bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct photoaffinity labeling of tubulin with colchicine.
Authors: Wolff J , Knipling L , Cahnmann H J , Palumbo G
Published: May 2006

Ultraviolet irradiation of the [3H]colchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to greater than 90%) of the labeling occurs on beta-tubulin and appears early after irradiation, whereas alpha-tubulin is labeled later. The labeling ratio of beta-tubulin to alpha-tubulin (beta/alpha ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the beta/alpha ratio. Limited data with [3H]podophyllotoxin show that it covalently bound with a similar beta/alpha distribution. Vinblastine, on the other hand, exhibits preferential attachment to alpha-tubulin. The possibilities that colchicine binds at the interface between alpha-tubulin and beta-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.
Inositol 1,4,5-trisphosphate receptors: distinct neuronal and nonneuronal forms derived by alternative splicing differ in phosphorylation.
Authors: Danoff S K , Ferris C D , Donath C , Fischer G A , Munemitsu S , Ullrich A , Snyder S H , Ross C A
Published: May 2006

We have identified two distinct transcripts of inositol 1,4,5-trisphosphate receptor by using the PCR on first-strand cDNAs from various rat tissues. The longer form, corresponding to the previously cloned adult rat brain inositol 1,4,5-trisphosphate receptor, contains a 120-nucleotide insert between the two cAMP-dependent protein kinase phosphorylation consensus sequences. The shorter form (lacking the insert) predominates in fetal brain and peripheral tissues and appears to represent a nonneuronal receptor, whereas the longer form is found in adult brain and appears to be exclusively neuronal. The phosphorylation kinetics by cAMP-dependent protein kinase and the phosphopeptide maps differ for inositol 1,4,5-trisphosphate receptors purified from tissues predominantly expressing different forms of the transcript.
Intracellular sites for storage and recycling of C3b receptors in human neutrophils.
Authors: Berger M , Wetzler E M , Welter E , Turner J R , Tartakoff A M
Published: May 2006

Upon activation of human neutrophils, C3b receptors (type 1 complement receptors, CR1) are rapidly translocated from an intracellular pool to the surface, increasing plasma membrane expression 6- to 10-fold. This is followed by reinternalization and degradation of the receptors, even in the absence of ligand. Upregulation of surface CR1 may occur without exocytosis of primary or secondary granules, and intracellular CR1 in resting neutrophils does not sediment with these granules on density gradient fractionation. To directly localize the intracellular pools of CR1, we used immunoelectron microscopy of fixed, permeabilized cells. In resting neutrophils, CR1 is associated with the membranes of smooth-surfaced, empty-appearing vesicles whose irregular borders clearly distinguish them from primary and secondary granules. After activation by fMet-Leu-Phe, the intracellular pool is primarily found in large, conspicuous, multivesicular bodies. These bodies also incorporate colloidal gold from the extracellular fluid, suggesting that they are formed by endocytosis. Thus, the sites in which CR1 is stored in resting cells, and recycled in activated cells, are structurally unique and morphologically distinguishable.
Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.
Authors: Satoh T , Nakafuku M , Miyajima A , Kaziro Y
Published: May 2006

The protooncogene ras acts as a component of signal-transduction networks in many kinds of cells. The ras gene product (p21) is a GTP-binding protein, and the activity of the protein is regulated by bound GDP/GTP. Recent studies have shown that a certain class of growth factors stimulates the formation of active p21-GTP complexes in fibroblasts and that oncogene products with enhanced tyrosine kinase activities have a similar effect on ras p21. We have measured the ratio of active GTP-bound p21 to total p21 in several lymphoid and myeloid cell lines in order to understand the role of ras in the proliferation of these cells. Interleukin 2 (IL-2), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) enhance the formation of the active p21.GTP, whereas IL-4 has no effect on p21-bound GDP/GTP. These results strongly suggest that ras p21 acts as a transducer of signals from IL-2, IL-3, and GM-CSF, but not from IL-4.
A transition metal complex (Venus flytrap cluster) for radioimmunodetection and radioimmunotherapy.
Authors: Paxton R J , Beatty B G , Hawthorne M F , Varadarajan A , Williams L E , Curtis F L , Knobler C B , Beatty J D , Shively J E
Published: May 2006

A novel transition metal complex, Venus flytrap cluster (VFC), is described for the preparation of radio-labeled antibodies. VFC contained 57Co, which was held tightly between the faces of two covalently bridged carborane ligands by cluster bonding of the metal with appropriate ligand orbitals. Anti-carcinoembryonic antigen monoclonal antibody T84.66 was conjugated to 57Co-VFC with full retention of immunological activity. Biodistribution studies in nude mice bearing carcinoembryonic antigen-producing tumors showed excellent tumor localization of 57Co-VFC-T84.66. The accumulation of radionuclide in normal liver was low and independent of dose, which may reflect the stability of the radionuclide complex. These results presage the use of VFC systems for binding transition metals that are clinically useful for radioimmunodiagnosis and radioimmunotherapy.
Migratory arrest of gonadotropin-releasing hormone neurons in transgenic mice.
Authors: Radovick S , Wray S , Lee E , Nicols D K , Nakayama Y , Weintraub B D , Westphal H , Cutler G B , Wondisford F E
Published: May 2006

Gonadotropin-releasing hormone (GnRH) is important in reproduction, although the mechanism of central hypogonadism in humans remains unclear. Because the GnRH neuron originates from the olfactory placode and migrates to the hypothalamus during development, central hypogonadism in humans could be caused by failure in normal migration of GnRH neurons to the hypothalamus. We report that in transgenic mice expression of the simian virus 40 T antigen, driven by the promoter of human GnRH gene, resulted in central hypogonadism due to an arrest in neuronal migration during development and tumor formation along the migratory pathway. This system appears to be an important animal model of hypogonadotropic hypogonadism in humans. Additionally, olfactory bulb tumors from these animals were dispersed, and a GnRH-secreting neuronal cell line (GN cell line) was established.
Cloning of a leucine-zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration.
Authors: Fathallah-Shaykh H , Wolf S , Wong E , Posner J B , Furneaux H M
Published: May 2006

Antibody-associated paraneoplastic cerebellar degeneration (the Yo syndrome) is an uncommon disorder in which an immune response is specifically directed against tumor tissue and the cerebellum. Screening of a lambda expression library has resulted in the isolation of cDNA clones that encode the major antigen recognized by serum from these patients. The fusion protein produced by the cDNA clones provides the basis of a simple diagnostic assay for this neurological syndrome. The occurrence of leucine-zipper and zinc-finger motifs in the predicted open reading frame suggests that this protein plays a role in the regulation of gene expression.
Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells.
Authors: Kwon Y K , Hecht N B
Published: May 2006

The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA.
Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor.
Authors: Atassi M Z , Manshouri T , Sakata S
Published: May 2006

Two regions of human thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) (residues 12-44 and 308-364) were selected on the basis that they exhibit no sequence resemblance to luteinizing hormone/chorionic gonadotropin receptor. Five synthetic overlapping peptides (12-30, 24-44, 308-328, 324-344, and 339-364) were studied for their ability to bind 125I-labeled human TSH (hTSH), its isolated alpha and beta subunits, bovine TSH, ovine TSH, human luteinizing hormone, and human follicle-stimulating hormone. The human TSHR peptides 12-30 and 324-344 exhibited remarkable binding activity to human, bovine, and ovine TSH and to the beta chain of hTSH. Lower binding activity resided in the adjacent overlapping peptides, probably due to the contribution of the shared overlap to the binding. The specificity of TSH binding to these peptides was confirmed by their inability to bind human luteinizing hormone, human follicle-stimulating hormone, and the alpha chain of hTSH. Thyrotropins did not bind to bovine serum albumin or to peptide controls unrelated to the TSHR system. Furthermore, the binding of hTSH to TSHR peptides 12-30 and 324-344 was almost completely (approximately 90%) inhibited by rabbit antibodies against hTSH but not by antisera against unrelated proteins. It is concluded that the binding of TSH to its receptor involves extensive contacts and that the TSHR peptides 12-30 and 324-344 contain specific binding regions for TSH that might be either independent sites or two faces (subsites) within a large binding site.
The different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3, and HNF-4, protein factors that govern liver-specific transcription.
Authors: Xanthopoulos K G , Prezioso V R , Chen W S , Sladek F M , Cortese R , Darnell J E
Published: May 2006

The transcription factors that act in hepatocyte-specific gene expression include proteins that are present mainly in liver cells (HNF-1/LFB1, C/EBP, HNF-3, HNF-4) (HNF, hepatocyte nuclear factor; C/EBP, rat enhancer binding protein) and proteins that are widely distributed (AP-1, NF-1, NF-Y/ACF). We show here that the genes encoding each of these liver-enriched factors exhibit different patterns of transcriptional control in different tissues. In addition, there were several instances in which transcription was detected (e.g., for HNF-1) when no mRNA or specific DNA binding protein was found, suggesting the importance of posttranscriptional control in some instances for these factors. These experiments identify C/EBP, HNF-3, and HNF-4, and perhaps also HNF-1, as targets for the study of cascades of transcriptionally controlled transcription factors in differentiated cells.
Alternative splicing contributes to K+ channel diversity in the mammalian central nervous system.
Authors: Luneau C J , Williams J B , Marshall J , Levitan E S , Oliva C , Smith J S , Antanavage J , Folander K , Stein R B , Swanson R
Published: May 2006

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.
Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection.
Authors: Woisetschlaeger M , Jin X W , Yandava C N , Furmanski L A , Strominger J L , Speck S H
Published: May 2006

During latent Epstein-Barr virus (EBV) infection of human B lymphocytes, six viral nuclear antigen (EBNAs) are expressed from long primary transcripts by means of alternative splicing and alternative polyadenylylation sites. These transcripts initiate from one of two promoters, Cp or Wp, that function in a mutually exclusive fashion. Wp is exclusively utilized during the initial stages of infection of primary B lymphocytes, followed by a switch to Cp usage. These studies have been extended to show that (i) a mutant EBV strain lacking the gene encoding EBNA 2 fails to switch from Wp to Cp usage in primary B lymphocytes, although the virus contains a functional Cp; (ii) a region from -429 to -245 base pairs upstream of Cp is essential for Cp activity in B lymphocytes, but only in the context of upstream and downstream sequences; (iii) this region contains an EBNA 2-dependent enhancer; and (iv) DNase I protection employing nuclear extracts from B and T lymphocytes revealed a B-cell-specific footprint in the region of the EBNA 2-dependent enhancer. These results support a model for viral promoter switching during the initial stages of infection in which Wp activity leads to the expression of EBNA 2, followed by activation of Cp through the EBNA 2-dependent enhancer.
Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells.
Authors: Owens R J , Dubay J W , Hunter E , Compans R W
Published: May 2006

In polarized epithelial cells, the release of enveloped viruses by budding at the cell surface is restricted to a specific cell membrane domain, either the apical or basolateral domain. To investigate the role of the envelope glycoprotein and the capsid proteins of human immunodeficiency virus type 1 (HIV-1) in determining the site of virus assembly, we analyzed virus maturation in a polarized monkey kidney cell line. A line of cells harboring the HIV-1 provirus (VERO-pFN) was found to differentiate into polarized epithelial cell monolayers upon reaching confluency. By electron microscopy, virus maturation was observed predominantly at the basolateral membranes of VERO-pFN cells. Analysis of HIV-1 proteins revealed that virtually all of glycoprotein gp120 and capsid protein p24 were found in the basolateral medium, while no HIV-1 proteins were detected apically. A recombinant vaccinia virus (VV) expressing the HIV-1 gag polyprotein (VVgag) was used to determine the site of release of HIV-1 core particles in polarized epithelial cells in the presence or absence of envelope glycoproteins. When cells were infected with VVgag in the absence of envelope proteins, similar amounts of the p24 capsid protein were released into virus particles at the apical or basolateral surface. In contrast, when cells were doubly infected with VVgag and a recombinant VV expressing the HIV-1 envelope glycoprotein (VVenv), 94% of p24 and all of gp120 were found to be associated with particles released into the basolateral medium. These results indicate that the HIV-1 envelope glycoprotein directly influences the site of release of virus particles containing the gag protein, probably via a specific interaction between the envelope protein and the gag protein.
Splicing of juvenile and adult tau mRNA variants is regulated by thyroid hormone.
Authors: Aniello F , Couchie D , Bridoux A M , Gripois D , Nunez J
Published: May 2006

The effect of thyroid hormone on the expression of tau transcripts was studied during postnatal brain development. The level of tau mRNA was only slightly changed postnatally in the cerebral hemispheres of hypothyroid rats, whereas the level of tau mRNA in the cerebellum was maintained at a higher level than in the euthyroid controls. As shown by in situ hybridization studies, such an alteration in tau mRNA expression can be ascribed to an effect of thyroid hormone on the rate of migration of the granule cells in the cerebellum; that tau mRNAs remain high in the cerebellum as long as the granule cells are migrating correlates with the observation that hypothyroidism slows the rate of migration of granule cells. RNase protection assays also showed that thyroid hormone deficiency delays the transition between the immature and mature tau transcripts in both brain regions. Thus, one of the effects of thyroid hormone is to regulate the splicing mechanism that allows replacement of the juvenile tau variants by the adult entities during neuronal differentiation.
Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins.
Authors: Murray M T , Schiller D L , Franke W W
Published: May 2006

Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation.
Pyridine nucleotide redox state parallels production of aldosterone in potassium-stimulated adrenal glomerulosa cells.
Authors: Pralong W F , Hunyady L , Várnai P , Wollheim C B , Spät A
Published: May 2006

Extracellular potassium ions (K+) raise the intracellular concentration of free Ca2+ ([Ca2+]i) by gating voltage-dependent Ca2+ channels and stimulate aldosterone production in adrenal glomerulosa cells. The pathway leading from calcium influx to increased steroid synthesis has not been completely elucidated. In the present study we demonstrate that the reduction of pyridine nucleotides known to be required for steroid hydroxylation is enhanced by K+ (4.1-8.4 mM) in single rat glomerulosa cells. The action of K+ was strictly dependent on the presence of extracellular Ca2+. Amytal, a blocker of site I of the mitochondrial respiratory chain, abolished the K+ effect, indicating a mitochondrial origin for the recorded changes. Supraphysiological K+ concentration (18 mM) resulted in a further increase in [Ca2+]i, while steroidogenesis was decreased as measured in cell suspensions. However, a possible explanation for this dichotomy is provided by the finding that the level of reduced pyridine nucleotides also decreased at supraphysiological K+ concentration.
Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation.
Authors: Klingel K , Hohenadl C , Canu A , Albrecht M , Seemann M , Mall G , Kandolf R
Published: May 2006

Coxsackievirus B3-induced myocarditis in different immunocompetent mouse strains was used as a model to investigate interrelationships between virus replication and development of chronic enteroviral heart disease. Using in situ hybridization to detect enteroviral RNA, we show that heart muscle infection is not only detected in acute myocarditis but is also detected during the chronic phase of the disease. Coxsackievirus B3 could evade immunological surveillance in a host-dependent fashion, thus inducing a persistent infection of the myocardium in association with ongoing inflammation. Patterns of acute and persistent myocardial infection were quantitatively assessed in one representative mouse strain (A.CA/SnJ, H-2f) by applying computer-assisted digital image processing; these patterns were then related to the extent of myocardial tissue damage as well as to inflammation. We observed a strong correlation, both spatial and temporal, between viral replication and development of myocardial lesions, indicating that acute and chronic myocardial injuries are a consequence of multifocal organ infection. Analysis of strand-specific in situ hybridization revealed that viral replication in persistent infection is restricted at the level of RNA synthesis. The described procedure for quantitating organ infection provides a powerful tool for evaluating virus-host interactions and will be of particular interest to those studying human enterovirus-induced cardiomyopathies.
Dynamics of single-motor molecules: the thermal ratchet model.
Authors: Córdova N J , Ermentrout B , Oster G F
Published: May 2006

We present a model for single-motor molecules--myosin, dynein, or kinesin--that is powered either by thermal fluctuations or by conformational change. In the thermally driven model, the cross-bridge fluctuates about its equilibrium position against an elastic restoring force. The attachment and detachment of the cross-bridge are determined by modeling the electrostatic attraction between the cross-bridge and the fiber binding sites, so that binding depends on the strain in the cross-bridge and its velocity with respect to the fiber. The model correctly predicts the empirical force-velocity characteristics for populations of motor molecules. For a single motor, the apparent cross-bridge step size per ATP hydrolysis depends nonlinearly on the load. When the elastic energy driving the cross-bridge is generated by a conformational change, the velocity and duty cycle are much larger than is observed experimentally for myosin.
Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells.
Authors: Shiota G , Rhoads D B , Wang T C , Nakamura T , Schmidt E V
Published: May 2006

Hepatocyte growth factor (HGF) is a potent mitogen for primary hepatocytes. Therefore, we examined HGF as a possible autocrine growth factor in hepatocellular carcinoma (HCC). We introduced an albumin-HGF expression vector into Fao HCC cells and transgenic mice. Expression of the albumin-HGF vector in Fao HCC cells inhibited their growth in vitro. In vivo, FaoHGF cells produced tumors that averaged 10% of the sizes of G418-resistant controls when transplanted into nude mice. In contrast, hepatocytes from transgenic mice expressing HGF grew more rapidly than did those from normal siblings. Further, growth of eight additional HCC cell lines was inhibited by the addition of recombinant HGF. Finally, of 35 tumor cell lines surveyed, only 6 cell lines expressed HGF mRNA, and no HCC cell line expressed HGF. Although HGF stimulates normal hepatocytes, it is a negative growth regulator for HCC cells.
Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.
Authors: Walker G T , Little M C , Nadeau J G , Shank D D
Published: May 2006

An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.
Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells.
Authors: Resnitzky D , Tiefenbrun N , Berissi H , Kimchi A
Published: May 2006

One approach to identify postreceptor molecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G0/G1-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical G0/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G0/G1-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor beta, reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon- or IL-6-induced G0/G1-specific arrest requires at least these two receptor-generated events.
Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants.
Authors: Cibotti R , Kanellopoulos J M , Cabaniols J P , Halle-Panenko O , Kosmatopoulos K , Sercarz E , Kourilsky P
Published: May 2006

We have produced transgenic mice expression hen egg-white lysozyme (HEL) under the control of a ubiquitous promoter, so that in transgenic animals, HEL is presumably present in the serum and thymus throughout the period of establishment of the T-cell repertoire. We show that HEL transgenic H-2d mice with HEL blood levels greater than 10 ng/ml are tolerant to HEL as well as to the immunodominant peptide 108-116. Thus, their T lymphocytes do not proliferate in response to the immunodominant peptide 108-116 after in vivo immunization with HEL or peptide 108-116. In contrast, in transgenic mice tolerant to HEL, the state of tolerance to subdominant peptides 1-18 and 74-96 appears variable and highly depended on HEL blood levels. Complete unresponsiveness is seen when HEL serum levels are high, and this unresponsiveness is reached at a lower HEL concentration for peptide 1-18 than for peptide 74-96. Thus, a hierarchy exists among the three peptides (108-116 much greater than 1-18 greater than 74-96) for induction of a response to HEL and for HEL tolerance induction in T cells specific for these peptides. Persistence in the periphery of autoreactive T cells recognizing subdominant peptides of self-proteins, as shown in this transgenic model, indicates that self-tolerance is limited to a subset of dominant self-peptides and suggests a role for T lymphocytes specific for subdominant determinants in autoimmunity.
S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds.
Authors: Stamler J S , Simon D I , Osborne J A , Mullins M E , Jaraki O , Michel T , Singel D J , Loscalzo J
Published: May 2006

Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.
Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase.
Authors: Nunn D N , Lory S
Published: May 2006

In the Gram-negative pathogen Pseudomonas aeruginosa, mutants in the gene for the prepilin peptidase (pilD) are pleiotropic, as they not only fail to process pilin but also accumulate in the periplasm, in their mature form, several toxins and hydrolytic enzymes that are normally exported to the external medium (excreted). We have suggested that this excretion defect is due to the lack of PilD-dependent processing of proteins that share sequences in common with the prepilin subunit and that are components of a protein-excretion machinery. In this paper we report the isolation and characterization of transposon-induced excretion mutants with phenotypes similar to that of a pilD gene mutant. Using oligonucleotide probes designed to recognize sequences encoding the cleavage site of the type IV prepilins, we have isolated four linked genes with the predicted putative PilD-dependent cleavage site. Site-specific mutations within these genes have shown that they are required for protein excretion, and PilD-dependent processing of at least one of the four encoded proteins was demonstrated. Evidence suggests that similar components play a role in protein excretion in a wide variety of Gram-negative bacteria.
Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.
Authors: Lem J , Flannery J G , Li T , Applebury M L , Farber D B , Simon M I
Published: May 2006

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.
Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo.
Authors: Lee R C , River L P , Pan F S , Ji L , Wollmann R L
Published: May 2006

Victims of major electrical trauma frequently suffer extensive skeletal muscle and nerve damage, which is postulated to be principally mediated by electroporation and/or thermally driven cell membrane permeabilization. We have investigated the efficacy of two blood-compatible chemical surfactants for sealing electroporated muscle membranes. In studies using cultured skeletal muscle cells, poloxamer 188 (P188; an 8.4-kDa nonionic surfactant) blocks, and neutral dextran (10.1 kDa) substantially retards, carboxyfluorescein release from electropermeabilized cell membranes. To test whether P188 administered intravenously could have the same therapeutic effect in vivo, the rat biceps femoris muscle flap attached by its arteriovenous pedicle was electropermeabilized until its electrical resistivity dropped to 50% of the initial value. P188 (460 mg/kg) administered intravenously 20 min postshock restored the resistivity to 77% of the initial value. When P188 was administered intravenously 5 min before shock, a dose-dependent impedance recovery rate was observed. Neither neutral dextran (460 mg/kg) nor sterile saline was effective. Histopathologic studies indicated that postshock poloxamer administration reduced tissue inflammation and damage in comparison with dextran-treated or control tissues. Electrophysiologic evidence of membrane damage was not observed in flaps of animals pretreated with poloxamer. These results suggest that it may be possible to seal in vivo tissue membranes injured by electrical, thermal, or other membrane-damaging forces.
Evidence that Mos protein may not act directly on cyclin.
Authors: Xu W , Ladner K J , Smith L D
Published: May 2006

Using affinity-purified antiserum we have examined cyclin B2 levels in Xenopus oocytes at various stages of oogenesis. We found that cyclin B2 is detected from stage 2 to stage 6 as two bands, one of which is phosphorylated, and that cyclin B2 mass increases about 28-fold between stage 2 and stage 6. To examine the effect of Mos protein on cyclin phosphorylation, we microinjected synthetic Xenopus c-mos (c-mosxe) RNA into stages 4, 5, and 6 Xenopus oocytes. In stage 6 oocytes, maturation was induced by c-mosxe RNA, and, as is the case with progesterone treatment, all cyclin B2 was shifted to the phosphorylated form. However, c-mosxe RNA injected into stage 4 or 5 oocytes did not induce maturation or cause a shift in the relative proportion of the two cyclin B2 bands. These data suggest that Mos does not act directly to phosphorylate cyclin B2, causing the band shift during maturation. Cyclin B2 synthesis increases about 2-fold during maturation, in concert with total protein synthesis. Data from experiments on cyclin B2 stability indicate that the half-life of cyclin B2 is about 85 hr in stage 6. This suggests that if Mos protein has a direct effect on cyclin stability, it does so only at a later stage in oocyte maturation, but not at the onset.
Region-specific expression of a K+ channel gene in brain.
Authors: Rudy B , Kentros C , Weiser M , Fruhling D , Serodio P , Vega-Saenz de Miera E , Ellisman M H , Pollock J A , Baker H
Published: May 2006

Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions.
Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.
Authors: Soldatov N M
Published: May 2006

The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of these sites correlate with RNA splice sites, indicating that the molecular diversity of the transcripts is a result of alternative splicing. The fourth diversity region is located at the C-terminal region and comprises insertions and deletions. It is suggested that these variations may give rise to multiple subforms of HFCC with altered electrophysiological properties.
Dodeca satellite: a conserved G+C-rich satellite from the centromeric heterochromatin of Drosophila melanogaster.
Authors: Abad J P , Carmena M , Baars S , Saunders R D , Glover D M , Ludeña P , Sentis C , Tyler-Smith C , Villasante A
Published: May 2006

To identify sequences from the centromeric region, we have constructed a Drosophila melanogaster yeast artificial chromosome (YAC) library and screened it with purified DNA from the minichromosome Dp(1;f)1187 derived from the X chromosome. We describe the structure of one clone isolated in this way. This YAC is structurally unstable and contains tandemly repeated G+C-rich 11-mer and 12-mer units, which we call dodeca satellite. Most of this satellite is located near the centromere of an autosome. Cross-hybridizing sequences are found in the genomes of organisms as distant as Arabidopsis thaliana and Homo sapiens.
Mechanism of dioxin action: Ah receptor-mediated increase in promoter accessibility in vivo.
Authors: Wu L , Whitlock J P
Published: May 2006

We have analyzed dioxin-inducible, Ah receptor-dependent changes in protein-DNA interactions at the CYP1A1 transcriptional promoter in intact mouse hepatoma cells. Our findings indicate that in uninduced cells, the promoter is inaccessible to its cognate binding proteins, which are known to be expressed constitutively. Dioxin induces, in Ah receptor-dependent fashion, an increase in promoter accessibility, which occurs rapidly and does not require ongoing transcription of the CYP1A1 gene. The change in promoter accessibility is not due to an altered pattern of cytosine methylation at the promoter; it probably reflects a 2,3,7,8-tetrachlorodibenzo-p-dioxin- induced change in the chromatin structure. These findings provide new insight into the mechanism of dioxin action and contribute to a better understanding of the regulation of inducible gene transcription in mammalian cells.
The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes.
Authors: Saccone S , De Sario A , Della Valle G , Bernardi G
Published: May 2006

Chromosome in situ suppression hybridization has been carried out on human metaphase chromosomes to localize the G+C-richest human DNA fraction (which only represents 3.5% of the genome), as isolated by preparative equilibrium centrifugation in Cs2SO4/3,6-bis(acetatomercurimethyl)-1,4-dioxane density gradient. This fraction essentially corresponds to isochore family H3. The rationale for carrying out this experiment is that this isochore family has, by far, the highest gene concentration, the highest concentration in CpG islands, the highest transcriptional and recombinational activity, and a distinct chromatin structure. The in situ hybridization results obtained show that the H3 isochore family is localized in two coincident sets of bands of human metaphase chromosomes: telomeric bands and chromomycin A3-positive 4',6-diamidino-2-phenylindole-negative bands. This result is the first step toward a complete compositional map of the human karyotype. Because the G+C gradient across isochore families is paralleled by a gene concentration gradient, such a map has structural, functional, and evolutionary relevance.
Cholecystokinin cells purified by fluorescence-activated cell sorting respond to monitor peptide with an increase in intracellular calcium.
Authors: Liddle R A , Misukonis M A , Pacy L , Balber A E
Published: May 2006

Cholecystokinin (CCK) is secreted from specific enteroendocrine cells of the upper small intestine upon ingestion of a meal. In addition to nutrients, endogenously produced factors appear to act within the gut lumen to stimulate CCK release. One such factor is a trypsin-sensitive CCK-releasing peptide found in pancreatic juice, known as monitor peptide. This peptide is active within the intestinal lumen and is hypothesized to stimulate CCK secretion by interacting directly with the CCK cell. We have found that monitor peptide releases CCK from isolated rat intestinal mucosal cells and that this effect is dependent upon extracellular calcium. In the present study, we used monitor peptide as a tool for isolating CCK cells from a population of small intestinal mucosal cells. Dispersed rat intestinal mucosal cells were loaded with the calcium-sensitive fluorochrome Indo-1, and CCK secretory cells were identified spectrofluorometrically by their change in fluorescence when stimulated with monitor peptide. Cells demonstrating a change in their emission fluorescence ratio were sorted using a fluorescence-activated cell sorter. More than 90% of the sorted cells stained positively for CCK with immunohistochemical staining. Furthermore, sorted cells secreted CCK when stimulated with membrane-depolarizing concentrations of potassium chloride, dibutyryl cAMP, calcium ionophore, and monitor peptide. These findings indicate that functional intestinal CCK cells can be highly enriched using fluorescence-activated cell sorting. Furthermore, monitor peptide appears to interact directly with CCK cells to signal CCK release through an increase in intracellular calcium.
The gene for an inherited form of deafness maps to chromosome 5q31.
Authors: Leon P E , Raventos H , Lynch E , Morrow J , King M C
Published: May 2006

Primary--i.e., nonsyndromal-postlingual deafness is inherited as an autosomal dominant phenotype in a large kindred in Costa Rica. Genetically susceptible individuals begin to lose hearing at low frequencies at about age 10 years, after language and speaking are learned. Deafness inevitably progresses by age 30 years to bilateral hearing loss of all frequencies. Intelligence, fertility, and life expectancy are normal. The family traces its ancestry to an affected founder born in Costa Rica in 1754. We have mapped the gene for deafness in this kindred to chromosome 5q31, between the markers IL9 and GRL, by linkage analysis involving 99 informative relatives.
A 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues.
Authors: Lederfein D , Levy Z , Augier N , Mornet D , Morris G , Fuchs O , Yaffe D , Nudel U
Published: May 2006

The known Duchenne muscular dystrophy (DMD) gene products, the muscle- and brain-type dystrophin isoforms, are 427-kDa proteins translated from 14-kilobase (kb) mRNAs. Recently we described a 6.5-kb mRNA that also is transcribed from the DMD gene. Cloning and in vitro transcription and translation of the entire coding region show that the 6.5-kb mRNA encodes a 70.8-kDa protein that is a major product of the DMD gene. It contains the C-terminal and the cysteine-rich domains of dystrophin, seven additional amino acids at the N terminus, and some modifications formed by alternative splicing in the C-terminal domain. It lacks the entire large domain of spectrin-like repeats and the actin-binding N-terminal domain of dystrophin. This protein is the major DMD gene product in brain and other nonmuscle tissues but is undetectable in skeletal muscle extracts.
Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis.
Authors: Antoniades H N , Neville-Golden J , Galanopoulos T , Kradin R L , Valente A J , Graves D T
Published: May 2006

Macrophages are thought to play an important role in the pathologic changes associated with idiopathic pulmonary fibrosis (IPF). The mechanisms for increased monocyte/macrophage recruitment in IPF are unknown. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cell types in culture. We examined the expression of MCP-1 mRNA and its protein product in vivo in IPF and non-IPF lung specimens by in situ hybridization and immunocytochemistry. The cell types expressing MCP-1 in vivo were identified by immunostaining with specific antibodies. We demonstrated the expression of MCP-1 mRNA in pulmonary epithelial cells, in monocytes/macrophages, and in vascular endothelial and smooth muscle cells. Lung epithelial cells in patients with IPF strongly expressed MCP-1 mRNA and its protein product. In contrast, epithelial cells in non-IPF specimens did not express MCP-1 mRNA. Macrophages and vascular endothelial and smooth muscle cells were shown to express MCP-1 in both IPF and non-IPF lung specimens. These findings provide a basis for the understanding of the in vivo physiologic processes that mediate monocyte/macrophage recruitment and infiltration in the lung interstitium and the pathologic state contributing to an increased alveolar monocyte/macrophage population and inflammation in IPF.