bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of a cDNA encoding the rat high molecular weight neurofilament peptide (NF-H): developmental and tissue expression in the rat, and mapping of its human homologue to chromosomes 1 and 22.
Authors: Lieberburg I , Spinner N , Snyder S , Anderson J , Goldgaber D , Smulowitz M , Carroll Z , Emanuel B , Breitner J , Rubin L
Published: May 2006

Neurofilaments (NFs) are the intermediate filaments specific to nervous tissue. They are probably essential to the tensile strength of the neuron, as well as to transport of molecules and organelles within the axon. Three peptides with apparent molecular masses of approximately 68 (NF-L), 145 (NF-M), and 200 (NF-H) kDa appear to be the major components of NF. The expression of these peptides is specific to nervous tissue and is developmentally regulated. Recently, complete cDNAs encoding NF-L and NF-M, and partial cDNAs encoding NF-H, have been described. To better understand the normal and pathophysiology of NFs we chose to clone the cDNA encoding the rat NF-H peptide. Using monoclonal antibodies that recognized NF-H, we screened a rat brain lambda gt11 library and identified a clone that contained a 2100-nucleotide cDNA insert representing the carboxyl-terminal portion of the NF-H protein. Anti-fusion protein antibodies recognized the NF-H peptide on immunoblots and stained fibrillar structures only in neurons. The cDNA recognized a 4500-nucleotide polyadenylated mRNA that was present only in nervous tissue and a 3500-nucleotide mRNA in adrenal. Brain NF-H mRNA levels were tightly developmentally regulated and paralleled the levels of NF-H peptide on immunoblots. Nuclear runoff studies showed that the 20-fold developmental increase in the NF-H message was due only in part to a 4-fold increase in its transcription rate. Levels of NF-H mRNA varied 20-fold among brain regions, with highest levels in pons/medulla, spinal cord, and cerebellum, and lowest levels in olfactory bulb and hypothalamus. Transcription studies revealed only a 2-fold difference in the transcription rates among these brain regions. Based on these results, we infer that half of the developmental increase and most of the interregional variation in the levels of the NF-H mRNA are mediated through message stabilization. Sequence information revealed that the carboxyl-terminal region of the NF-H peptide contained a unique serine-, proline-, alanine-, glutamic acid-, and lysine-rich repeat. The serine residues are likely sites of phosphorylation in the mature peptide. Genomic blots revealed a single copy of the gene in the rat genome and two copies in the human genome. In situ hybridizations performed on human chromosomes mapped the NF-H gene to chromosomes 1 and 22. Whether one copy is a pseudogene remains to be determined.
An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes.
Authors: Tuan D Y , Solomon W B , London I M , Lee D P
Published: May 2006

We have identified an erythroid-specific enhancer element far upstream of the human "beta-like globin" genes, at 10.2-11.0 kilobases 5' of the embryonic epsilon-globin gene, and thus at 53-54 kilobases 5' of the adult beta-globin gene. It is capable of enhancing the expression of a cis-linked test gene by up to 300-fold. This enhancer element is apparently developmental-stage-independent, as it is functional at the embryonic and the adult developmental stages in erythroid cells that are expressing the respective beta-like globin genes. The enhancer and globin promoter sequences work in synergy and are capable of conferring on a cis-linked gene the high transcriptional efficiency (enhancer function), erythroid specificity (enhancer and promoter functions), and developmental-stage specificity (promoter function) that are characteristic of the in vivo transcription of the beta-like globin genes in erythroid cells.
Tissue cooperation in a proteolytic cascade activating human interstitial collagenase.
Authors: HE C S , Wilhelm S M , Pentland A P , Marmer B L , Grant G A , Eisen A Z , Goldberg G I
Published: May 2006

We present a cascade of proteolytic events catalyzed by the proteases secreted by cultured keratinocytes and fibroblasts that results in the activation of interstitial procollagenase. Cultured human skin fibroblasts constitutively secrete interstitial collagenase and stromelysin as proenzymes. In contrast, interstitial collagenase found in serum-free skin organ culture conditioned medium is activated. Cocultivation of the major cellular components of skin organ culture, dermal fibroblasts and epidermal keratinocytes, induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a urokinase-dependent pathway where added keratinocytes secrete the plasminogen activator urokinase, which converts plasminogen into plasmin. Plasmin is capable of activating purified procollagenase and prostromelysin. Plasmin-dependent activation of procollagenase generates an enzyme species, by amino-terminal processing, identical to those generated by limited proteolysis with trypsin or treatment with organomercurial compounds. Catalytic amounts of activated stromelysin can in turn convert plasmin- or trypsin-activated collagenase into a fully active enzyme by removal of approximately 15 amino acid residues from the carboxyl end of the enzyme. This results in a 5- to 8-fold increase in collagenase specific activity that is due to its proteolytic cleavage and not to the presence of the activator stromelysin. Stromelysin alone in both pro- and activated forms is not capable of efficient activation of human fibroblast interstitial procollagenase.
Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity.
Authors: Phillips J P , Campbell S D , Michaud D , Charbonneau M , Hilliker A J
Published: May 2006

The role of copper/zinc-containing superoxide dismutase (cSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) in metabolic defense against O2 toxicity in Drosophila is examined through the properties of a mutant strain carrying a cSOD-null mutation, cSODn108. Homozygotes are viable as larvae, which indicates that cSOD is not essential for cell viability per se. cSODn108 confers recessive sensitivity to the superoxide anion (O2-)-generator paraquat and to the transition metal compound CuSO4, which indicates that the cSOD-null condition in fact leads to impaired O2- metabolism. The primary biological consequences of the reduced O2- dismutation capacity of cSODn108 Drosophila are realized in the adult as infertility and reduction in life-span. We conclude that the infertility and reduced life-span of cSODn108 adults arise as a consequence of the reduced capacity of embryos, larvae, and pupae to adequately protect developing preimaginal cells from O2- -initiated cytotoxic damage.
Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.
Authors: Orita M , Iwahana H , Kanazawa H , Hayashi K , Sekiya T
Published: May 2006

We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of single-stranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.
Characterization of a presecretory phase in B-cell differentiation.
Authors: King L B , Corley R B
Published: May 2006

We have identified and characterized an inducible in vitro subclone of the CH12 B-cell lymphoma, CH12-LBK, which appears to represent a transitional phase in the B-cell differentiation pathway. This phase, which we call the "presecretory" phase, falls between replicating B cells that are not secreting antibodies and B cells that secrete antibody at a high rate. Presecretory cells are characterized by abundant steady-state levels of immunoglobulin and joining (J) chain transcripts and of protein but low levels of mouse mammary tumor virus envelope transcripts and low rates of immunoglobulin secretion. Additional stimulation is required for presecretory cells to differentiate into cells that secrete antibodies at a high rate. The existence of cells with this phenotype suggests that high-level expression of immunoglobulin and J-chain protein does not necessarily commit a B cell to polymerize and secrete multimeric immunoglobulin. Rather, other gene products, expressed after immunoglobulin and J-chain transcripts have been upregulated late in B-cell differentiation, appear responsible for inducing high rates of antibody secretion.
Membrane docosahexaenoate is supplied to the developing brain and retina by the liver.
Authors: Scott B L , Bazan N G
Published: May 2006

Docosahexaenoic acid [22:6 omega 3; 22:6(4, 7, 10, 13, 16, 19)] is concentrated in phospholipids of cellular membranes from brain and retina. Although linolenic acid [18:3 omega 3; 18:3(9, 12, 15)] is the major omega 3 fatty acid of mouse dams' milk, 22:6 is the prevalent omega 3 fatty acid in serum and tissues. Intraperitoneal injection of [1-14C]18:3 into 3-day-old mouse pups resulted in liver and serum lipid labeling that was initially high, followed by a rapid decline. In contrast, labeling of brain and retinal lipids were initially low and increased with time. Labeled 22:6 first appeared in liver 2 hr after injection and later in brain and retina. We suggest that 22:6 synthesized from 18:3 by the liver is secreted into the bloodstream in lipoproteins, taken up by brain and retina, and incorporated into cell membranes. We hypothesize that the 22:6 requirements of membranes (e.g., during synaptogenesis, photoreceptor membrane biogenesis, or repair after ischemic injury or neurodegenerative disorders) are met by a signal that is sent by the appropriate tissues to the liver to evoke the secretion of 22:6-containing lipoproteins.
T-cell interleukin 1 receptor cDNA expressed in Chinese hamster ovary cells regulates functional responses to interleukin 1.
Authors: Curtis B M , Gallis B , Overell R W , McMahan C J , DeRoos P , Ireland R , Eisenman J , Dower S K , Sims J E
Published: May 2006

We have cloned a cDNA encoding a receptor identical to the native Mr 80,000 glycoprotein that binds interleukin (IL) 1 alpha and -beta in murine T cells. Chinese hamster ovary (CHO) cells transfected with this T-cell IL-1 receptor (IL-1R) [CHO(IL-1R)] cDNA express approximately 100,000 IL-1Rs per cell, compared to the less than 100 receptors present on control CHO cells. For two functional responses to IL-1, prostaglandin synthesis and cytokine secretion, CHO(IL-1R) cells were 1000 times more sensitive to IL-1 alpha than were control CHO cells. Northern blot analysis and antibody precipitation demonstrated that one of the cytokines induced was granulocyte colony-stimulating factor and that mRNA levels for this cytokine were increased in CHO(IL-1R) cells by IL-1 alpha concentrations that had no effect on control cells. To establish the role of the recombinant receptors in signal transduction, an IL-1R cDNA modified by deletion of the predicted cytoplasmic domain was expressed in the CHO cell line termed CHO(IL-1R delta CT). CHO(IL-1R delta CT) cells expressed approximately 100,000 high-affinity IL-1 binding sites per cell, but these cells were less sensitive than control lines to IL-1, as measured by prostaglandin and cytokine release. These results show that the IL-1R cDNA encodes the entire functional receptor and that the cytoplasmic domain is required for signal transduction but not ligand binding.
Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain.
Authors: Prasad V R , Goff S P
Published: May 2006

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.
Gene for proliferating-cell nuclear antigen (DNA polymerase delta auxiliary protein) is present in both mammalian and higher plant genomes.
Authors: Suzuka I , Daidoji H , Matsuoka M , Kadowaki K , Takasaki Y , Nakane P K , Moriuchi T
Published: May 2006

Proliferating-cell nuclear antigen (PCNA; also called cyclin) was originally described in proliferating mammalian cells as a nuclear protein with an apparent Mr of 33,000-36,000 and recently was found to be a DNA polymerase delta auxiliary protein. To elucidate whether PCNA/cyclin is a universal protein necessary for proliferation of eukaryotes, a search was conducted for PCNA/cyclin homologues in higher plants. In Southern blot-hybridization analysis, a rat PCNA/cyclin cDNA probe hybridized with homologous sequences in genomic DNAs from rice, soybean, and tobacco. A PCNA/cyclin-related molecular clone (pCJ-1) was isolated from rice DNA and was partially sequenced. The pCJ-1 probe hybridized with a 1.2-kilobase transcript in RNA from rice root tips and shoots. Immunoblot analysis of the soluble extract of soybean root tips with monospecific anti-PCNA/cyclin identified an immunoreactive protein with an apparent Mr of 34,000. Immunohistochemical analysis revealed the presence of an immunoreactive PCNA/cyclin protein in the nuclei of cells in the meristem of soybean root tips. The highly homologous nature of the gene for PCNA/cyclin throughout the animal and plant kingdoms suggests that the product of the gene plays an essential role in DNA replication in eukaryotes.
Dual promoter of
Agrobacterium tumefaciens
mannopine synthase genes is regulated by plant growth hormones
Authors: Langridge W. H. R. , Fitzgerald K. J. , Koncz C. , Schell J. , Szalay A. A.
Published: May 2006

Temporal and spacial distribution of mannopine synthase (
mas
) promoter activity was determined throughout the development of transgenic tobacco plants using bacterial luciferase
luxA
and
luxB
as reporter genes. Luciferase activity was determined by luminometry
in vitro
and visualized by computer-enhanced single-photon video imaging
in vivo
. The activity of the
mas
dual promoters increased basipetally in developing plants and was wound-inducible in leaf and stem tissue. Hormone bioassays with isolated plant tissues and tumors deficient in the transferred DNA (T-DNA)-encoded genes
iaaM, iaaH
, and
ipt
indicated that activity of the
mas
dual promoters is regulated by auxin and enhanced by cytokinin in both differentiated and tumorous plant cells.
The Tc2 transposon in Caenorhabditis elegans.
Authors: Levitt A , Emmons S W
Published: May 2006

A second family of transposons, named Tc2 elements, has been identified in the nematode Caenorhabditis elegans. Tc2 elements are polymorphic in sequence and are present in different numbers in different strains. Like the transposon Tc1, Tc2 is active in the germ line of some C. elegans strains. A high rate of transposition has been observed in the progeny of certain interstrain crosses, where transposition events are frequent enough to be detected in blot hybridization experiments, without the use of a genetic screen. Our data suggest that transposition of Tc1 and Tc2 may be regulated by the same genomic factors.
The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.
Authors: Barber E K , Dasgupta J D , Schlossman S F , Trevillyan J M , Rudd C E
Published: May 2006

Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.
Decreased messenger RNA translation in herpesvirus-infected arterial cells: effects on cholesteryl ester hydrolase.
Authors: Hajjar D P , Nicholson A C , Hajjar K A , Sando G N , Summers B D
Published: May 2006

Herpes simplex viruses (HSVs) contain a function that can cause the degradation of host mRNA and mediate the shutoff of host protein synthesis. Previously, we observed that HSV infection causes a 40-fold increase in cholesteryl ester (CE) accretion in arterial smooth muscle cells due, in part, to a substantial decrease in CE hydrolysis. In studies reported herein, we found that HSV infection leads to reduced immunoprecipitable lysosomal (acid) CE hydrolase (ACEH) and beta-galactosidase, another lysosomal enzyme in vascular smooth muscle cells. The HSV-induced reduction was greater with respect to ACEH than beta-galactosidase. To determine whether degradation of host cellular mRNA or inhibition of cellular translation was responsible for decreased CE hydrolysis in HSV-infected smooth muscle cells, we utilized an in vitro translation system that permitted us to compensate for any mRNA degradation during viral infection. Reduced ACEH activity was observed in the total cellular RNA translation products of HSV-infected smooth muscle cells compared to uninfected cells owing to posttranscriptional modification. We conclude that the decrease in CE hydrolysis in HSV-infected smooth muscle cells is caused primarily by decreased ACEH synthesis and activity, which can contribute to CE accretion in these vascular cells.
[Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist.
Authors: Fuhlendorff J , Gether U , Aakerlund L , Langeland-Johansen N , Thøgersen H , Melberg S G , Olsen U B , Thastrup O , Schwartz T W
Published: May 2006

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31, Pro34]NPY is also active in vivo as it is even more potent than NPY in increasing blood pressure in anesthetized rats. It is concluded that [Leu31, Pro34]NPY is a specific Y1 receptor agonist and that the analog or variants of it can be useful in delineating the physiological importance of Y1 receptors.
Metastable intermediates in myoglobin at low pH.
Authors: Han S , Rousseau D L , Giacometti G , Brunori M
Published: May 2006

Resonance Raman and optical absorption spectra of ligand-free (deoxy) myoglobin and CO-bound myoglobin (MbCO) at pH 2.6 have been measured by using continuous-flow/rapid-mixing techniques. The spectra of deoxy myoglobin at low pH within 6 ms of the pH drop demonstrate that the iron-histidine bond has been ruptured but that the heme is still five-coordinate. Comparison with data from model complexes indicates that a weak-field ligand, such as a water molecule, is coordinated at the fifth position. The Raman spectrum of MbCO at low pH has an Fe-CO stretching mode that is characteristic of a six-coordinate heme with an unhindered Fe-CO moiety. Immediately following the pH drop in this case, there is no indication that the iron-proximal histidine bond is broken. Three different structural changes are detected at low pH: (i) the iron-proximal histidine (F8) bond in ligand-free myoglobin is broken and replaced by a weak-field ligand, (ii) the distal pocket in MbCO is opened, and (iii) protein constraints on the heme group in MbCO are relaxed. Previous conclusions that the kinetics of CO-binding in hemoproteins at low pH is modified by rupturing the iron-proximal histidine bond are supported by these new results which, however, demand a more complete reevaluation of the phenomenon.
Drosophila glutathione S-transferase 1-1 shares a region of sequence homology with the maize glutathione S-transferase III.
Authors: Toung Y P , Hsieh T S , Tu C P
Published: May 2006

We have characterized a Drosophila glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) cDNA encoding a protein of 209 amino acids. The cDNA was expressed in Escherichia coli harboring the expression plasmid construct pGTDml-KK. The active enzyme, designated as Drosophila glutathione S-transferase 1-1, had a specific activity toward 1-chloro-2,4-dinitrobenzene comparable to that for the mammalian glutathione S-transferases but did not have as broad a substrate specificity pattern. There is a region of 44 amino acids in this enzyme that shares 66% identity with an analogous region of maize glutathione S-transferase III. Drosophila glutathione S-transferase 1-1 had no obvious homology to any mammalian or parasitic glutathione S-transferases. The gene was found to be a member of a multigene family.
Cytotoxic T-cell precursors with low-level CD8 in the diabetes-prone Biobreeding rat: implications for generation of an autoimmune T-cell repertoire.
Authors: Bellgrau D , Lagarde A C
Published: May 2006

Lymphocytes from diabetes-prone Biobreeding rats consistently fail to generate T-cell-mediated cytotoxicity under conditions where cytotoxic T lymphocyte activity is readily demonstrated in normal rats. The failure is associated with generalized T-cell lymphopenia and marked reduction in the frequency of CD8+ cells. The few remaining CD8+ cells are widely held to be natural killer cells rather than class I major histocompatibility complex-restricted T lymphocytes. In this report we show that a detectable percentage of CD8+ lymphocytes express the T-cell receptor for antigen, thus identifying them as part of the T-cell lineage. The failure of these CD8+ T-cell-receptor-positive T cells to lyse target cells that are susceptible to T-cell mediated cytotoxicity is associated with markedly reduced expression of cell-surface CD8. Targets expressing higher than normal levels of class I major histocompatibility complex target antigen could be lysed, suggesting that reduction in CD8 has decreased T-cell activity for target antigen. We discuss the derivation of T cells that express low levels of CD8 and the role they could play in generating autoimmune diabetes.
Constitutive expression of growth-related mRNAs in proliferating and nonproliferating lung epithelial cells in primary culture: evidence for growth-dependent translational control.
Authors: Clement A , Campisi J , Farmer S R , Brody J S
Published: May 2006

We describe the control of proliferation and growth-related gene expression in primary cultures of epithelial cells derived from rat lung. Type 2 epithelial cells line the gas-exchange surface of the alveoli where they produce and secrete surfactant. When isolated from adult animals, type 2 cells do not proliferate in culture, although they have a limited ability to do so in vivo. We show that type 2 cells isolated from neonatal rats proliferate in culture and that growth can be reversibly arrested by withdrawing serum from the medium. We studied the expression of five genes whose mRNA levels fluctuate with the state of proliferation in several cell systems: the c-myc and c-Ha-ras protooncogenes and the genes encoding actin, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17), and histone 3.2. All five mRNAs were constitutively expressed at identical levels in proliferating and nonproliferating (serum deprived) neonatal cells and in adult cells. Thus, at the level of mRNA abundance, the expression of these five genes was uncoupled from the growth state of the cells. By contrast, synthesis of the replication-dependent histones and the activity of ornithine decarboxylase were detectable only in proliferating neonatal cells and not in serum-deprived neonatal cells or in adult cells. The results suggest that, in type 2 cells, growth factors might regulate the translation, rather than the mRNA abundance, of at least some growth-related genes and that the ability to respond to this translational control may be developmentally regulated.
Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: implications for regulation of actin-myosin interaction.
Authors: Sweeney H L , Stull J T
Published: May 2006

Myosin light chain phosphorylation in permeable skeletal muscle fibers increases isometric force and the rate of force production at submaximal levels of calcium activation; myosin light chain phosphorylation may underlie the increased rate and extent of force production associated with isometric twitch potentiation in intact fibers. To understand the mechanism by which myosin light chain phosphorylation manifests these effects, we have measured isometric force, isometric stiffness, rate of isometric force redevelopment after isotonic shortening, and isometric ATPase activity in permeabilized rabbit psoas muscle fibers. These measurements were made in the presence and absence of myosin light chain phosphorylation over a range of calcium concentrations that caused various levels of activation. The results were analyzed with a two-state cross-bridge cycle model as suggested by Brenner [Brenner, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3265-3269]. The results indicate that myosin light chain phosphorylation exerts its effect on force generation and the isometric rate of force redevelopment in striated muscle through a single mechanism, namely, by increasing the rate constant describing the transition from non-force-generating cross-bridges to force-generating states (fapp). gapp, the reverse rate constant, is unaffected by phosphorylation as are the number of cycling cross-bridges. Since both calcium and myosin light chain phosphorylation increase fapp, the possibility is considered that modulation of fapp may represent a general mechanism for regulating force in actin-myosin systems.
Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and cell type-specific antibodies.
Authors: Galileo D S , Gray G E , Owens G C , Majors J , Sanes J R
Published: May 2006

We used a recombinant retrovirus to study cell lineage in the chicken optic tectum. The virus inserts the Escherichia coli lacZ (beta-galactosidase) gene into the genome of an infected cell; a histochemical stain marks the progeny of infected cells with a blue precipitate. We had previously shown that individual clones frequently contain diverse neuronal types. Now we asked whether individual clones contain glia as well as neurons. To this end, we constructed a virus in which lacZ is fused to a nuclear localization signal sequence from the simian virus 40 large tumor antigen. Cells infected with this virus are marked with blue nuclei instead of blue somata. In embryos injected with a mixture of the two retroviruses, individual clusters contained cells with only one label type (nuclear or cytoplasmic), thus verifying that clusters of cells were clones. Furthermore, it was possible to immunostain the somata of cells that had blue nuclei, whereas the blue cytoplasmic precipitate hampered immunostaining. Together, these methods allowed us to show that some clones contained neurons (neurofilament-positive) and two types of glia (glutamine synthetase-positive and glial fibrillary acidic protein-positive). This result demonstrates the existence of a common progenitor for neurons and glia in optic tectum.
Maize regulatory gene opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA.
Authors: Schmidt R J , Burr F A , Aukerman M J , Burr B
Published: May 2006

The opaque-2 locus (o2) in maize regulates the expression of many members of the zein multigene family of storage proteins. cDNA clones for a wild-type allele of the (o2) locus (O2) were isolated from a maize endosperm cDNA library and sequenced. We found a 258-nucleotide 5' leader sequence containing three short open reading frames followed by a sequence specifying a protein of 437 amino acids. The presumptive amino acid sequence of the protein (O2) specified by the O2 cDNA contains a "leucine-zipper" domain characteristic of some mammalian and fungal transcription activation factors. lacZ-O2 fusion constructs, using nearly the entire coding region of O2 or only a fragment specifying the leucine-zipper domain, were expressed in Escherichia coli. In an in vitro binding assay, the beta-galactosidase-O2 fusion proteins bound to two specific regions on the 5' side of the coding sequence in a zein genomic clone. This suggests that the O2 protein affects zein transcription through direct interaction with one or more zein promoter elements.
Capelli bitableaux and Z-forms of general linear Lie superalgebras.
Authors: Brini A , Teolis A G
Published: May 2006

The combinatorics of the enveloping algebra UQ(pl(L)) of the general linear Lie superalgebra of a finite dimensional Z2-graded Q-vector space is studied. Three non-equivalent Z-forms of UQ(pl(L)) are introduced: one of these Z-forms is a version of the Kostant Z-form and the others are Lie algebra analogs of Rota and Stein's straightening formulae for the supersymmetric algebra Super[L P] and for its dual Super[L* P*]. The method is based on an extension of Capelli's technique of variabili ausiliarie to algebras containing positively and negatively signed elements.
Do thylakoids really contain phosphatidylcholine?
Authors: Dorne A J , Joyard J , Douce R
Published: May 2006

Isolated intact spinach chloroplasts were incubated with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) under mild experimental conditions in which only the phosphatidylcholine localized in the cytosolic leaflet of the outer envelope membrane can be hydrolyzed. Thylakoids, which were protected from phospholipase C degradation, were subsequently prepared from the phospholipase C-treated chloroplasts and found to be devoid of phosphatidylcholine. Previously reported occurrences of phosphatidylcholine in thylakoid preparations probably reflect contamination of the thylakoids by envelope membranes. In the present work, contamination of thylakoids by envelope membranes was determined by measuring the 1,2-diacylglycerol 3-beta-galactosyltransferase [monogalactosyldiacylglycerol (MGDG) synthase; UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyltransferase, EC 2.4.1.46] in the different chloroplast subfractions. We conclude that phosphatidylcholine is not present in highly purified thylakoids. Phosphatidylcholine is also absent from prokaryotic cyanobacterial membranes, and our results are in agreement with the endosymbiotic origin of higher plant chloroplasts.
An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes.
Authors: Driggers P H , Ennist D L , Gleason S L , Mak W H , Marks M S , Levi B Z , Flanagan J R , Appella E , Ozato K
Published: May 2006

Interferons (IFNs) induce transcription of major histocompatibility complex (MHC) class I genes through the conserved IFN consensus sequence (ICS) that contains an IFN response motif shared by many IFN-regulated genes. By screening mouse lambda ZAP expression libraries with the ICS as a probe, we isolated a cDNA clone encoding a protein that binds the ICS, designated ICSBP. Protein blot analysis with labeled oligonucleotide probes showed that ICSBP binds not only the MHC class I ICS but also IFN response motifs of many IFN-regulated genes, as well as a virus-inducible element of the IFN-beta gene. The ICSBP cDNA encodes 424 amino acids and a long 3' untranslated sequence. The N-terminal 115 amino acids correspond to a putative DNA-binding domain and show significant sequence similarity with other cloned IFN response factors (IRF-1 and IRF-2). Because of the structural similarity and shared binding specificity, we conclude that ICSBP is a third member of the IRF gene family, presumably playing a role in IFN- and virus-mediated regulation of many genes. Although IRF-1 and IRF-2 share some similarity in their C-terminal regions, ICSBP shows no similarity to IRF-1 or IRF-2 in this region, suggesting that it is more distantly related. We show that ICSBP mRNA is expressed predominantly in lymphoid tissues and is inducible preferentially by IFN-gamma. The induction by IFN-gamma appears to be predominant in lymphocytes and macrophages, implying that ICSBP plays a regulatory role in cells of the immune system. The presence of multiple factors that bind common IFN response motifs may partly account for the complexity and diversity of IFN action as well as IFN-regulated gene expression.
Activated type I phosphatidylinositol kinase is associated with the epidermal growth factor (EGF) receptor following EGF stimulation.
Authors: Bjorge J D , Chan T O , Antczak M , Kung H J , Fujita D J
Published: May 2006

We have shown that a type I phosphatidylinositol (PI) kinase activity is associated with the epidermal growth factor (EGF) receptor in a mouse fibroblast cell line expressing human EGF receptors (NRHER5) and that this activity increases dramatically upon treatment of cells with physiologically relevant concentrations of EGF. EGF stimulated a time-dependent increase in EGF receptor-associated PI kinase activity measured in EGF receptor immunoprecipitates. Activation was detected 15 min after the addition of EGF, and it peaked between 1 and 2 hr. Activation of PI kinase was detected with EGF concentrations as low as 10 pM and maximal stimulation occurred at approximately 1 nM. Analysis of deacylated PI phosphate products, and inhibition of the PI kinase activity by nonionic detergent, indicated that the PI kinase described here was type I or PI 3' kinase. These results demonstrate the regulation of a type I PI kinase by EGF and suggest a potential role in the EGF receptor signal transduction pathway.
Low density lipoprotein rich in oleic acid is protected against oxidative modification: implications for dietary prevention of atherosclerosis.
Authors: Parthasarathy S , Khoo J C , Miller E , Barnett J , Witztum J L , Steinberg D
Published: May 2006

Oxidative modification of low density lipoprotein (LDL) enhances its potential atherogenicity in several ways, notably by enhancing its uptake into macrophages. In vivo studies in the rabbit show that inhibition of LDL oxidation slows the progression of atherosclerotic lesions. In the present studies, rabbits were fed either a newly developed variant sunflower oil (Trisun 80), containing more than 80% oleic acid and only 8% linoleic acid, or conventional sunflower oil, containing only 20% oleic acid and 67% linoleic acid. LDL isolated from the plasma of animals fed the variant sunflower oil was highly enriched in oleic acid and very low in linoleic acid. These oleate-rich LDL particles were remarkably resistant to oxidative modification. Even after 16-hr exposure to copper-induced oxidation or 24-hr incubation with cultured endothelial cells, macrophage uptake of the LDL was only marginally enhanced. The results suggest that diets sufficiently enriched in oleic acid, in addition to their LDL-lowering effect, may slow the progression of atherosclerosis by generating LDL that is highly resistant to oxidative modification.
Asymmetric distribution of dystrophin in developing and adult Torpedo marmorata electrocyte: evidence for its association with the acetylcholine receptor-rich membrane.
Authors: Jasmin B J , Cartaud A , Ludosky M A , Changeux J P , Cartaud J
Published: May 2006

Dystrophin has been shown to occur in Torpedo electrocyte [Chang, H. W., Bock, E. & Bonilla, E. (1989) J. Biol. Chem. 264, 20831-20834], a highly polarized syncytium that is embryologically derived from skeletal muscle and displays functionally distinct plasma membrane domains on its innervated and noninnervated faces. In the present study, we investigated the subcellular distribution of dystrophin in the adult electrocyte from Torpedo marmorata and the evolution of its distribution during embryogenesis. Immunofluorescence experiments performed on adult electrocytes with a polyclonal antibody directed against chicken dystrophin revealed that dystrophin immunoreactivity codistributed exclusively with the acetylcholine receptor along the innervated membrane. At the ultrastructural level, dystrophin immunoreactivity appears confined to the face of the subsynaptic membrane exposed to the cytoplasm. In developing electrocytes (45-mm embryo), dystrophin is already detectable at the acetylcholine receptor-rich ventral pole of the cells before the entry of the electromotor axons. Furthermore, we show that dystrophin represents a major component of purified membrane fractions rich in acetylcholine receptor. A putative role of dystrophin in the organization and stabilization of the subsynaptic membrane domain of the electrocyte is discussed.
Evolutionary origin of human and simian immunodeficiency viruses.
Authors: Gojobori T , Moriyama E N , Ina Y , Ikeo K , Miura T , Tsujimoto H , Hayami M , Yokoyama S
Published: May 2006

From what viruses the human immunodeficiency viruses (HIVs) originated is an extremely controversial question. To address this question, we have analyzed nucleotide sequences of simian immunodeficiency viruses (SIVs) and HIVs by using the techniques for understanding molecular evolution. In particular, we compared the nucleotide sequences of whole genomes, gene region by gene region, between a given pair of viruses, including four types of SIVs--isolated from mandrills (Papio sphinx), African green monkeys (Cercopithecus aethiops), sooty mangabeys (Cercocebus atys), and rhesus macaques (Macaca mulatta)--as well as HIVs. Phylogenetic trees for all gene regions examined showed that the present HIVs may have emerged as different variants of SIVs of Old World monkeys, possibly from recombination between viruses related to SIVs.
Antibody-targeted photolysis: in vitro studies with Sn(IV) chlorin e6 covalently bound to monoclonal antibodies using a modified dextran carrier.
Authors: Rakestraw S L , Tompkins R G , Yarmush M L
Published: May 2006

A monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugate was prepared by a technique involving the site-specific covalent modification of the monoclonal antibody oligosaccharide moiety. Dextran carriers were synthesized with a single chain-terminal hydrazide group, which was used as the coupling point between the carrier and the monoclonal antibody carbohydrate. Selective in vitro photolysis of SK-MEL-2 human malignant melanoma cells was accomplished using several conjugates prepared from anti-melanoma 2.1 (chromophore:antibody molar ratios, 6.8 and 11.2). Phototoxicity, as measured by clonogenic assay, was dependent on the delivered dose of 634-nm light and was observed only for conjugates that bound SK-MEL-2 cells. As judged by competitive inhibition radioimmunoassay, conjugates prepared in this fashion showed excellent retention of antigen binding activity relative to the unmodified antibody.
Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents.
Authors: Albertsen H M , Abderrahim H , Cann H M , Dausset J , Le Paslier D , Cohen D
Published: May 2006

Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, we performed a series of preliminary experiments aimed at developing a suitable protocol. We found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.
Transport of phosphocholine in higher plant cells: 31P nuclear magnetic resonance studies.
Authors: Gout E , Bligny R , Roby C , Douce R
Published: May 2006

Phosphocholine (PC) is an abundant primary form of organic phosphate that is transported in plant xylem sap. Addition of PC to the perfusate of compressed Pi-starved sycamore cells monitored by 31P NMR spectroscopy resulted in an accumulation of PC and all the other phosphate esters in the cytoplasmic compartment. Addition of hemicholinium-3, an inhibitor of choline uptake, to the perfusate inhibited PC accumulation but not inorganic phosphate (Pi). When the Pi-starved cells were perfused with a medium containing either Pi or PC, the resulting Pi distribution in the cell was the same. Addition of choline instead of PC to the perfusate of compressed cells resulted in an accumulation of PC in the cytoplasmic compartment from choline kinase activity. In addition, PC phosphatase activity has been discovered associated with the cell wall. These results indicate that PC was rapidly hydrolyzed outside the cell and that choline and Pi entered the cytosolic compartment where choline kinase re-forms PC.
Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate.
Authors: Pek S B , Usami M , Bilir N , Fischer-Bovenkerk C , Ueda T
Published: May 2006

We have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, we considered the possibility that 3-phosphoglycerate may act as a coupling factor, and we searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [gamma-32P]ATP and appropriate test substances and were subjected to NaDodSO4/PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of a 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion.
In vivo expression of the lacY gene in two segments leads to functional lac permease.
Authors: Bibi E , Kaback H R
Published: May 2006

The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus). Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation. In contrast, cells expressing either half of the permease independently do not transport lactose. As judged by [35S]methionine labeling and immunoblotting, intact permease is completely absent from the membrane of cells expressing lacY fragments either individually or together. Thus, transport activity must result from an association between independently synthesized pieces of lac permease. When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed. When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane. It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex.