bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of c-mos RNA in germ cells of male and female mice.
Authors: Goldman D S , Kiessling A A , Millette C F , Cooper G M
Published: May 2006

We have investigated the cell types in mouse testis and ovary in which the c-mos protooncogene is normally transcribed. Blot hybridization analysis of electrophoretically fractionated RNAs from testes of mice with defects in germ-cell development and from prepubertal and adult mice indicated that c-mos was transcribed during male germ-cell development. Analysis of purified populations of spermatogenic cell types detected c-mos RNA in the earliest haploid postmeiotic germ cell, the round spermatid, indicating that c-mos was expressed transiently during spermatogenesis. c-mos RNA was detected by blot hybridization in the ovaries of prepubertal mice and decreased in relative concentration following gonadotropin-stimulated proliferation of granulosa cells. These results suggested that c-mos was transcribed in oocytes and were confirmed by detection of high levels of c-mos RNA in isolated grown oocytes. Thus, c-mos is expressed in both male and female germ cells, suggesting possible roles for this protooncogene in meiosis, germ-cell development, fertilization, and early embryogenesis.
Production of a monoclonal antibody useful in the molecular characterization of murine T-cell-replacing factor/B-cell growth factor II.
Authors: Harada N , Takahashi T , Matsumoto M , Kinashi T , Ohara J , Kikuchi Y , Koyama N , Severinson E , Yaoita Y , Honjo T
Published: May 2006

T-cell-replacing factor (TRF) is a T-cell-derived factor required for terminal differentiation of activated B cells to immunoglobulin-secreting cells. Previous studies have shown that a murine T-cell hybrid (B151K12) produces factor(s) that (i) induce immunoglobulin secretion by the B-cell leukemia line BCL1 and secondary anti-2,4-dinitrophenyl IgG synthesis in vitro by dinitrophenyl-primed B cells (TRF activity) and (ii) cause proliferation of the BCL1 cells [B-cell growth factor II (BCGF-II) activity]. Both activities appear to be associated with the same molecule. Here we report the production of a monoclonal antibody to murine TRF. The monoclonal antibody, designated TB13, strongly inhibited both TRF and BCGF-II activities and absorbed TRF- as well as BCGF-II-active molecules produced by B151K12 and by Xenopus oocytes that had been injected with mRNA transcribed from plasmid pSP6K-mTRF23. Inhibition was linearly dependent on the concentration of both TB13 and TRF. Monoclonal antibody TB13 did not inhibit the activities of B-cell stimulatory factor 1, interleukin 1, interleukin 2, or interleukin 3. TRF activity in dissolved samples of immunoprecipitates obtained with TB13 was recovered after NaDodSO4/PAGE, in the fractions corresponding to a protein band at Mr 46,000. Our results indicate that monoclonal antibody TB13 recognizes a molecule that has both TRF and BCGF-II activities.
Human T-cell lymphotropic virus IIIB glycoprotein (gp120) bound to CD4 determinants on normal lymphocytes and expressed by infected cells serves as target for immune attack.
Authors: Lyerly H K , Matthews T J , Langlois A J , Bolognesi D P , Weinhold K J
Published: May 2006

The lymphocyte differentiation antigen CD4 serves as a receptor for human retroviruses associated with acquired immunodeficiency syndrome (AIDS) through its interaction with the major envelope virion glycoprotein, gp120, which is also expressed on the surface of infected cells. In these experiments, purified gp120 was shown to bind to normal human T-lymphocyte populations. The gp120-CD4 complex served as a target antigen for antibody-dependent complement-mediated cytolysis by a goat serum raised against native gp120. However, patient sera that bound to gp120-adsorbed cells failed to direct their destruction in the presence of complement. In contrast, these sera were potent mediators of antibody-dependent cellular cytotoxicity. These studies demonstrate that gp120 situated on the cell surface can serve as an effective target for immune destruction by patient antibodies and effector lymphocytes. The possible contribution of this type of immunity to control of disease progression, on the one hand, and to lymphocyte destruction and immunopathology observed in AIDS, on the other, is discussed.
Distinct angiotensin II receptor in primary cultures of glial cells from rat brain.
Authors: Raizada M K , Phillips M I , Crews F T , Sumners C
Published: May 2006

Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Ang-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of 125I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 125I-Ang-II binding was as follows: Ang-II = [sarcosine1,Ala8]Ang-II greater than [sarcosine1,Ile8]Ang-II much greater than Ang-III greater than Ang-I. Scatchard analysis revealed a homogeneous population of high-affinity (Kd = 1.1 nM) binding sites with a Bmax of 110 fmol/mg of protein. Light-microscopic autoradiography of 125I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence [3H]norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors.
Polymeric amphiphiles at solid-fluid interfaces: Forces between layers of adsorbed block copolymers
Authors: Tirrell Matthew , Patel Sanjay , Hadziioannou Georges
Published: May 2006

Block copolymers are macromolecular amphiphiles and as such have a wide variety of interesting interfacial behavior. We present here a case of block copolymer adsorption from solution studied by the direct measurement of forces between two adsorbed layers. The case is polystyrene (PS)-poly(2-vinylpyridine) (PVP) adsorbed on mica from toluene. Under these circumstances a layer of firmly anchored PS cilia (or loops in the case of PVP-PS-PVP tri-blocks) is assembled on the surface. Furthermore, the PS cilia adopt an extended chain configuration due to lateral crowding imposed on them by the high graft density. The long-range repulsive forces measured in toluene are direct evidence for the well-known efficacy of this sort of macromolecule in the stabilization of colloidal dispersions. The picture of the adsorbed configuration for di-block copolymers is reasonably clear; that for tri-blocks is less so. Some thoughts are offered on new experiments and technological uses of these materials.
Factors determining the average atomic volumes in intermetallic compounds
Authors: Pauling Linus
Published: May 2006

In formation of an intermetallic compound from the elementary metals there is usually a contraction in volume. Electron transfer leading to the charge states M
+
and M
-
with increase in valence and decrease in volume explains the more than 2-fold range in contraction for different compounds in the same binary system. In a more thorough analysis, the better packing of atoms of different sizes also needs to be considered.
DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.
Authors: Tabor S , Richardson C C
Published: May 2006

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.
Chicken intestinal 28-kilodalton calbindin-D: complete amino acid sequence and structural considerations.
Authors: Fullmer C S , Wasserman R H
Published: May 2006

The complete primary structure of the cholecalciferol-induced chicken intestinal 28-kDa calbindin-D is reported. It is a single-chain polypeptide consisting of 261 amino acid residues (computed Mr = 30,042) and is blocked at the amino terminus. Tryptic digestion of the S-pyridylethylated protein followed by HPLC peptide mapping and automated sequence determination provided the bulk of the sequence information. Subsequent chymotryptic and Staphylococcus aureus V8 protease cleavages yielded the sequences of several additional regions as well as extensive overlapping of the tryptic peptides. The primary structure shows six homologous regions of sequence based on the EF-hand concept of calcium binding, four of which are predicted to actually bind calcium. Aside from these regions, there is no overall structural identity or apparent similarity with the mammalian calbindins (9 kDa), calmodulin, or troponin C. It is predicted that the secondary structure of 28-kDa calbindin-D is significantly different from the other proteins of this class, which bind four calcium atoms.
Specific cellular immune response and neutralizing antibodies in goats immunized with native or recombinant envelope proteins derived from human T-lymphotropic virus type IIIB and in human immunodeficiency virus-infected men.
Authors: Krohn K , Robey W G , Putney S , Arthur L , Nara P , Fischinger P , Gallo R C , Wong-Staal F , Ranki A
Published: May 2006

Animals immunized with native or recombinant envelope proteins from the human immunodeficiency virus (HIV, formerly referred to as human T-lymphotropic virus type III) human T-lymphotropic virus type IIIB and naturally HIV-infected men were assessed for neutralizing antibodies and cell-mediated immunity toward the virus. Immunization of rabbits or goats with the native external envelope glycoprotein gp120 or with corresponding recombinant proteins elicited strictly type-specific neutralizing antibodies. A broad, group-specific cellular immune response to gp120 and to three different HIV isolates was seen in goats immunized with the native gp120 but not in animals immunized with the nonglycosylated recombinant envelope proteins. In HIV-infected people, no T-cell response was seen, even though their T-cell response toward other foreign antigens was intact. The results show type- and group-specific epitopes on gp120, some of which may be of importance for the development of a vaccine against HIV infection.
Conformation of a 16-residue zervamicin IIA analog peptide containing three different structural features: 3(10)-helix, alpha-helix, and beta-bend ribbon.
Authors: Karle I L , Flippen-Anderson J , Sukumar M , Balaram P
Published: May 2006

Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-Ala-Aib-Pro-Aib-Pro-Phe-OMe (where Boc is t-butoxycarbonyl and Aib is alpha-aminoisobutyric acid), a synthetic apolar analog of the membrane-active fungal peptide antibiotic zervamycin IIA, crystallizes in space group P1 with Z = 1 and cell parameters a = 9.086 +/- 0.002 A, b = 10.410 +/- 0.002 A, c = 28.188 +/- 0.004 A, alpha = 86.13 +/- 0.01 degrees, beta = 87.90 +/- 0.01 degrees, and gamma = 89.27 +/- 0.01 degrees; overall agreement factor R = 7.3% for 7180 data (F0 greater than 3 sigma) and 0.91-A resolution. The peptide backbone makes a continuous spiral that begins as a 3(10)-helix at the N-terminus, changes to an alpha-helix for two turns, and ends in a spiral of three beta-bends in a ribbon. Each of the beta-bends contains a proline residue at one of the corners. The torsion angles phi i range from -51 degrees to -91 degrees (average value -64 degrees), and the torsion angles psi i range from -1 degree to -46 degrees (average value -31 degrees). There are 10 intramolecular NH...OC hydrogen bonds in the helix and two direct head-to-tail hydrogen bonds between successive molecules. Two H2O and two CH3OH solvent molecules fill additional space with appropriate hydrogen bonding in the head-to-tail region, and two additional H2O molecules form hydrogen bonds with carbonyl oxygens near the curve in the helix at Pro-10. Since there is only one peptide molecule per cell in space group P1, the molecules repeat only by translation, and consequently the helices pack parallel to each other.
Tumor necrosis factor type alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo.
Authors: Fràter-Schröder M , Risau W , Hallmann R , Gautschi P , Böhlen P
Published: May 2006

Tumor necrosis factor type alpha (TNF-alpha) inhibits endothelial cell proliferation in vitro. Basal cell growth (in the absence of exogenously added growth factor) and fibroblast growth factor (FGF)-stimulated cell proliferation are inhibited in a dose-dependent manner from 0.1 to 10 ng/ml with half-maximal inhibition occurring at 0.5-1.0 ng of TNF-alpha per ml. Bovine aortic and brain capillary endothelial and smooth muscle cells are similarly affected. TNF-alpha is a noncompetitive antagonist of FGF-stimulated cell proliferation. Its action on endothelial cells is reversible and noncytotoxic. Surprisingly, TNF-alpha does not seem to inhibit endothelial cell proliferation in vivo. In the rabbit cornea, even a high dose of TNF-alpha (10 micrograms) does not suppress angiogenesis induced by basic FGF. On the contrary, in this model system TNF-alpha stimulates neovascularization. The inflammatory response that is seen in the cornea after TNF-alpha implantation suggests that the angiogenic properties of this agent may be a consequence of leukocyte infiltration.
Distinct developmental patterns of c-mos protooncogene expression in female and male mouse germ cells.
Authors: Mutter G L , Wolgemuth D J
Published: May 2006

The protooncogene c-mos is expressed in murine reproductive tissues, producing transcripts of 1.7 and 1.4 kilobases in testis and ovary, respectively. In situ hybridization analysis of c-mos expression in histological sections of mouse ovaries revealed that oocytes are the predominant if not exclusive source of c-mos transcripts. c-mos transcripts accumulate in growing oocytes, increasing 40- to 90-fold during oocyte and follicular development. c-mos transcripts were also detected in male germ cells and are most abundant after the cells have entered the haploid stage of spermatogenesis. This developmentally regulated pattern of c-mos expression in oocytes and spermatogenic cells suggests that the c-mos gene product may have a function in normal germ-cell differentiation or early embryogenesis.
Generation of specific cytotoxic T cells with a fragment of the Epstein-Barr virus-encoded p63/latent membrane protein.
Authors: Thorley-Lawson D A , Israelsohn E S
Published: May 2006

Human B lymphocytes, transformed by the herpesvirus Epstein-Barr virus, are known to express a characteristic antigen(s) recognized by the cellular immune response. This structure has been termed lymphocyte-determined membrane antigen. Because of the significance of this structure in controlling Epstein-Barr virus infection in vivo, the molecular nature of lymphocyte-determined membrane antigen has been long sought. In this paper, we show that a sequence of 10 amino acids (residues 43-53) from the Epstein-Barr virus-encoded membrane protein p63/latent membrane protein can induce Epstein-Barr virus-specific cytotoxic T cells and, therefore, bears at least one of the lymphocyte-determined membrane antigenic determinants.
Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons.
Authors: Unsicker K , Reichert-Preibsch H , Schmidt R , Pettmann B , Labourdette G , Sensenbrenner M
Published: May 2006

Embryonic and neonatal neurons require specific trophic supplements for their survival and the induction of transmitter-synthesizing enzymes in vivo and in vitro. Acidic and basic fibroblast growth factor (aFGF, bFGF) and the closely related astroglial growth factors AGF-1 and AGF-2 were studied for putative neurotrophic functions using dissociated, highly neuron-enriched cultures from chick and rat peripheral ganglia and central nervous system tissues. Embryonic chick ciliary ganglion neurons were the only peripheral neurons that responded to bFGF and AGF-2 by enhanced survival equivalent to that obtained with ciliary neurotrophic factor. Half-maximal effects were achieved with bFGF at 360 pg/ml or AGF-2 at 3 ng/ml. Small effects seen with aFGF could be potentiated by adding heparin at 1 microgram/ml. bFGF, but not ciliary neurotropic factor, also promoted neuron survival after the factor was bound to polyornithine and laminin. Both AGF-2 and ciliary neurotropic factor induced choline acetyltransferase activity during 48 hr. AGFs and FGFs also enhanced the long-term survival of embryonic chick spinal cord neurons, including motoneurons that had been retrogradely labeled with rhodamine isothiocyanate. These results demonstrate the potency of a class of mitogenic growth factors as neurotrophic agents for embryonic ciliary ganglion and spinal cord neurons--adding to the emerging evidence that mitogenic and neuronal growth factors are not strictly separate entities.
Behavioral feeding specialization in
Pinaroloxias inornata
, the “Darwin's Finch” of Cocos Island, Costa Rica
Authors: Werner Tracey K. , Sherry Thomas W.
Published: May 2006

As a population, Cocos Finches exhibit a broad range of feeding behaviors spanning those of several families of birds on the mainland, while individuals feed as specialists year-round. Although this extreme intraspecific variability occurs as predicted in a tropical oceanic island environment, these specializations challenge contemporary ecological theory in that they are not attributable to individual differences in age, sex, gross morphology, or opportunistic exploitation of patchy resources. Instead, they appear to originate and be maintained behaviorally, possibly via observational learning. This phenomenon adds another direction to the evolutionary radiation of the Darwin's Finches and underscores the necessity for detailed behavioral and ecological studies at the individual level for understanding animal feeding systems and the causation of phenotypic variation.
Molecular cloning of rat renin cDNA and its gene.
Authors: Burnham C E , Hawelu-Johnson C L , Frank B M , Lynch K R
Published: May 2006

Renin (EC 3.4.23.15) is an aspartyl protease that cleaves its only known substrate, angiotensinogen, to release the vasopressor hormone angiotensin. We have isolated full-length cDNAs for renin from a rat kidney cDNA library. The cDNAs are complementary to a 1434-nucleotide rat kidney mRNA that encodes preprorenin, the 402-amino acid precursor of renin. This rat cDNA was used to isolate the complete copy of a renin gene from a rat genomic library, and a comparison of this genomic clone with rat genomic DNA showed that renin is a single-copy gene in the rat. Rat renin is 85% identical to one mouse renin isozyme (renin-1) and 81% identical to the second mouse renin isozyme (renin-2), suggesting that the duplication of the mouse renin genes is a more recent event than the speciation of rats and mice. Analyses of rat, human, and mouse renin sequences revealed that the potential to form two-chain renin is apparently peculiar to mouse renin, and the expression of a tenth exon (which results in a three-amino acid insertion) is observed only in the human renin gene.
Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein.
Authors: Lopez J A , Chung D W , Fujikawa K , Hagen F S , Papayannopoulou T , Roth G J
Published: May 2006

Glycoprotein Ib is a surface membrane glycoprotein of platelets that functions as a receptor for von Willebrand factor. It is a heterodimer composed of an alpha and a beta chain linked by a disulfide bond(s). A phage lambda gt11 cDNA expression library prepared from mRNA from a human erythroleukemia cell line, HEL, was screened using an affinity-purified antibody to the glycocalicin portion of the alpha chain of glycoprotein Ib. Eleven positive clones were isolated and plaque-purified. The largest cDNA insert was 2420 nucleotides in length and coded for a leader sequence of 16 amino acids, a mature protein of 610 amino acids, and a stop codon. It also contained 42 nucleotides of 5' noncoding sequence and 497 nucleotides of 3' noncoding sequence, including a poly(A) tail. The amino acid sequence of the alpha chain of GPIb predicted from the cDNA agreed completely with the sequence of 156 amino acids that was determined by Edman degradation of peptides isolated from human platelet glycocalicin after digestion with trypsin or Staphylococcus aureus V8 protease. The extracytoplasmic domain of the alpha subunit of GPIb contains several noteworthy structural features, including a region of seven tandem repeats of 24 amino acids that are homologous with those present in leucine-rich alpha 2-glycoprotein. The extracytoplasmic domain also contains two hydrophilic regions, one rich in charged amino acids and a second rich in serine and threonine residues. The region rich in serine and threonine includes five repeats of nine amino acids as well as the majority of the O-linked carbohydrate sites present in the molecule. The extracytoplasmic domain is followed by a potential transmembrane segment of approximately 29 amino acids and a potential intracellular domain of approximately 100 amino acids located at the carboxyl end of the molecule.
The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins.
Authors: Johnson P R , Spriggs M K , Olmsted R A , Collins P L
Published: May 2006

Two major antigenic subgroups (designated A and B) have been described for human respiratory syncytial virus (RSV). Previously, on the basis of reactivity patterns with monoclonal antibodies, the greatest intersubgroup variation was shown to occur in the G protein, the putative attachment glycoprotein. To delineate the molecular basis for this variation, we have determined the nucleotide and deduced amino acid sequences of the G mRNAs and proteins representing a subgroup A (Long strain) and a subgroup B (18537 strain) virus. These sequences were compared to the available G mRNA sequence for another subgroup A (A2 strain) virus. The Long G protein shared 94% amino acid identity with the A2 G protein. In contrast, the 18537 G protein shared only 53% amino acid identity with the A2 sequence; interestingly, most of the sequence divergence occurred in the proposed extracellular domain of the G protein. This extensive divergence for the G protein was significantly greater than that observed for other RSV proteins. Despite this considerable divarication, the proposed extracellular domains of the G proteins contained a single region of highly conserved sequence and secondary structure that may represent a conserved structural or function domain, perhaps involved in attachment to cellular receptors. Furthermore, this conserved region may comprise part of an epitope that is shared between the two subgroup G proteins and may significantly contribute to the fact that, despite extensive overall amino acid sequence divergence, the RSV G proteins maintain significant antigenic relatedness.
Identification of an essential upstream element in the nopaline synthase promoter by stable and transient assays
Authors: Ebert Paul R. , Ha Sam Bong , An Gynheung
Published: May 2006

We studied the fine structure of the nopaline synthase (
nos
) promoter, which is active constitutively in a wide range of plant tissues, by both transient and stable transformation expression analyses. 3′ and 5′ deletion fragments were linked to form a set of internal deletion and duplication mutants that scanned the
nos
promoter. These mutated promoters were linked to the gene for the marker chloramphenicol acetyltransferase (CATase) as a means to readily assay promoter strength. The stable transformation analysis revealed the functional importance of an extended CCAAT box region (-97 to -63). Deletion of an upstream region (-112 to -101) containing an octameric repeated element resulted in a reduction in promoter strength by a factor of 30. A further deletion (-119 to -101) disrupted a potential Z-DNA-forming element as well, totally eliminating promoter function. Thus, a 19-base deletion across a repeated octamer and a potential Z-DNA-forming element identifies an essential upstream activator in the
nos
promoter. Duplication of the upstream element tripled promoter activity. Electroporation-mediated transient analysis was unable to distinguish downstream promoter elements. However, the upstream element behaved similarly in both assays in that deletion of the entire upstream element resulted in no promoter activity and that duplication of the element significantly enhanced the promoter strength.
Vasoactive intestinal peptide enhances aromatase activity in the neonatal rat ovary before development of primary follicles or responsiveness to follicle-stimulating hormone.
Authors: George F W , Ojeda S R
Published: May 2006

We have investigated the factors that regulate aromatase activity in fetal-neonatal rat ovaries. Ovarian aromatase activity (assessed by measuring the amount of 3H2O) formed from [1 beta-3H]testosterone) is low prior to birth (less than 0.5 pmol/hr per mg of protein) and increases to values greater than 30 pmol/hr per mg of protein between days 8 and 12 after birth. The appearance of ovarian aromatase (postnatal days 2-4) coincides with the development of primordial follicles. Fetal-neonatal ovaries maintained in serum-free organ culture do not develop aromatase activity at the expected time. Ovine follicle-stimulating hormone (0.1-1 microgram/ml), ovine luteinizing hormone (0.1 microgram/ml), or their combination failed to induce the enzyme activity in cultured fetal ovaries, whereas follicle-stimulating hormone is effective in preventing the decline in aromatase activity when postnatal day 8 ovaries are placed in culture. In contrast to follicle-stimulating hormone, dibutyryl-cAMP markedly enhances ovarian aromatase in cultured fetal ovaries. Likewise, enhancement of endogenous cAMP formation with forskolin or cholera toxin caused an increase in enzyme activity within 24 hr. Vasoactive intestinal peptide, a peptide known to occur in ovarian nerves, caused a dose-dependent increase in aromatase activity in fetal ovaries prior to folliculogenesis. Of related peptides tested, only the peptide having N-terminal histidine and C-terminal isoleucine amide was capable of inducing aromatase activity in fetal ovaries. The fact that VIP can induce aromatase activity in fetal rat ovaries prior to follicle formation and prior to responsiveness to follicle-stimulating hormone suggests that this neuropeptide may play a critical role in ovarian differentiation.
Specific stimulation of simian virus 40 late transcription in vitro by a cellular factor binding the simian virus 40 21-base-pair repeat promoter element.
Authors: Kim C H , Heath C , Bertuch A , Hansen U
Published: May 2006

We have identified a cellular transcription factor from uninfected HeLa cells that stimulates the simian virus 40 (SV40) late mode of transcription and specifically binds the SV40 21-base-pair repeat promoter element. In particular, the late SV40 transcription factor (LSF) stimulates transcription at initiation sites L325 and L264 of the SV40 late promoter, which are the major transcription sites utilized after DNA replication during the SV40 lytic cycle. In addition, LSF appears to stimulate transcription to a lesser extent from the late-early initiation site of the early promoter. LSF binds specifically to the 21-base-pair repeats of the SV40 promoters, forming specific protein-DNA complexes, which migrate more rapidly through nondenaturing polyacrylamide gels than that formed by the previously identified transcription factor Sp1. Thus, LSF is distinguishable from Sp1 in both its transcriptional and DNA-binding properties. These findings suggest a potential role of LSF in the early to late transcriptional switch during a SV40 lytic infection.
Early promoter-binding factor from vaccinia virions.
Authors: Yuen L , Davison A J , Moss B
Published: May 2006

A factor, present in transcriptionally active extracts prepared from purified vaccinia virus particles, binds to vaccinia early promoter sequences. The specificity of binding was demonstrated by electrophoretic mobility shift assays using the 5'-terminal segments of two early genes and related and unrelated competitor DNA fragments. DNase I "footprint" analysis indicated that the factor formed a complex with promoter regions of both genes and protected sequences of 10-15 nucleotides centered 21-24 nucleotides upstream of the RNA start sites. The lack of protection of a late regulatory sequence and of an early promoter with transcriptionally inactivating single-nucleotide substitutions suggested that the protein is an early transcription factor. When subjected to glycerol gradient centrifugation, the DNA-binding factor was resolved from RNA polymerase and sedimented as a 7.5S species with an estimated molecular weight of 130,000.
Coat protein gene sequence of tobacco mosaic virus encodes a host response determinant.
Authors: Saito T , Meshi T , Takamatsu N , Okada Y
Published: May 2006

The common strain and tomato strain of tobacco mosaic virus (TMV) are known to be closely related to each other. However, plants with the N' gene, such as Nicotiana sylvestris and Nicotiana tabacum L. cv. Bright Yellow, respond differently to infections by these viruses. In the N' plants, TMV-OM (common strain) spreads systemically with mosaic symptoms, whereas TMV-L (tomato strain) induces the necrotic response of plants, causing local lesions. To reveal the viral factor of TMV-L inducing the necrotic response, we have constructed several recombinant viruses between the two strains, in which TMV-L RNA was partly replaced by TMV-OM RNA. The recombinant viruses having the coat protein gene sequence of TMV-OM in place of TMV-L produced no necrotic local lesions but spread systemically with mosaic symptoms in the N' plants. On the other hand, the recombinant viruses having TMV-OM-derived sequences other than the coat protein gene sequence, and in which the coat protein gene sequence of TMV-L still remained, produced necrotic local lesions. These observations indicate that the viral factor of TMV-L responsible for the necrotic response of the N' plants is coded in the coat protein gene sequence.
The actinorhizal root-nodule symbiont
Frankia
sp. strain CpI1 has two glutamine synthetases
Authors: Edmands Janet , Noridge Nancy A. , Benson David R.
Published: May 2006

Frankia
sp. strain CpI1 has two glutamine synthetases. Glutamine synthetase I (GSI) is present during growth on ammonium or N
2
and is similar to classical prokaryotic glutamine synthetases. Gel-filtration chromatography gave a molecular weight estimate of about 680,000 for the GSI holoenzyme, and denaturing polyacrylamide gel electrophoresis yielded a subunit molecular weight of about 59,000, indicating that GSI is most likely a dodecamer. GSI is regulated by adenylylation, as shown by the presence of two spots on two-dimensional polyacrylamide gel electrophoresis and by its behavior during treatment with snake venom phosphodiesterase. GSII is derepressed during nitrogen starvation and accounts for about 95% of the glutamine synthetase activity in nitrogen-starved cells. It is heat-labile and has a subunit molecular weight of about 43,000.
Frankia
GSII is similar to GSII enzymes found in all but one member of the Rhizobiaceae analyzed to date. The presence of a second glutamine synthetase in
Frankia
lends support to the proposal that symbiotic organisms have unique modes of nitrogen nutrition but reopens questions about the origins and uniqueness of GSII genes in members of the Rhizobiaceae.
Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits.
Authors: Allen J P , Feher G , Yeates T O , Komiya H , Rees D C
Published: May 2006

The three-dimensional structure of the protein subunits of the reaction center (RC) of Rhodobacter sphaeroides has been determined by x-ray diffraction at a resolution of 2.8 A with an R factor of 26%. The L and M subunits each contain five transmembrane helices and several helices that do not span the membrane. The L and M subunits are related to each other by a 2-fold rotational symmetry axis that is approximately the same as that determined for the cofactors. The H subunit has one transmembrane helix and a globular domain on the cytoplasmic side, which contains a helix that does not span the membrane and several beta-sheets. The structural homology with RCs from other purple bacteria is discussed. A structure of the complex formed between the water soluble cytochrome c2 and the RC from Rb. sphaeroides is proposed.
Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: demonstration of cleavage within clathrin-coated (pro)secretory vesicles.
Authors: Steiner D F , Michael J , Houghten R , Mathieu M , Gardner P R , Ravazzola M , Orci L
Published: May 2006

Polyclonal antibodies reactive with a cleavage site in human proinsulin (HPI) (C-peptide-A-chain junction) have been raised (rabbit, guinea pig) using a synthetic peptide antigen coupled with keyhole limpet hemocyanin. These antisera recognize native HPI and des-31,32-HPI equally well but react 20-50 times less well with des-64,65-HPI, the intermediate cleaved at the C-peptide-A-chain junction and lacking the Lys-Arg pair. The guinea pig antisera did not recognize insulin but reacted weakly with C peptide at high concentrations; the rabbit antisera reacted with neither insulin nor C peptide. Immunocytochemical studies with human islet tissue localized the immunoreactivity of these antisera to clathrin-coated (pro)secretory vesicles derived from the trans Golgi, indicating that cleavage of the C-peptide-A-chain junction of proinsulin occurs mainly, if not exclusively, in this compartment of the beta cell.
Self-excitation of a nonlinear scalar field in a random medium
Authors: Zeldovich Ya. B. , Molchanov S. A. , Ruzmaikin A. A. , Sokoloff D. D.
Published: May 2006

We discuss the evolution in time of a scalar field under the influence of a random potential and diffusion. The cases of a short-correlation in time and of stationary potentials are considered. In a linear approximation and for sufficiently weak diffusion, the statistical moments of the field grow exponentially in time at growth rates that progressively increase with the order of the moment; this indicates the intermittent nature of the field. Nonlinearity halts this growth and in some cases can destroy the intermittency. However, in many nonlinear situations the intermittency is preserved: high, persistent peaks of the field exist against the background of a smooth field distribution. These widely spaced peaks may make a major contribution to the average characteristics of the field.
Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells.
Authors: Matzuk M M , Krieger M , Corless C L , Boime I
Published: May 2006

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunits. The beta subunit of hCG (hCG beta) is unique among the beta subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either the hCG beta gene alone or together with the hCG alpha gene were transfected into a mutant Chinese hamster ovary cell line, IdID, which exhibits a reversible defect in O-glycosylation. Our results reveal that hCG beta can be secreted normally in the absence of its O-linked oligosaccharides. hCG beta devoid of O-linked carbohydrate can also combine efficiently with hCG alpha and be secreted as an intact dimer. We conclude that in Chinese hamster ovary cells, the hCG beta O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.
Posttranscriptional gene regulation and specific binding of the nonhistone protein HMG-I by the 3' untranslated region of bovine interleukin 2 cDNA.
Authors: Reeves R , Elton T S , Nissen M S , Lehn D , Johnson K R
Published: May 2006

The 3' untranslated tail region (3'-UTR) of the cDNA of bovine interleukin 2 (bIL-2) acts as a lymphoid cell-specific gene regulatory element in vivo when ligated to the 3' end of the "marker" bacterial gene coding for chloramphenicol acetyltransferase (CAT) and the hybrid fusion gene is introduced into bovine lymphoid cells by transfection. Evidence is also presented that the 3'-UTR with its conserved (TATT)n motif probably has multiple functions in lymphoid cells operating both at the chromosomal level, where the sequence may be involved in the specific binding of the nonhistone chromatin high mobility group protein HMG-I, and at the RNA level, where the conserved sequence is involved in selective posttranscriptional mRNA degradation by a lymphocyte-specific nuclease(s). These results suggest a complex in vivo role for the 3'-UTR of bIL-2 cDNA and the conserved (TATT)n sequences found within it. They also offer a plausible explanation for the high degree of conservation of similar A + T-rich sequences in the 3'-UTRs of many of the other immune-response and growth-regulatory genes of mammals.
Purified membrane and soluble folate binding proteins from cultured KB cells have similar amino acid compositions and molecular weights but differ in fatty acid acylation.
Authors: Luhrs C A , Pitiranggon P , da Costa M , Rothenberg S P , Slomiany B L , Brink L , Tous G I , Stein S
Published: May 2006

A membrane-associated folate binding protein (FBP) and a soluble FBP, which is released into the culture medium, have been purified from human KB cells using affinity chromatography. By NaDodSO4/PAGE, both proteins have an apparent Mr of approximately 42,000. However, in the presence of Triton X-100, the soluble FBP eluted from a Sephadex G-150 column with an apparent Mr of approximately 40,000 (similar to NaDodSO4/PAGE) but the membrane-associated FBP eluted with an apparent Mr of approximately 160,000, indicating that this species contains a hydrophobic domain that interacts with the detergent micelles. The amino acid compositions of both forms of FBP were similar, especially with respect to the apolar amino acids. In addition, the 18 amino acids at the amino termini of both proteins were identical. The membrane FBP, following delipidation with chloroform/methanol, contained 7.1 mol of fatty acid per mol of protein, of which 4.7 mol was amide-linked and 2.4 mol was ester-linked. The soluble FBP contained only 0.05 mol of fatty acid per mol of protein. These studies indicate that the membrane FBP of KB cells contains covalently bound fatty acids that may serve to anchor the protein in the cell membrane.
Site-directed serology with synthetic peptides representing the large glycoprotein G of respiratory syncytial virus.
Authors: Norrby E , Mufson M A , Alexander H , Houghten R A , Lerner R A
Published: May 2006

A set of 23 nested 15-amino-acid-long peptides with overlaps of 5 amino acids, representing the complete extramembranous part of the large glycoprotein G of respiratory syncytial (RS) virus, was analyzed in ELISA against different sera containing virus-specific antibodies. Seven of the peptides reacted with rabbit hyperimmune sera against purified virions. In contrast, only one of these seven peptides reacted with murine monoclonal antibodies specific for G. In connection with RS virus infections in humans, increase of antibody titers against three peptides was found in about one-third of the cases. These three peptides were included among those identified by both murine and rabbit antibodies. The present findings may open possibilities for site-directed clinical serology in the case of RS virus infections.
Missing contact probing of DNA-protein interactions.
Authors: Brunelle A , Schleif R F
Published: May 2006

We have examined the positions of contact between lambda phage repressor protein and operator OR1 DNA by scanning populations of lightly depurinated or depyrimidated DNA for bases essential to or irrelevant to repressor binding. This global scanning technique delineates the apparent contact region between lambda repressor and operator and shows bases previously demonstrated or predicted to be contacted plus some additional bases. A mutant repressor, previously shown to contact DNA as wild-type repressor does with the exception of a missing contact to guanosine G4' [Hochschild, A. & Ptashne, M. (1986) Cell 44, 925-933], similarly failed to contact G4' when assayed by this method. Coupled with altering a test residue of a DNA-contacting protein to glycine or alanine so as to eliminate a specific contact, the method appears to provide an efficient means of scanning for specific residue-base contacts.
Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the arginine repressor.
Authors: Lim D B , Oppenheim J D , Eckhardt T , Maas W K
Published: May 2006

In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis. We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated. The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ("ARG boxes"). The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined. Antibodies prepared against the fusion protein react with the repressor. The repressor is precipitable by L-arginine, which facilitates its purification. The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500. To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR.
Purification of a membrane-derived human erythroid growth factor.
Authors: Feldman L , Cohen C M , Riordan M A , Dainiak N
Published: May 2006

We have purified erythroid burst-promoting activity (BPA) from human lymphocyte plasma membranes by detergent extraction followed by gel-filtration, ion-exchange, and hydroxylapatite chromatography. BPA is a heat-stable integral membrane glycoprotein of Mr 28,000 by gel filtration whose activity is eluted from NaDodSO4/polyacrylamide gels as a broad band at Mr 25,000-29,000. The growth stimulator appears to be erythroid-specific, stimulating proliferation of the human erythroid burst-forming unit (BFU-E) by up to 600% of control values when tested in serum-free bone marrow culture. In contrast, it is devoid of granulocyte/macrophage colony-stimulating factor activity and has a negligible effect on the formation of human megakaryocyte and mixed hematopoietic colonies. Polyclonal anti-lymphocyte membrane IgG, which neutralizes BPA expression in culture, completely absorbs BPA from all lymphocyte-derived sources [solubilized lymphocyte plasma membranes, membrane-containing vesicles shed into lymphocyte conditioned medium (LCM) and soluble vesicle-free LCM supernatants], suggesting that soluble and membrane-derived lymphocyte BPA are antigenically related. This membrane glycoprotein may be an important mediator of proximal cellular interactions that are known to promote erythropoiesis in vitro.
Isolation and characterization of three endosomal fractions from the liver of estradiol-treated rats.
Authors: Belcher J D , Hamilton R L , Brady S E , Hornick C A , Jaeckle S , Schneider W J , Havel R J
Published: May 2006

Three distinct endosomal fractions were isolated in high purity from livers of estradiol-treated rats. Each fraction had characteristic physical and ultrastructural properties, but the lipid composition and major proteins of their membranes were similar and differed from those derived from the Golgi apparatus. Injected radioiodinated low density lipoproteins accumulated first in the fraction of intermediate density and later in the low density fraction. The latter was composed almost exclusively of lipoprotein-filled multivesicular bodies, most of which had a single membranous appendage. The fraction of intermediate density was composed of lipoprotein-filled vesicles that were smaller than multivesicular bodies and also had membranous appendages. The high density fraction was composed of membranes resembling the appendages of the two vesicular fractions. All three fractions were enriched in receptors for low density lipoproteins and asialoglycoproteins, but receptor concentrations were considerably reduced in multivesicular bodies. The fraction of intermediate density may represent the compartment of uncoupling of receptor and ligand (CURL) described by Geuze et al. [Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, H. F. & Schwartz, A. L. (1983) Cell 32, 277-287]. CURL vesicles may lose some of their appendage as multivesicular bodies are formed. The high density fraction then may represent a receptor-recycling compartment.
Delta-crystallin genes become hypomethylated in postmitotic lens cells during chicken development.
Authors: Sullivan C H , Grainger R M
Published: May 2006

Although it has been argued that the loss of 5-methylcytosine from specific sites in DNA plays an important role in activation of specific genes, the mechanism of hypomethylation is not well understood. One model links the process to DNA replication, proposing that it occurs by not remethylating cytosine on newly synthesized DNA. An alternative model argues that hypomethylation results from excision of part or all of the 5-methylcytosine. We were able to test whether hypomethylation can occur without replication by analysis of methylation changes in the delta-crystallin genes of the chicken lens. During embryonic development a large fraction of cells in the lens stops dividing as part of the differentiation process. Shortly after this stage, the delta-crystallin genes in samples of the whole lens become hypomethylated, suggesting the possibility that this process might be occurring in the subset of cells that is no longer dividing. We found that hypomethylation of these genes does occur in postmitotic lens cells, a result that implicates an excision mechanism in this tissue.