bluuebunny/crossref_metadata_2025 huggingface dataset:
Preliminary representation of world population by spherical harmonics.
Authors: Tobler W
Published: May 2006

The geographical arrangement of people on the surface of the earth is approximated by a mathematical equation of 361 terms. This is a convenient form for comparison with other distributions and for use in global change studies, and it has other advantages, but it must be considered preliminary because of data limitations.
Developmental segregation in the afferent projections to mammalian auditory hair cells.
Authors: Echteler S M
Published: May 2006

The mammalian ear contains two types of auditory receptors, inner and outer hair cells, that lie in close proximity to each other within the sensory epithelium of the cochlea. In adult mammals, these two classes of auditory hair cells are innervated by separate populations of afferent neurons that differ strikingly in their cellular morphology and their pattern of arborization within the cochlea. At present, it is unclear when or how these distinctive patterns of cochlear innervation emerge and become segregated during development. In the present study, an in vitro horseradish peroxidase labeling method was used to examine the formation of individual auditory neuron arbors at the same location within the apex of the developing gerbil cochlea. At birth, most cochlear neurons displayed peripheral arbors that embraced both inner and outer hair cell receptors. During the next 6 days, however, the arbors of individual cochlear afferents become confined to either the inner or outer hair cell zone, and thus there is a complete segregation of afferent innervation. This neural segregation occurs principally through the withdrawal of inappropriate connections to the outer hair cell system and is completed well before hearing commences.
Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.
Authors: Sganga M W , Aksamit R R , Cantoni G L , Bauer C E
Published: May 2006

The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulatus, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medium; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis.
An NK1.1+ CD4+8- single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor V beta family.
Authors: Arase H , Arase N , Ogasawara K , Good R A , Onoé K
Published: May 2006

In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha beta T-cell antigen receptors (TCRlo) from the majority of CD4+8- high-density TCR (TCRhi) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCRlo population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCRhi thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (V beta) usage was analyzed, this thymic population expressed predominantly products of V beta 7 and V beta 8.2 TCR gene families. Interestingly, cells with V beta 8.1 TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCRlo subpopulation but had been eliminated from the major CD4+8- HSA- TCRhi subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCRlo thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCRlo cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3hi cells. However, the CD4+8- HSA- TCRlo cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.
Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.
Authors: Chan S J , Oliva A A , LaMendola J , Grens A , Bode H , Steiner D F
Published: May 2006

A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed.
The partially folded conformation of the Cys-30 Cys-51 intermediate in the disulfide folding pathway of bovine pancreatic trypsin inhibitor.
Authors: van Mierlo C P , Darby N J , Creighton T E
Published: May 2006

The best-characterized protein folding pathway is that of bovine pancreatic trypsin inhibitor, which folds from the reduced form through a series of disulfide bond intermediates. The crucial one-disulfide intermediate of bovine pancreatic trypsin inhibitor with the disulfide bond between Cys-30 and Cys-51 is shown here to have a partially folded conformation in which the major elements of secondary structure interact via a core of apolar side chains, which resembles part of the native conformation. The stability of this structure can account for the predominance of this one-disulfide intermediate during folding. Much of the remaining one-third of the polypeptide chain, in particular the N-terminal 14 residues, is largely disordered; this accounts for the ability of this intermediate to form readily any of the three possible second disulfide bonds involving Cys-5, -14, and -38. The partially folded conformation of this intermediate provides direct evidence for the importance of native-like interactions between elements of secondary structure in directing protein folding, which is assumed in many studies.
Evidence for a Ras-dependent extracellular signal-regulated protein kinase (ERK) cascade.
Authors: Robbins D J , Cheng M , Zhen E , Vanderbilt C A , Feig L A , Cobb M H
Published: May 2006

The small GTP-binding protein Ras appears to be required for transformation and differentiation induced by tyrosine kinases. The Ras requirement may be limited to a few tyrosine kinase-regulated signaling pathways or may be universal for all tyrosine kinase actions. Because both Ras and the microtubule-associated protein 2 kinases ERK1 and ERK2 have been implicated in events that lead to neurite outgrowth, we explored the possibility that Ras and ERKs may lie on the same signaling pathway. Utilizing PC-12 rat adrenal pheochromocytoma cell lines that contain a dominant inhibitory Ras mutant (S17N-Ras(H)), we found that Ras was required for stimulation of the ERK cascade by nerve growth factor but apparently not by the heterotrimeric G protein activator AlF4-. Within this cascade, Ras appears to be upstream of an ERK activator, raising the intriguing possibility that Ras may directly regulate a serine/threonine protein kinase.
Genetic and molecular analysis of recessive alleles at the pink-eyed dilution (p) locus of the mouse.
Authors: Lyon M F , King T R , Gondo Y , Gardner J M , Nakatsu Y , Eicher E M , Brilliant M H
Published: May 2006

Recessive mutant alleles at the pink-eyed dilution (p) locus on mouse chromosome 7 reduce pigmentation of both the coat and eyes. Here we describe the properties and complementation interactions of 10 p alleles, including 6 not previously reported. Several alleles that cause additional phenotypes affecting development, reproduction, and behavior were shown to be deletions by using DNA probes derived from the p region. An alignment of functional and marker-defined units is proposed, giving a linear complementation map that orders at least four functional loci. The characterization of a nested set of deletions around p will facilitate detailed molecular analyses of the genes and developmental functions associated with this part of the mouse genome.
Quantitation of immunoglobulin mu-gamma 1 heavy chain switch region recombination by a digestion-circularization polymerase chain reaction method.
Authors: Chu C C , Paul W E , Max E E
Published: May 2006

B lymphocytes expressing surface IgM with or without IgD may switch to the expression of other isotypes (IgG, IgA, or IgE) in the course of immune responses. Analyses of genomic DNA from cloned myelomas and hybridomas have shown that the isotype switch is accompanied by a rearrangement characterized by deletion of DNA between the switch (S) region of the mu gene and that associated with the new isotype, resulting in the formation of a composite S region. Measurement of this deletional rearrangement has been difficult in populations of normal B cells but would be useful for investigating the mechanism of the rearrangement and determining whether deletional rearrangement is responsible for all instances of class switching. We have developed a sensitive assay for deletional rearrangement that we designate the digestion-circularization polymerase chain reaction (PCR). In this assay, genomic DNA is digested with a restriction enzyme that recognizes sites that flank the recombined composite S region. The digested DNA is then ligated at low concentrations to favor the formation of circles. The ligation joins the 5' and 3' ends of each restriction fragment, making it possible to amplify by PCR across the ligated restriction site by using appropriate primers. From DNA that has undergone deletional rearrangement, a single-sized PCR product is produced and can be quantitated. We demonstrate here that the digestion-circularization PCR assay can detect S mu-S gamma 1 rearrangements in B cells cultured with lipopolysaccharide and interleukin 4. The assay is sensitive enough to quantitate switched cells constituting only 1-2% of the population.
Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides.
Authors: Citro G , Perrotti D , Cucco C , D'Agnano I , Sacchi A , Zupi G , Calabretta B
Published: May 2006

Exposure of human leukemia HL-60 cells to an oligodeoxynucleotide complementary to an 18-base sequence (codons 2-7) of c-myb-encoded mRNA has previously been shown to result in inhibition of cell proliferation. Because HL-60 cells express high levels of transferrin receptor we adapted a DNA delivery system based on receptor-mediated endocytosis to introduce myb oligomers complexed with a transferrin-polylysine conjugate into those cells. A DNA.RNA duplex resistant to S1 nuclease digestion was detected as early as 12 hr after culture of HL-60 cells in the presence of the myb antisense/transferrin-polylysine complex. Exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in rapid and profound inhibition of proliferation and loss of cell viability much more pronounced than that occurring in cells exposed to free myb antisense oligodeoxynucleotides. The transferrin-polylysine/myb sense complex or the transferrin-polylysine conjugate alone had no effect on HL-60 cell proliferation and viability. These findings indicate that myb synthetic oligodeoxynucleotides enter efficiently into HL-60 by transferrin receptor-mediated endocytosis and exert a profound biological effect. Such a delivery system could exploit other ligand-receptor interactions for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides.
Disruption of circadian regulation by brain grafts that overexpress Alzheimer beta/A4 amyloid.
Authors: Tate B , Aboody-Guterman K S , Morris A M , Walcott E C , Majocha R E , Marotta C A
Published: May 2006

Alzheimer disease patients exhibit irregularities in the patterns of normally circadian (daily) rhythms. Alzheimer-type pathology has been reported in the hypothalamus and in the suprachiasmatic nuclei, the putative site of the circadian oscillator. We examined the relationship between the neuropathology of Alzheimer disease, as modeled by an animal system, and circadian dysregulation by grafting genetically transformed cells that overexpress beta/A4 amyloid into the suprachiasmatic nuclei of adult rats. Grafts of beta/A4-positive cells, but not of control cells, significantly altered the pattern of activity of implanted rats. Although experimental conditions included light-dark cycles that normally tend to drive rats to 24-h rhythms, animals with grafts of beta/A4-positive cells showed abnormally high levels of activity during the light phase in addition to a disrupted circadian pattern. Periodogram analysis demonstrated significant rhythms outside of a circadian range. The body temperature rhythm of these animals was also weak 6 weeks after grafting; however, unlike activity patterns, body temperature regained a circadian period by 8 weeks after cell implantation. These data indicate that disruption of circadian activity is a behavioral measure of the consequences of beta/A4 accumulation in brain implants.
Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I.
Authors: Williamson R , Lee D , Hagaman J , Maeda N
Published: May 2006

Atherosclerosis is a major cause of morbidity and mortality in developed countries. In humans the risk of atherosclerosis is inversely correlated with plasma levels of high density lipoprotein (HDL). As a step in determining whether the experimental reduction of plasma HDL level will increase susceptibility to atherosclerosis, we have used gene targeting in embryonic stem cells to produce mice lacking apolipoprotein A-I, the major protein component of HDL particles. Mice homozygous for the disrupted gene have no plasma apolipoprotein A-I detectable by double immunodiffusion; their total plasma cholesterol and HDL-cholesterol levels after overnight fasting are reduced to about one-third and one-fifth of normal levels, and they are grossly deficient in alpha-migrating HDL particles.
A distinct cytoplasmic domain of CD2 regulates ligand avidity and T-cell responsiveness to antigen.
Authors: Hahn W C , Rosenstein Y , Calvo V , Burakoff S J , Bierer B E
Published: May 2006

The T-cell glycoprotein CD2 not only contributes to intercellular adhesion but also plays a direct role in T-cell activation. Here we demonstrate that the interaction of CD2 with its ligand lymphocyte function-associated antigen 3 (CD58) is regulated by T-cell receptor-CD3 signaling. T-cell receptor-CD3 crosslinking by specific antigen or monoclonal antibodies rapidly increases the avidity with which cell-surface CD2 binds immunoaffinity-purified CD58. Mutational analysis of the CD2 cytoplasmic domain demonstrates that the carboxyl-terminal asparagine is essential for T-cell receptor-induced changes in CD2 avidity but is not essential for CD2-mediated signaling, establishing that the cytoplasmic portion of CD2 consists of distinct functional domains. Furthermore, cell lines expressing CD2 molecules incapable of avidity regulation exhibit a marked deficiency in an antigen-specific response. Thus, the regulation of CD2 adhesiveness has a profound effect on the ability of CD2 to enhance antigen responsiveness. These observations demonstrate that adhesion strengthening resulting from increased CD2 avidity contributes directly to T-cell responsiveness independently of CD2-mediated signal transduction.
Analysis of gamma-ray energies for 56 excited superdeformed rotational bands of nuclei of lanthanons La to Dy and of Hg, Tl, and Pb on the basis of the two-revolving-cluster model, with evaluation of moments of inertia and radii of revolution and assignment of nucleonic compositions to the clusters and the central sphere.
Authors: Pauling L
Published: May 2006

Analysis of the gamma-ray energies of 28 excited superdeformed bands of lanthanon nuclei by application of the two-revolving-cluster model yields the result that the central sphere for all 28 has the semimagic-magic composition p40n50, with the range p8n12 to p14n18 for the clusters and the radius of revolution increasing from 7.31 to 7.76 fm. Similar analysis of 28 excited bands of Hg, Tl, and Pb nuclei leads to p56n82 (semimagic-magic) for the central sphere of 24 bands, p64n82 (semimagic-magic) for 2, and p64n90 (doubly semimagic) for 2, with cluster range p8n12 to p14n16 and values of the radius of revolution from 8.70 to 8.92 fm for 26 bands and 9.2 fm for 2.
Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor.
Authors: Kuhn A , Grummt I
Published: May 2006

In a reconstituted system consisting of partially purified RNA polymerase I (pol I) and the initiation factors TIF-IA, TIF-IB, and TIF-IC, the nucleolar factor UBF (upstream binding factor) stimulates transcription from the rRNA-encoding DNA (rDNA) promoter at least 50-fold. This activation is not observed at high template concentrations or in the presence of highly purified pol I. Template commitment experiments suggest that UBF activates transcription by relieving inhibition exerted by a negative-acting factor(s) in the polymerase fraction that competes for TIF-IB binding to the rDNA promoter and prevents the formation of preinitiation complexes. Using purified histone H1 bound to DNA as a model for the repressed state of the rDNA promoter, we show that UBF counteracts H1-mediated repression of pol I transcription. The implications of these findings are discussed with respect to the protein-protein and protein-DNA interactions at the rDNA promoter and the possible involvement of UBF in control of ribosomal gene transcription.
Development of a quasispecies of human immunodeficiency virus type 1 in vivo.
Authors: Cichutek K , Merget H , Norley S , Linde R , Kreuz W , Gahr M , Kurth R
Published: May 2006

During treatment with one specific batch of blood clotting factor IX, a number of hemophilia B patients in Germany recently became infected with human immunodeficiency virus type 1 (HIV-1). The nucleotide sequences of cloned HIV-1 envelope gene regions including the variable V3 loop and the V4 region derived from short-term virus cultures and directly from peripheral blood cells of these patients were shown to be highly homologous. Based on the assumption that the corresponding consensus sequence (termed HIV-1MBK) was identical to the genotype of the initially infecting virus, we were able to construct phylogenetic trees of the developing quasispecies in two patients studied in detail. True intermediates between input and multiply mutated genotypes were found in individual blood samples. Except for the initially infecting variant HIV-1MBK, variants found at 11 months postinfection had replaced those seen at 5 months postinfection. Variability early after infection was shown to cluster in two small regions located 3' of the V3 loop (i.e., outside the loop) and within the V4 region. This communication therefore describes the evolution of an HIV-1 quasispecies in humans starting from a single genotype.
MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination.
Authors: Bossone S A , Asselin C , Patel A J , Marcu K B
Published: May 2006

ME1a1, a 16-base-pair nuclear factor binding site residing between the c-MYC P1 and P2 transcription initiation sites, is required for P2 activity. A cDNA encoding a 477-amino acid zinc finger protein designated MAZ (MYC-associated zinc finger protein) was cloned from a HeLa lambda gt11 library by screening with a concatamerized ME1a1 binding site probe. In addition to six potential zinc fingers of the Cys2His2 type, MAZ contains an amino-terminal proline-rich domain and several polyalanine tracts. Its mRNA was present in all human tissues tested except for kidney, as a doublet of approximately 2.5 and 2.7 kilobases, along with differentially expressed minor species. MAZ bound specifically to the wild-type ME1a1 sequence but not to a ME1a1 mutant that also failed to yield P2 activity. When expressed as a fusion protein in a pMAL-c vector, MAZ binds with specificity to a GA box sequence (GGGAGGG) found in the c-MYC P2 promoter, to the P2 attenuator region within the gene's first exon, and to a related sequence involved in the transcriptional termination of the C2 gene. MAZ may encode a transcription factor with dual roles in transcription initiation and termination.
Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.
Authors: Vuori K , Pihlajaniemi T , Marttila M , Kivirikko K I
Published: May 2006

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.
Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type 1 E2 protein confers redox regulation of the DNA-binding activity in vitro.
Authors: McBride A A , Klausner R D , Howley P M
Published: May 2006

The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfhydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfhydryl group(s) may be protected by DNA binding. Sensitivity to sulfhydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.
Extensive peptide ligand exchange by surface class I major histocompatibility complex molecules independent of exogenous beta 2-microglobulin.
Authors: Smith J D , Lie W R , Gorka J , Myers N B , Hansen T H
Published: May 2006

Certain class I major histocompatibility complex molecules expressed on live cells have been shown to bind exogenous peptide ligands. However, it remains controversial whether this binding occurs by peptide exchange or to empty surface class I molecules. In this report we compare the surface binding and dissociation of two virus-derived ligands of the Ld class I molecule of the mouse. The peptide ligands were previously identified in immune responses to cytomegalovirus or lymphochoriomeningitis virus as immunodominant, optimally sized, and Ld restricted. Ligand dissociation was monitored on live cells indirectly by measuring the surface turnover of Ld-peptide complexes or directly by using labeled peptides. The cytomegalovirus-derived and lymphochoriomeningitis virus-derived peptides appeared to dissociate relatively rapidly; however, the cytomegalovirus-derived peptide had a more rapid off-rate than the lymphochoriomeningitis-derived peptide. Furthermore, these rates of dissociation appear to span that seen with endogenous Ld-associated peptides expressed by cells at 37 degrees C. Exploiting the extraordinary accessibility of the surface Ld ligand binding site we developed an assay to quantitate peptide ligand exchange. Cells were precoated with saturating amounts of unlabeled peptide by overnight incubation and were then tested for secondary binding of labeled peptides in a 4-h assay. Our results unequivocally demonstrate the potential for surface class I molecules to undergo peptide exchange. Furthermore, peptide exchange was found to be largely independent of exogenous beta 2-microglobulin. This result implies that beta 2-microglobulin association and not beta 2-microglobulin exchange is the critical factor in peptide exchange by surface class I molecules. Because of the exquisite ability of T cells to discriminate different amounts of ligand bound to class I, the binding of exogenous peptides could play a critical role in normal or aberrant immune responses.
Marked variation in the size of genomic plasmids among members of a family of related Epstein-Barr viruses.
Authors: Kolman J L , Kolman C J , Miller G
Published: May 2006

Epstein-Barr virus (EBV) genomes in the P3J-HR-1 (HR-1) Burkitt lymphoma cell line rearrange at a high rate. Previously described deletions and rearrangements in HR-1 cells have been found at sites of EBV replication in vivo, suggesting that DNA rearrangement may be an integral aspect of EBV biology and pathogenesis. We examined the structure of linear EBV genomes in subcultures of HR-1 cells using contour-clamped homogenous electric field gel electrophoresis. We developed a second pulsed electrophoretic technique to separate intracellular circular EBV plasmids. The standard, linear HR-1 EBV genome was approximately 155 kilobases in length. Linear molecules of less than unit length, presumably defective genomes, were seen in numerous subcultures. Linear intracellular genomes greater than 155 kilobases were also detected, but only linear genomes of 155 kilobases or less were packaged into virions. The size of circular EBV plasmids also varied greatly among HR-1 subcultures, some of which contained two plasmids of different size. The progeny of the unusual circular plasmids could be either standard or nonstandard linear genomes. No aberrant linear or circular form was detected in a subculture carrying the previously described het fragments. Pulsed-gel electrophoresis has provided two additional characteristics of mutant EBVs: abnormal linear and circular genome configurations.
Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha.
Authors: Schindler C , Fu X Y , Improta T , Aebersold R , Darnell J E
Published: May 2006

ISGF-3 is an interferon-dependent positive-acting transcription factor that is cytoplasmically activated, possibly through direct interaction with the interferon receptor. The factor has been purified, its component proteins have been separated, and its peptide sequences have been obtained. From the sequences, degenerate oligonucleotide probes were constructed to screen for cDNA clones. Sequencing of the selected clones shows that the 91- and 84-kDa components represent two forms of a previously unknown (to our knowledge) protein. Several antibodies raised against these proteins prove that they indeed do encode protein components of ISGF-3. This work provides reagents to explore the modification of this cytoplasmically activated transcription factor.
Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.
Authors: Skopek T R , Walker V E , Cochrane J E , Craft T R , Cariello N F
Published: May 2006

We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine (guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hprt- mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-gradient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprt- mutant frequency of 7.3 x 10(-5) in splenic T cells, approximately 35-fold above background levels. Sixty-nine of the 521 Hprt- mutants analyzed contained mutations in exon 3 (13%). Transversions and transitions at A.T base pairs dominated the spectrum; 62 of the 69 exon 3 mutations were at A.T base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at A.T bases) were located on the nontranscribed strand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G.C base pair. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse Hprt- T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.
Catalysis of serine and tyrosine autophosphorylation by the human insulin receptor.
Authors: Baltensperger K , Lewis R E , Woon C W , Vissavajjhala P , Ross A H , Czech M P
Published: May 2006

The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.
A trinucleotide can promote metal ion-dependent specific cleavage of RNA.
Authors: Kazakov S , Altman S
Published: May 2006

Nucleotide sequence and metal ion requirements for Mn(2+)-dependent self-cleavage of an RNA 31 nucleotides long [Dange, V., Van Atta, R. B. & Hecht, S. M. (1990) Science 248, 585-588] were examined by analysis of the site-specific cleavage activity of substitution and deletion mutants as well as complexes assembled from fragments of this RNA. A complex of UUU and GAAACp allows specific cleavage between G and A at 37 degrees C and pH 7.5. Additional nucleotides flanking the oligonucleotides in the minimal complex are not necessary for the cleavage reaction to take place but can affect the rate of the reaction. The 2'-OH groups of uridine residues do not participate in catalysis since both poly(U) and poly(dU) can promote the specific cleavage reaction in trans. Cd2+ ions can also promote the specific cleavage reaction and Mg2+ ions (which are inactive alone), under certain conditions, can enhance the Mn(2+)-induced cleavage of RNA.
Heterotetrameric structure of the human progesterone receptor.
Authors: Rehberger P , Rexin M , Gehring U
Published: May 2006

Nonactivated progesterone receptors in extracts of human T47D mammary carcinoma cells were investigated. Chemical cross-linking with dimethyl suberimidate resulted in complete stabilization of the A and B receptors with an average molecular mass of 340 kDa. For analyzing the subunit structure, we concentrated on the larger B receptor, which was separated from the A form by immunoaffinity chromatography. Progressive cross-linking of the photoaffinity-labeled receptor resulted in patterns of labeled bands in SDS gels, which are indicative of a heterotetrameric structure. It consists of one receptor polypeptide in association with two 90-kDa subunits and one polypeptide of approximately 60 kDa. The completely cross-linked B receptor has a molecular mass of approximately 390 kDa. To identify the subunits, the oligomeric B receptor was cross-linked with a cleavable bisimidate, highly purified by immunoaffinity chromatography, and analyzed by gel electrophoresis and immunoblotting. The receptor polypeptide has a mass of 116.5 kDa. The 90-kDa band was identified as the heat shock protein hsp90 and was roughly twice as intense as the receptor polypeptide. By use of specific antibodies, we identified the fourth receptor subunit as a 59-kDa protein (p59); we did not obtain any evidence for the heat shock protein hsp70 being a receptor component. We suggest an analogous heterotetrameric structure for the nonactivated A receptor.
A homolog of Escherichia coli RecA protein in plastids of higher plants.
Authors: Cerutti H , Osman M , Grandoni P , Jagendorf A T
Published: May 2006

Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.
Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3.
Authors: Demay M B , Kiernan M S , DeLuca H F , Kronenberg H M
Published: May 2006

1,25-dihydroxyvitamin D3 [1,25(OH)2D3], plays an important role in the regulation of mineral ion homeostasis. As well as being the major steroid hormone that regulates calcium metabolism, 1,25(OH)2D3 suppresses transcription of the gene encoding parathyroid hormone, a peptide that plays a dominant role in regulating extracellular calcium levels. To identify DNA sequences that may mediate this transcriptional repression, nuclear extracts containing the 1,25(OH)2D3 receptor were examined for binding to sequences in the 5'-flanking region of the human parathyroid hormone gene. A 25-base-pair (bp) oligonucleotide containing the sequences from -125 to -101 from the start of exon I binds nuclear proteins recognized by monoclonal antibodies against the 1,25(OH)2D3 receptor. The sequences in this region contain a single copy of a motif (AGGTTCA) homologous to the motifs repeated in the up-regulatory 1,25(OH)2D3-response elements. When placed upstream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediate transcriptional repression in response to 1,25(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. This down-regulatory element, therefore, differs from the up-regulatory 1,25(OH)2D3-response elements both in sequence composition and in the requirement for particular cellular factors other than the 1,25(OH)2D3 receptor for repressing transcription.
Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter.
Authors: Zimmer M , Medcalf R L , Fink T M , Mattmann C , Lichter P , Jenne D E
Published: May 2006

The human neutrophil and monocyte-derived serine protease homologues neutrophil elastase (NE), proteinase 3 (PR3), and azurocidin (AZU) are involved in a variety of immune defense reactions. NE and PR3 assist in the destruction of phagocytosed microorganisms, cleave the important connective-tissue protein elastin, and generate chemotactic activities by forming alpha 1-proteinase inhibitor complexes and elastin peptides. AZU is cytotoxic to certain microorganisms and chemotactic for monocytes. All three proteins are produced and packaged into azurophil granules in large quantities during neutrophil differentiation. We have isolated several cosmid clones each of which contains the functional genes for AZU, PR3, and NE in this order. The PR3 gene is separated by 8 kilobases from the 3' end of the AZU gene and by 3 kilobases from the 5' end of the NE gene. We report a physical map of the gene cluster, its location on chromosome 19pter, and the exon-intron organization of the AZU and PR3 genes. Our fluorescence in situ hybridization studies disprove the previous chromosomal assignment of the human NE gene to 11q14. The five exons of AZU and PR3 are organized like those of NE and other granule-associated serine proteases of hematopoietic cells. NE, PR3, and AZU are coordinately downregulated in the premonocytic cell line U937 during induced terminal differentiation. The cluster-like physical organization of these genes and concerted regulation during hematopoietic differentiation suggests that they are located in a developmentally activated chromatin domain promoting high-level, cell-specific expression in the monocyte-myelocyte lineage.
Role for major histocompatibility complex class I in regulating natural killer cell-mediated killing of virus-infected cells.
Authors: Kaufman D S , Schoon R A , Leibson P J
Published: May 2006

Target structures important for natural killer (NK) cell recognition of virally infected cells are not well defined. Since major histocompatibility complex (MHC) class I molecules bind viral peptides during acute infection, we evaluated whether an interaction between MHC and virus might influence the susceptibility of infected cells to NK cell-mediated lysis. To control for MHC class I expression on target cells, we used either HLA class I-deficient C1R cells or C1R sublines expressing transfected HLA class I gene products. Human NK cells were unable to preferentially lyse class I-deficient C1R cells after infection with herpes simplex virus (HSV). In contrast, HLA class I transfectants were significantly more susceptible to NK cell-mediated cytotoxicity after HSV infection. This occurred for HSV-infected C1R cells expressing any of the three HLA class I gene products tested (i.e., HLA-B27, HLA-A3, or HLA-Aw68), indicating that NK cell recognition in this system does not require "self" MHC and is not unique for a single haplotype. Productive HSV infection is required for the increased killing, since inoculation with UV-inactivated virus did not lead to increased lysis. In addition, since HSV infection of the transfectants did not significantly alter the level of class I expression, the change in susceptibility appears to be due to qualitative changes in the target structures on HSV-infected, HLA class I+ targets. These results demonstrate a role for MHC class I in regulating NK cell-mediated killing of virus-infected cells.
The major histocompatibility complex class I antigen-binding protein p88 is the product of the calnexin gene.
Authors: Galvin K , Krishna S , Ponchel F , Frohlich M , Cummings D E , Carlson R , Wands J R , Isselbacher K J , Pillai S , Ozturk M
Published: May 2006

A 90-kDa phosphoprotein (p90) of the endoplasmic reticulum was identified by a monoclonal antibody generated against human hepatoma cells. Pulse-chase experiments with [32P]phosphate and [35S]methionine demonstrated that p90 formed both stable and transient complexes with other cellular proteins, suggesting its role as a molecular chaperone. This protein associates with heavy chains of major histocompatibility complex class I proteins, suggesting that it is the human homolog of the recently described 88-kDa protein that transiently associates with murine class I molecules in the endoplasmic reticulum. The p90 protein also associates in B lymphocytes with membrane immunoglobulin mu heavy chains and may serve as a chaperone for many membrane-bound polypeptides. A partial human p90 cDNA was cloned from a lambda gt11 expression library and identified as the human homolog of calnexin, a major canine calcium-binding protein found to be associated with the signal-sequence receptor in endoplasmic reticulum membranes.
HEN1 and HEN2: a subgroup of basic helix-loop-helix genes that are coexpressed in a human neuroblastoma.
Authors: Brown L , Espinosa R , Le Beau M M , Siciliano M J , Baer R
Published: May 2006

An important family of regulatory molecules is made up of proteins that possess the DNA-binding and dimerization motif known as the basic helix-loop-helix (bHLH) domain. The bHLH family includes subgroups of closely related proteins that share common functional properties and overlapping patterns of expression (e.g., the MyoD1 and achaete-scute subgroups). In this report we describe HEN1 and HEN2, mammalian genes that encode a distinct subgroup of bHLH proteins. The HEN1 gene was identified on the basis of cross-hybridization with TAL1, a known bHLH gene implicated in T-cell acute lymphoblastic leukemia. In situ fluorescence hybridization was used to localize the human HEN1 gene to chromosome band 1q22. HEN1 and HEN2 are coexpressed in the IMR-32 human neuroblastoma cell line, and they encode highly related proteins of 133 and 135 residues, respectively, that share 98% amino acid identity in their hHLH domains. These data imply that the bHLH protein subgroup encoded by HEN1 and HEN2 may serve important regulatory functions in the developing nervous system.
An enzyme releasing lacto-N-biose from oligosaccharides.
Authors: Sano M , Hayakawa K , Kato I
Published: May 2006

alpha-L-Fucosidase (alpha-L-fucoside fucohydrolase; EC 3.2.1.51) preparations from Streptomyces sp. 142 were found to contain an enzyme specific for lacto-N-biosidic (Gal beta 1-3GlcNAc beta 1-) linkages (type 1 structure) in oligosaccharides. The enzyme preparation, which was eluted after alpha-fucosidase from a CM-Sepharose column, contained some alpha-fucosidase activity but was free from other glycosidases and proteases. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine showed that the enzyme specifically hydrolyzed lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) but did not hydrolyze lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, or poly-N-acetyllactosamine. Structural analysis of the enzyme digest of the N-acetyllactosamine type of triantennary sugar chain with type 1 structure showed that lacto-N-biose (Gal beta 1-3GlcNAc) and the N-acetyllactosamine type of biantennary sugar chain were produced. Thus this enzyme was tentatively named lacto-N-biosidase, because it hydrolyzes oligosaccharides containing a type 1 structure at the nonreducing terminus and produces lacto-N-biose.