35 was achieved. The major cause of death in the control animals was massive bacteremia consisting of enteric organisms. The rSCF-treated animals had a much lower frequency of septicemia, due primarily to a rapid hematopoietic recovery of bone marrow function not evident in control animals.
Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258.
Authors: Ji G , Silver S
Published: May 2006

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 consists of three genes, arsR (encoding the repressor regulatory protein), arsB (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsC (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). ArsC has now been shown to be an arsenate reductase, converting intracellular arsenate [As(V)] to arsenite [As(III)], which is then exported from the cells by an energy-dependent efflux process. The arsenate reductase activity was found in the soluble cytoplasmic fraction in Escherichia coli (and not associated with the periplasmic fraction or the sedimentable cell envelope). Purified ArsC protein coupled in vitro with thioredoxin plus dithiothreitol (but not 2-mercaptoethanol or reduced glutathione) to reduce arsenate to arsenite.
Association of protein-tyrosine kinase with phospholipase C-gamma 1 in bone marrow-derived mouse mast cells.
Authors: Fukamachi H , Kawakami Y , Takei M , Ishizaka T , Ishizaka K , Kawakami T
Published: May 2006

Bone marrow-derived mouse mast cells contain phospholipase C-gamma 1 (PLC-gamma 1), which is phosphorylated at tyrosine residues upon cross-linking of cell-bound IgE antibodies with multivalent antigen. It was found that immune complexes formed from digitonin lysates of the mast cells by monoclonal anti-PLC-gamma 1 antibodies contained protein-tyrosine kinase (PTK), which phosphorylated PLC-gamma 1 in vitro. The tyrosine kinase activity coprecipitated with PLC-gamma 1-anti-PLC-gamma 1 complexes markedly increased when the cell lysates were obtained immediately after antigen challenge. The results indicate that PTK is associated with PLC-gamma 1 in the mast cells and that the kinase is activated upon cross-linking of Fc epsilon RI. Neither beta nor gamma subunit of Fc epsilon RI nor src family PTK was coprecipitated with the PLC-gamma 1-anti-PLC-gamma 1 complexes. In situ denaturation/renaturation experiments, which detect autophosphorylated kinases, indicated that the PTK associated with PLC-gamma 1 was a 44-kDa protein.
O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA.
Authors: Chanock S J , Faust L R , Barrett D , Bizal C , Maly F E , Newburger P E , Ruedi J M , Smith R M , Babior B M
Published: May 2006

The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells.
Alpha-crystallin can function as a molecular chaperone.
Authors: Horwitz J
Published: May 2006

The alpha-crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.
Heat shock induces the release of fibroblast growth factor 1 from NIH 3T3 cells.
Authors: Jackson A , Friedman S , Zhan X , Engleka K A , Forough R , Maciag T
Published: May 2006

Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene encoding FGF-1 secrete FGF-1 into their conditioned medium in response to heat shock. The form of FGF-1 released by NIH 3T3 cells in response to increased temperature (42 degrees C, 2 hr) in vitro is not biologically active and does not associate with either heparin or the extracellular NIH 3T3 monolayer matrix. However, it was possible to derive biologically active FGF-1 from the conditioned medium of heat-shocked NIH 3T3 cell transfectants by ammonium sulfate fractionation. The form of FGF-1 exposed by ammonium sulfate fractionation is similar in size to cytosolic FGF-1 and can bind and be eluted from immobilized heparin similarly to the recombinant human FGF-1 polypeptide. Further, the release of FGF-1 by NIH 3T3 cell transfectants in response to heat shock is reduced significantly by both actinomycin D and cycloheximide. These data indicate that increased temperature may upregulate the expression of a factor responsible for the secretion of FGF-1 as a biologically inactive complex that requires an activation step to exhibit the biological activity of the extracellular polypeptide mitogen.
Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer.
Authors: Rettig W J , Garin-Chesa P , Healey J H , Su S L , Jaffe E A , Old L J
Published: May 2006

Cell surface antigens of transformed cells are the traditional targets for antibody-guided detection and therapy of solid neoplasms. Alternative targets may be found in the tumor stroma, which contains newly formed blood vessels, reactive fibroblasts, and extracellular matrix proteins. The F19 cell surface glycoprotein of reactive fibroblasts is a prototypic stromal antigen since it is found in the stroma of > 90% of common epithelial cancers but is absent or expressed at low levels in normal tissues and benign epithelial tumors. In the present study, we define an additional tumor stromal antigen, FB5, that is selectively expressed in vascular endothelial cells of malignant tumors. Immunohistochemical analysis of 128 tumors identified FB5 in endothelial cells in 67% of the samples (including 41 of 61 sarcomas, 26 of 37 carcinomas, and 18 of 25 neuroectodermal tumors) whereas normal blood vessels and other adult tissues tested lacked FB5 expression. In vitro studies showed that FB5 is a M(r) 165,000 cell surface glycoprotein, comprised of a M(r) 95,000 core polypeptide and highly sialyated O-linked oligosaccharides but few if any N-linked sugars, and that the FB5 gene is located on chromosome 11q13-qter. The restricted tissue distribution of the FB5 protein, which we refer to as endosialin, suggests strategies for tumor imaging and therapy that are aimed primarily at the tumor vasculature.
Intact and amino-terminally shortened forms of insulin-like growth factor I induce mammary gland differentiation and development.
Authors: Ruan W , Newman C B , Kleinberg D L
Published: May 2006

Growth hormone (GH) plays a role in regulating growth and differentiation of immature glandular structures in the mammary gland, but the mechanisms by which the hormone exerts these effects are unknown. We have previously found that GH stimulates insulin-like growth factor I (IGF-I) I mRNA production within the mammary glands of hypophysectomized rats. In this study we set out to determine if IGF-I administration could mimic the action of GH in initiating mammary gland differentiation and development. Two forms of IGF-I, intact and amino-terminally shortened [des-(1-3)-IGF-I], were found to induce the development of terminal end buds and the formation of alveolar structures in the mammary glands of hypophysectomized, castrated, and estradiol-treated sexually immature male rats. The effect of both forms of IGF-I was similar to that obtained with human GH, but the truncated form was at least 5 times more potent than intact IGF-I. These findings suggest that the inductive effect of GH on glandular differentiation is mediated by the GH-induced production of IGF-I or a related molecule within the mammary gland itself.
Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid.
Authors: Gasset M , Baldwin M A , Lloyd D H , Gabriel J M , Holtzman D M , Cohen F , Fletterick R , Prusiner S B
Published: May 2006

By comparing the amino acid sequences of 11 mammalian and 1 avian prion proteins (PrP), structural analyses predicted four alpha-helical regions. Peptides corresponding to these regions of Syrian hamster PrP were synthesized, and, contrary to predictions, three of the four spontaneously formed amyloids as shown by electron microscopy and Congo red staining. By IR spectroscopy, these amyloid peptides exhibited secondary structures composed largely of beta-sheets. The first of the predicted helices is the 14-amino acid peptide corresponding to residues 109-122; this peptide and the overlapping 15-residue sequence 113-127 both form amyloid. The most highly amyloidogenic peptide is AGAAAAGA, which corresponds to Syrian hamster PrP residues 113-120 and is conserved across all species for which the PrP sequence has been determined. Two other predicted alpha-helices corresponding to residues 178-191 and 202-218 form amyloids and exhibit considerable beta-sheet structure when synthesized as peptides. These findings suggest the possibility that the conversion of the cellular isoform of PrP to the scrapie isoform of PrP involves the transition of one or more putative PrP alpha-helices into beta-sheets and that prion diseases are disorders of protein conformation.
Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor.
Authors: Daly J W , Caceres J , Moni R W , Gusovsky F , Moos M , Seamon K B , Milton K , Myers C W
Published: May 2006

A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans.