bluuebunny/crossref_metadata_2025 huggingface dataset:
Infidelity of DNA synthesis associated with bypass of apurinic sites.
Authors: Schaaper R M , Kunkel T A , Loeb L A
Published: May 2006

The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.
Natural immunity to Sindbis virus is influenced by host tissue sialic acid content.
Authors: Hirsch R L , Griffin D E , Winkelstein J A
Published: May 2006

Recent studies have shown that the sialic acid content of Sindbis virus influences both its ability to active the alternative pathway in vitro and its susceptibility to complement dependent clearance from the bloodstream in vivo. Other studies have shown that the sialic acid content of Sindbis virus is determined by the host in which it is propagated. Because individuals vary in their cell surface sialic acid content, it is possible they also vary in their ability to defend themselves against Sindbis virus infection by virtue of their ability to modify the virus sialic acid content and thereby the capacity of the virus to activate the alternative pathway. To test this hypothesis, outbred Swiss mice were injected subcutaneously with Sindbis virus. There was a significant positive correlation between the level of viremia 18 hr after infection and the sialic acid content of the host's erythrocytes. In addition, animals with erythrocyte sialic acid levels equal to or greater than the mean had a higher level of viremia than animals with erythrocyte sialic acid levels less than the mean. Finally, animals that had muscle sialic acid levels equal to or greater than the mean had a higher incidence of viremia than animals with muscle sialic acid levels less than the mean. These studies suggest that the amount of tissue sialic acid in an individual host influences its ability to resist Sindbis virus infection.
Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine.
Authors: Scherman D , Jaudon P , Henry J P
Published: May 2006

[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-11b-H-benzo [a]-quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane of purified bovine chromaffin granules. Specific binding was characterized by Kd and Bmax values of 3.1 nM and 62 pmol/mg of membrane protein, respectively. It was reversible, with association and dissociation rate constants of 0.22 x 10(6) M-1 s-1 and 1.8 x 10(-3) s-1, respectively. Binding sites were present in extracts of medulla but not in corticoadrenal extracts; in the medulla they were restricted to chromaffin granule membranes, [2-3H]Dihydrotetrabenazine binding occurred on the catecholamine carrier of the chromaffin granule membrane because it was clearly correlated with inhibition of norepinephrine uptake. In addition, inhibitors and substrates of the uptake reaction displaced [2-3H]dihydrotetrabenazine from its binding sites, and their potency as displacers was qualitatively correlated with their IC50 or Km. These results suggest that use of [2-3H]dihydrotetrabenazine binding might be an interesting technique in the study of the vesicular monoamine carrier.
Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae.
Authors: Nagata S , Tsunetsugu-Yokota Y , Naito A , Kaziro Y
Published: May 2006

A 3.1-kilobase Bgl II fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'- and 3'-flanking regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-1A transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-1 alpha. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu.
Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis.
Authors: Duke R C , Chervenak R , Cohen J J
Published: May 2006

Within minutes of exposure of target cells to cytotoxic T lymphocytes, their nuclear DNA begins to be fragmented. This phenomenon precedes 51Cr release by at least an hour. DNA fragmentation occurs only when appropriately sensitized cytotoxic T cells are used and is not merely a result of cell death because killing of target cells by heating, freeze/thawing, or lysing with antibody and complement did not yield DNA fragments. Agarose gel electrophoresis of target cell DNA showed discrete multiples of an approximately 200-base-pair subunit, suggesting that fragmentation was the result of activation of a specific endonuclease. A similar pattern of DNA fragments is observed during glucocorticoid-induced killing of mouse thymocytes. The endonuclease in that case is inhibited by zinc ions, and we find that Zn2+ also inhibits DNA fragmentation and 51Cr release induced by cytotoxic T cells, suggesting a final common biochemical pathway for both types of cell death.
Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ.
Authors: Nghiêm H O , Cartaud J , Dubreuil C , Kordeli C , Buttin G , Changeux J P
Published: May 2006

Subsynaptic membrane fragments prepared from Torpedo marmorata electric organ contain, in addition to the acetylcholine receptor polypeptides, a major protein band of apparent molecular mass 43,000 daltons. On two-dimensional gels, this band yields three spots referred to as v1, v2, and v3. Monoclonal antibodies against the 43,000-dalton proteins were developed in CBA mice. One of them reacted exclusively with the v1 polypeptide but not with v2 and v3. Staining by the "immunogold" reaction followed by observation by electron microscopy showed that this antibody exclusively labeled the innervated membrane of T. marmorata electroplaque on its cytoplasmic face. Electroblots of one-dimensional gels of membrane preparations from 80-mm embryo electric organ were prepared. After reaction with the anti-v1 monoclonal antibody, a strongly stained 43,000-dalton band was revealed.
Spatial patterns and double diffusion in chemical reactions
Authors: Dewel G. , Borckmans P. , Walgraef D.
Published: May 2006

Spatial ordering has been observed recently during various photochemical reactions. Convoluted concentration bands first appear near the surface of shallow irradiated solutions. They thereafter extend into the bulk, and finger-like structures spontaneously develop. We discuss here the possible role of double-diffusion effects in the onset of this phenomenon. Indeed, chemical reactions occurring near the surface or evaporation of the solvent, or both, induce in the bulk adverse gradients of a pair of properties (concentrations of solute or concentration and temperature) having different diffusivities. This difference can then destabilize the homogeneous solution and trigger the observed patterns.
The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system.
Authors: Zylicz M , LeBowitz J H , McMacken R , Georgopoulos C
Published: May 2006

The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The lambda O and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified lambda P protein and stimulated by purified lambda O protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the O and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.
Monoclonal antibodies against epidermal growth factor receptor induce prolactin synthesis in cultured rat pituitary cells (GH3).
Authors: Hapgood J , Libermann T A , Lax I , Yarden Y , Schreiber A B , Naor Z , Schlessinger J
Published: May 2006

The addition of epidermal growth factor (EGF) to cultured rat pituitary cells (GH3) leads to increased synthesis of prolactin and to partial inhibition of cell proliferation. Monoclonal antibodies generated against EGF receptor from human epidermoid carcinoma (A-431) cells were used to characterize the EGF receptor kinase system of GH3 cells and to investigate the role of the hormone-receptor complex in the expression of the prolactin gene in these cells. The EGF receptor of GH3 cells is a 170,000-dalton protein associated with a protein kinase. It is similar but not identical to the EGF receptor identified in other tissues. The immunoprecipitated membrane receptor is phosphorylated on both serine and tyrosine residues. The monoclonal antibody denoted 2G2-IgM binds to EGF receptor on GH3 cells. Like EGF, the monoclonal antibody induced the synthesis of prolactin and morphological changes in these cells. Hence, EGF receptor in GH3 cells, when properly triggered, contains all of the biological attributes necessary for the induction of EGF-induced gene expression and morphological changes in GH3 cells.
Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.
Authors: Logeat F , Vu Hai M T , Fournier A , Legrain P , Buttin G , Milgrom E
Published: May 2006

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.
Tissue-specific expression of a chicken calmodulin pseudogene lacking intervening sequences.
Authors: Stein J P , Munjaal R P , Lagace L , Lai E C , O'Malley B W , Means A R
Published: May 2006

An eel calmodulin cDNA probe has been used to isolate a calmodulin gene from a chicken DNA library. Sequence analysis revealed this calmodulin gene (cCM1) to contain the nucleotides that code for 148 amino acids, a termination codon, and 486 residues of 3'-noncoding sequence before an A-A-T-A-A-A poly(A) addition signal. The amino acid sequence derived from these nucleotides is 87% homologous to that of bovine brain calmodulin. cCM1 is one of two calmodulin genes in the chicken genome but is unique in that it does not contain intervening sequences to interrupt the structural segments of the protein. This suggests that cCM1 originated as a processed gene copy derived from the other calmodulin gene, cCL1, a circumstance usually associated with pseudogenes. In contrast, cCM1 appears to be a functional member of a multigene family whose expression is specific for muscle cells.
Biological activity of mRNA immobilized on nitrocellulose in NaI.
Authors: Bresser J , Hubbell H R , Gillespie D
Published: May 2006

In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells.
Homologue destabilization by a putative transposable element in Drosophila melanogaster.
Authors: Lim J K , Simmons M J , Raymond J D , Cox N M , Doll R F , Culbert T P
Published: May 2006

We postulate the presence of a transposable element, designated the L factor, to explain the properties of an unstable X chromosome and its derivatives. These chromosomes generate recessive lethal mutations at high rates, as does a stable X chromosome that has been associated with them for only one generation. The stable X chromosome does not become highly mutable in the absence of the unstable X chromosome, even when autosomes from the unstable stock are present. These facts suggest that the L factor is confined to the X chromosome and that it transposes to other X chromosomes paired with it. We propose the term "homologue destabilization" to denote the change in the stable chromosome brought about by this transposition. The lethal mutations caused by the L factor occur preferentially in the region around the cut wing locus (ct) and are sometimes associated with recognizable chromosome aberrations. The breakpoints of these aberrations are most often in the vicinity of ct, implying that the L factor is located near ct on the unstable chromosome, but it may reside at other sites as well. Alternately, the ct region may simply be a preferred target for the insertion of this transposable element.
Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation.
Authors: Storb U , Arp B
Published: May 2006

Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions. To assess the potential role of DNA methylation in the expression of immunoglobulin genes, which require complex rearrangements prior to expression, methylation patterns were examined in cell lines representing different stages of lymphocyte maturation. Methylation of the second cytosine in the sequence 5' C-C-G-G 3' was determined by using Hpa II/Msp I endonuclease digestion. Four CH genes (C mu, C delta, C gamma 2b, and C alpha), C kappa, V kappa, C lambda, and V lambda genes were analyzed. The results lead to the following conclusions: (i) transcribed immunoglobulin genes are undermethylated; (ii) the C gene allelic to an expressed C gene is always also undermethylated; and (iii) all immunoglobulin loci tend to become increasingly undermethylated as B cells mature.
A monoclonal anti-platelet antibody with decreased reactivity for autoimmune thrombocytopenic platelets.
Authors: Varon D , Karpatkin S
Published: May 2006

Two monoclonal anti-platelet antibodies, 3B2 and 8G11, have been raised that are specific for normal human platelets. 3B2 is unique in that it has decreased reactivity for platelets from 16 patients with autoimmune thrombocytopenic purpura [mean platelet count, 65,000 +/- 6,000 (SEM)]. With 8G11 in an enzyme-linked immunosorbent assay, the mean of the ratios of patient platelet OD to control platelet OD was 0.95 +/- 0.07, whereas with 3B2, the mean of the ratios of patient platelet OD to control OD was 0.24 +/- 0.04, P less than 0.001. With 3B2 the mean of the OD ratios of five patients with autoimmune thrombocytopenic purpura in remission (greater than 150,000 platelets per mm3) compared to controls was 0.80 +/- 0.14. 3B2 did not react with platelets from a patient with Glanzmann's thrombasthenia, in which membranes lack glycoproteins IIb and IIIa (GPIIb and GPIIIa). Platelet membranes were run on crossed immunoelectrophoresis against a rabbit polyclonal anti-human platelet membrane antibody with 125I-labeled purified 3B2 in an intermediate spacer gel. 3B2 reacted with the GPIIb-GPIIIa-Ca2+ complex in the presence of excess Ca2+ and with GPIIb alone in the presence of excess EGTA. When Triton X-100-solubilized platelet membranes were immunoprecipitated with 3B2 plus rabbit anti-mouse IgG, reduced, and run on NaDodSO4/polyacrylamide gel electrophoresis, a single protein band was obtained with a molecular weight of 120,000 (the molecular weight of GPIIb). Thus, the reactivity of monoclonal antibody 3B2 with GPIIb or the GPIIb-GPIIIa-Ca2+ complex appears to be inhibited by the presence of autoantibody on platelets.
Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.
Authors: Rodgers G M , Shuman M A
Published: May 2006

Vascular endothelial cells derived from adult bovine aorta (ABAE) treated with factor Xa and calcium were found to activate prothrombin. In contrast, nonvascular cells (human foreskin fibroblasts, bovine corneal endothelial cells, or human fetal lung cells) had either no or very little effect on prothrombin activation. In the presence of 6 X 10(5) ABAE cells, 20 ng of factor Xa converted 90 micrograms of prothrombin into 80 units of thrombin after 45 min at 37 degrees C. Exogenous factor V was not required for prothrombin activation, but thrombin generation was enhanced 2- to 4-fold by the addition of factor V (500-2,500 ng/ml). Treatment of ABAE cells with anti-bovine factor V IgG markedly inhibited prothrombin activation by factor Xa and calcium. In cells grown in serum-free medium for 3 months, the amount of factor V activity was equivalent to that found in cells grown with serum, which suggests that these cells probably synthesize factor V. Sparse ABAE cells increased prothrombin activation by factor Xa 6-fold compared to activation in confluent cells. Although previous thrombin treatment of ABAE cells did not enhance prothrombin activation, addition of dansyl arginine-4-ethyl piperidine amide markedly inhibited activation of 125I-labeled prothrombin by factor Xa, indicating that thrombin formation is necessary for optimal prothrombin activation. These data indicate that aortic endothelium may provide a physiologically important surface for activation of prothrombin as well as a mechanism for optimal formation of clots at sites of vascular injury.
Human T-cell leukemia virus type II transforms normal human lymphocytes.
Authors: Chen I S , Quan S G , Golde D W
Published: May 2006

A unique human retrovirus (human T-cell leukemia virus type II, HTLV-II), isolated from a patient with a T-cell variant of hairy-cell leukemia, has been shown to be distinct from the more common isolates of human T-cell leukemia virus. This virus was tested for its ability to transform normal human peripheral blood lymphocytes. The HTLV-II-infected T-cell line Mo-T was lethally x-irradiated and cocultivated with normal human peripheral blood lymphocytes. The cocultivation of normal cells with Mo-T cells resulted in the transformation of the normal cells as evidenced by the establishment of permanent cell lines. The transformed cells are infected with HTLV-II as shown by immunologic tests and molecular hybridization. The cells are of mature T-cell phenotype and constitutively produce lymphokines. An Epstein-Barr virus-transformed lymphoblast B-cell line established from peripheral blood cells of the patient Mo, designated Mo-B, also was found to be infected with HTLV-II. All HTLV-II-infected cells, including the Mo-B cells, were capable of transforming normal cells of T-cell phenotype by transmission of virus by cocultivation. These results indicate that HTLV-II infects both B and T cells but transforms normal human peripheral blood lymphocytes of T-cell phenotype.
Lipid metabolism of isolated oligodendrocytes maintained in long-term culture mimics events associated with myelinogenesis.
Authors: Szuchet S , Yim S H , Monsma S
Published: May 2006

Oligodendrocytes isolated from ovine white matter according to a published procedure [Szuchet, S., Stefansson, K., Wollmann, R. L., Dawson, G. & Arnason, B. G. W. (1980) Brain Res. 200, 151-164] were cultured for up to 35 days and their capacity to incorporate precursors into lipids was investigated. At various times, cultures were double labeled with [3H]glycerol/[14C]acetate or [3H]galactose/35SO2-4. The cells were harvested 72 hr later and lipids were fractionated using standard procedures. The time course of incorporation for each precursor was distinct. In the days after attachment to substratum, oligodendrocytes preferentially incorporated [3H]glycerol into phospholipids and [14C]acetate into cholesterol while uptake of 35SO2-4 and [3H]galactose into glycolipids was modest. A switch in phospholipid metabolism from preferential incorporation into phosphatidylcholine to incorporation into phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol occurred at about the 10th day in vitro. After 20 days, uptake of [3H]glycerol into phospholipids and [14C]acetate into cholesterol had stabilized but incorporation of 35SO2-4 into glycolipids had increased. 35SO2-4 incorporation into glycolipids was even greater at 35 than at 20 days. Uptake of [3H]galactose did not change over time. An attempt was made to correlate changes in lipid metabolism with morphologic developments. High incorporation into phospholipids and cholesterol coincided in time with the extensive membrane synthesis required for cell attachment and process extension. Differentiation of these newly formed membranes, as assessed by the incorporation of myelin-characteristic glycolipids, galactocerebrosides, and sulfatides, occurred at a time when an intricate network of processes had already been established. The sequence of metabolic events observed in vitro parallels that observed at the onset of myelinogenesis in vivo. We postulate that mature oligodendrocytes can reenact those early events associated with myelinogenesis.
Oxidation of methionine residues in proteins of activated human neutrophils.
Authors: Fliss H , Weissbach H , Brot N
Published: May 2006

A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins.
Nerve growth factor: stimulation of polymorphonuclear leukocyte chemotaxis in vitro.
Authors: Gee A P , Boyle M D , Munger K L , Lawman M J , Young M
Published: May 2006

Topical application of mouse nerve growth factor (NGF) to superficial skin wounds of mice has previously been shown to accelerate the rate of wound contraction. Results of the present study reveal that NGF in the presence of plasma is also chemotactic for human polymorphonuclear leukocytes in vitro, and the concentration of NGF required for this effect is similar to that which stimulates ganglionic neurite outgrowth. This property does not arise from liberation of the C5a fragment of complement, nor does it require the known enzymic activity of NGF. (NGF inactivated with diisopropyl fluorophosphate is equally active.) We conclude that NGF can display biological effects on cells of nonneural origin and function, and this feature might play a role in the early inflammatory response to injury.
Steady-state theory of the interference of GTP hydrolysis in the mechanism of microtubule assembly.
Authors: Hill T L , Carlier M F
Published: May 2006

A model is presented for the interference of GTP hydrolysis in the mechanism of microtubule assembly. This model is suggested by previous results showing that both GTP and GDP are present at microtubule ends because of GTP hydrolysis and that tubulin does not bind to a GDP-bound end. The analytical theory developed here is aimed at calculation of the steady-state subunit flux at one end of the polymer. The GTP/GDP features just mentioned result in a nonlinear plot of the flux versus tubulin concentration. Microtubules are predicted to exhibit a different kinetic behavior below and above the critical concentration, which can be considered as a transition between two regimes.
Phorbol esters, phospholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells.
Authors: Rozengurt E , Rodriguez-Pena M , Smith K A
Published: May 2006

Addition of biologically active phorbol esters to intact quiescent 3T3 mouse cells stimulates an extremely rapid (detectable within seconds) phosphorylation of a Mr 80,000 cellular protein (termed "80k"). Phorbol 12,13-dibutyrate enhances 80k phosphorylation in a dose-dependent manner; half-maximal effect is obtained at 32 nM. The possibility that this phosphorylation is related to the activation of Ca2+-activated phospholipid-dependent protein kinase is suggested by the fact that phospholipid breakdown induced by exogenous treatment of the cells with phospholipase C from Clostridium perfringens or with platelet-derived growth factor, which is a potent activator of endogenous phospholipase C activity, also causes a rapid enhancement of 80k phosphorylation. Moreover, prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which leads to a marked decrease in the number of specific phorbol ester binding sites, prevents the phosphorylation of 80k stimulated by phorbol esters, phospholipase C, and platelet-derived growth factor. These findings provide evidence obtained with intact cells that implicate the stimulation of Ca2+-activated phospholipid-dependent protein kinase in the action of phorbol esters and other growth factors.
DNA sequences near the site of reciprocal recombination between a c-myc oncogene and an immunoglobulin switch region.
Authors: Dunnick W , Shell B E , Dery C
Published: May 2006

The chromosomal translocations found in many B-cell tumors result in the joining of a c-myc oncogene with an immunoglobulin heavy chain switch region. This finding is striking because the natural function of switch regions is to mediate DNA rearrangements important to the maturation of immune responses. These normal switch rearrangements are probably mediated by specific enzymes. In this paper we report the isolation of the two reciprocal products of a recombination between a c-myc gene on murine chromosome 15 and an immunoglobulin switch region (S mu S gamma 2b) on chromosome 12. We have determined the sequences of these DNA molecules near the recombination sites and show that the recombination is nearly perfectly reciprocal, with a seven-nucleotide deletion. An examination of the sequences reported in this paper, and of sequences published by other authors, shows a correlation between the points of recombination for c-myc-S segment rearrangements and for normal heavy chain switches. We suggest that this correlation implies a role for switch recombination enzymes in creating substrates for the c-myc recombination. The c-myc gene also seems to share some limited homology to sequences thought to be important in heavy chain switching. Finally, we discuss a working model that accounts for some characteristics of c-myc-S segment recombinations. The model also suggests a mechanism for increased transcriptional activity of the rearranged c-myc oncogene in B-cell tumors.
Lysis of Chinese hamster embryo fibroblast mutants by human natural cytotoxic (NK) cells.
Authors: Dubey D P , Staunton D E , Smith B L , Yunis E J , Sager R
Published: May 2006

The nontransformed, nontumorigenic CHEF/18 Chinese hamster embryo fibroblast line, as well as nontumorigenic CHEF/18 mutants that had become anchorage independent or acquired a reduced serum requirement for growth, and fully transformed, tumorigenic CHEF cell lines were analyzed for their sensitivity to killing in vitro by human natural killer (NK) cells. Nontumorigenic but transformed anchorage-independent and low-serum-requiring mutants remained insensitive to NK-mediated lysis like the parent CHEF/18 line. Only fully tumorigenic CHEF lines were found to be sensitive to NK-mediated lysis, although a few tumorigenic lines were resistant to NK lysis. These results indicate that NK sensitivity is not the result of any cellular changes associated with acquisition of an anchorage-independent or low-serum-requiring phenotype but is the result of some additional change(s) found only in fully tumorigenic CHEF cells. Our studies also show that, whatever the NK target structure is, it is evolutionarily conserved so that human NK cells are able to distinguish between Chinese hamster tumorigenic and nontumorigenic cells.
Transcription modulation in vitro of the fibroin gene exerted by a 200-base-pair region upstream from the "TATA" box.
Authors: Tsuda M , Suzuki Y
Published: May 2006

We have previously reported that the 5'-flanking sequence upstream from the "TATA" box modulates the faithful transcription initiation of the fibroin gene in a homologous whole cell extract prepared from the silk glands, whereas such a modulating effect is not observed in a HeLa cell extract. Subsequently we have determined that major signals responsible for the modulating effect are located within a 200-base-pair region upstream from the TATA box, mainly in a distal region between nucleotide positions -238 and -116 and in a proximal one between -73 and -53. Inversion of the sequence element -234 to -66 did not alter its modulating effect. A similar modulating effect by the upstream region of the sericin gene was also observed in the silk gland extract but not in the HeLa cell extract. In contrast, a modulating effect by the upstream region of the adenovirus 2 major late gene was observed in the HeLa cell extract but not in the silk gland extract. Thus, the modulating effect by the upstream regions of these genes is exerted only in their own homologous extracts.
Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus.
Authors: Takegami T , Kuhn R J , Anderson C W , Wimmer E
Published: May 2006

A small nucleotidyl-protein has been synthesized in vitro in a membrane fraction of poliovirus-infected HeLa cells. Analyses of the nucleotides and polypeptide have shown that the nucleotidyl-protein is VPg-pUpU: the genome-linked protein of poliovirion RNA covalently bound to the first two 5'-terminal nucleotides of poliovirus RNA. Synthesis of VPg-pUpU in vitro was sensitive to nonionic detergent. We suggest that VPg-pUpU is part of the initiation complex in poliovirus RNA replication in a membranous environment.
Yeast mutants deficient in protein glycosylation.
Authors: Huffaker T C , Robbins P W
Published: May 2006

The synthesis of asparagine-linked oligosaccharides involves the formation of a lipid-linked precursor oligosaccharide that has the composition Glc3Man9GlcNAc2. We have used a [3H]mannose suicide selection to obtain mutants in yeast that are blocked in the synthesis of this precursor oligosaccharide. The alg1 mutant accumulated lipid-linked GlcNAc2, alg2 mutants accumulated Man1-2GlcNAc2, alg3 mutants accumulated Man5GlcNAc2, alg4 mutants accumulated Man1-8GlcNAc2, and alg5 and alg6 mutants accumulated Man9GlcNAc2. Some of these mutants appeared to transfer oligosaccharides other than Glc3Man9GlcNAc2 from the lipid carrier to invertase. These aberrant protein-linked oligosaccharides were processed by the addition of outer chain residues in the alg3, alg5, and alg6 mutants. There was virtually no outer chain addition in the alg2 and alg4 mutants. alg4 was the only mutant that failed to secrete invertase.
Dissociation of iron transport and heme biosynthesis from commitment to terminal maturation of murine erythroleukemia cells.
Authors: Tsiftsoglou A S , Nunez M T , Wong W , Robinson S H
Published: May 2006

Treatment of murine erythroleukemia (MEL) cells with imidazole in the presence of dimethyl sulfoxide (Me2SO) has been shown to dissociate hemoglobin accumulation from commitment to terminal maturation. To explore the mechanism(s) of this effect, we studied iron transport and heme and hemoglobin synthesis in Me2SO-induced MEL cells that were then exposed to imidazole. Imidazole treatment (i) causes moderate inhibition of 125I-labeled transferrin binding to both control and Me2SO-treated MEL cells; (ii) markedly suppresses Me2SO-induced activation of iron uptake into MEL cells; (iii) markedly decreases the incorporation of iron into ferritin; and (iv) abolishes heme biosynthesis from [2-14C]glycine and hemoglobin accumulation in Me2SO-treated cells. Imidazole treatment does not inhibit other aspects of cellular maturation; cells treated with Me2SO in the presence or absence of imidazole exhibit similar changes in proliferative activity and protein synthesis and, as shown previously, in cell morphology. Inhibition of hemoglobin accumulation in MEL cells is reversible on withdrawal of imidazole but is not altered by exogenous hemin. These data indicate that commitment to terminal maturation is regulated independently from the systems for iron transport and heme biosynthesis during early phases of erythroid cell differentiation.
Vitamin D metabolites change the phenotype of monoblastic U937 cells.
Authors: Dodd R C , Cohen M S , Newman S L , Gray T K
Published: May 2006

U937 is a human-derived lymphoma cell line that has monoblastic properties and high-affinity receptors for 1 alpha,-dihydroxyvitamin D3. Incubation of these cells with the vitamin D metabolite at 10 nM for 5 days produced marked stimulation in adherence and ingestion of Staphylococcus aureus (645% of control) and of C3b receptor (CR1) expression (292% of control) and a slight increase in hexose monophosphate shunt activity without changing cell growth rates or Fc fragment receptor expression. The changes in cellular association of S. aureus and the CR1 were detected as early as 48 hr of incubation and peaked between 3 and 5 days. Similar changes in the CR1 were induced by 25-hydroxy- and 24,25-dihydroxyvitamin D3 at micromolar concentrations. Dexamethasone, hydrocortisone, and progesterone had no effect on CR1 expression. U937 cells incubated in the presence of vitamin D metabolites exhibited a change in their phenotype. These results suggest that vitamin D metabolites may contribute to monocyte/macrophage differentiation.
Different exon-intron organization at the 5' part of a mouse class I gene is used to generate a novel H-2Kd-related mRNA.
Authors: Lalanne J L , Cochet M , Kummer A M , Gachelin G , Kourilsky P
Published: May 2006

A cDNA library constructed from liver mRNA of DBA/2 (H-2d) mice has been screened with H-2-specific probes. The nucleotide sequence of one clone (pH-2d-24) indicates that it derives from an H-2 gene with an unexpected exon-intron organization. Nucleotide sequence comparisons suggest that two distinct mRNAs are produced from a single H-2Kd gene by a mechanism involving the use of alternative splicing sites in its 5' region. pH-2d-24 carries an open reading frame encoding a thus-far-undescribed polypeptide product identical to an H-2Kd-molecule, except for the NH2-terminal half of the first domain.
Resistance of human cells to tumorigenesis induced by cloned transforming genes.
Authors: Sager R , Tanaka K , Lau C C , Ebina Y , Anisowicz A
Published: May 2006

The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse. The EJ gene, a mutant form of the cellular c-Ha-ras gene, actively transforms NIH/3T3 mouse cells and CHEF/18 hamster cells but is inactive in FS-2 cells. Morphological transformation, focus formation, and tumorigenicity in nude mice were not induced when EJ DNA was transfected into FS-2 cells by using the selectable vector pSVgptEJ. The intactness of the transfected EJ DNA was established by restriction fragment analysis. This result raises the question of what role, if any, the mutated gene derived from the EJ cells played in the origin of the EJ bladder carcinoma.
Procedure for rapid isolation of photosynthetic reaction centers using cytochrome
c
affinity chromatography
Authors: Brudvig G. W. , Worland S. T. , Sauer K.
Published: May 2006

Horse heart cytochrome
c
linked to Sepharose 4B is used to purify reaction centers from
Rhodopseudomonas sphaeroides
R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (
A
280
/
A
802
< 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type
Rps. sphaeroides
and
Rps. capsulata
also bind to a cytochrome
c
column. Cytochrome
c
affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.
Gene for staphylococcal protein A.
Authors: Löfdahl S , Guss B , Uhlén M , Philipson L , Lindberg M
Published: May 2006

The gene for protein A from Staphylococcus aureus was cloned into pBR322 in Escherichia coli. An immunoassay was used to detect production of the protein. Protein A produced in E. coli was found in the periplasmic space and was purified and concentrated by IgG-Sepharose affinity chromatography. DNA sequence assay of the gene revealed a region with the general features of a prokaryotic signal peptide and a fifth structural region homologous to the four repetitive regions found earlier by amino acid sequence determination of the mature protein. Upstream from the structural gene there is a possible promoter region and a ribosomal binding sequence typical of gram-positive bacteria. The initiation codon is TTG.
Structural and biological characteristics of connective tissue activating peptide (CTAP-III), a major human platelet-derived growth factor.
Authors: Castor C W , Miller J W , Walz D A
Published: May 2006

Connective tissue activating peptides (CTAPs) extracted from leukocytes and platelets stimulate glycolysis and synthesis of glycosaminoglycan and DNA in cultured human connective tissue cells. CTAP-III, isolated from fresh or outdated human platelets, is a low molecular weight single-chain protein with an isoelectric point of 8.5 that markedly stimulates DNA synthesis and multiple aspects of glycosaminoglycan and proteoglycan metabolism. This report presents a definitive comparison of CTAP-III prepared by two methods [one designated (A), alternative] with similar platelet proteins described by others, beta-thromboglobulin (beta-TG) and low-affinity platelet factor 4 (LA-PF-4). CTAP-III, CTAP-III(A), LA-PF-4, and beta-TG have common antigenic determinants documented by immunoprecipitation and radioimmunoassay. CTAP-III, CTAP-III(A), and LA-PF-4 are biologically active in that they stimulate DNA and glycosaminoglycan synthesis by human synovial cells; beta-TG is inactive. Carboxyl-terminal digestion gave identical terminal sequences for CTAP-III, CTAP-III(A), and beta-TG. Amino-terminal sequence data indicate that CTAP-III and CTAP-III(A) (also LA-PF-4) are identical and differ from beta-TG only by an additional amino-terminal tetrapeptide (Asn-Leu-Ala-Lys-). The biologically active molecule, CTAP-III, may be proteolytically converted to its inactive degradation product (beta-TG) in the course of platelet aging, platelet storage, release from the platelets, or initiation of biological activity.
Changes in mitochondrial structure and function in 9L rat brain tumor cells treated
in vitro
with α-difluoromethylornithine, a polyamine biosynthesis inhibitor
Authors: Oredsson Stina M. , Friend Daniel S. , Marton Laurence J.
Published: May 2006

Mitochondrial structure and function were studied in 9L rat brain tumor cells depleted of polyamines by α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Cells treated with methylglyoxal bis(guanylhydrazone), a reversible inhibitor of
S
-adenosylmethionine decarboxylase, were used for comparison because this polyamine biosynthesis inhibitor is known to cause structural and functional disruption of mitochondria. A significant increase in mitochondrial size, measured quantitatively, was found in α-difluoromethylornithine-treated cells (10 mM for 72 hr) compared with untreated cells (
P
< 0.001). This increase in mitochondrial size was reversed when putrescine was added to the cultures for 24 hr after α-difluoromethylornithine treatment. Putrescine alone had no effect on the size of mitochondria. Treatment of cells with methylglyoxal bis(guanylhydrazone) (80 μM for 48 hr) caused only a slight increase in mitochondrial size compared with mitochondria in untreated cells (
P
< 0.05) and failed to produce the dramatic ultrastructural changes reported in other cell lines. Ultrastructural examination revealed an increase in cytoplasmic and membrane-associated ribosomes in α-difluoromethylornithine-treated cells, an increase in cytoplasmic ribosomes in methylglyoxal bis(guanylhydrazone)-treated cells, and an increase in membrane-bound ribosomes in putrescine-treated cells. In cells treated first with α-difluoromethylornithine and then with putrescine, the distribution of ribosomes was normal. The distributions of ribosomes were not quantitatively assessed. Pyruvate utilization, a measure of mitochondrial function, was decreased in cells treated with 10 mM α-difluoromethylornithine for 72 hr, compared with untreated cells. Restoration of intracellular polyamine levels by the addition of putrescine 24 hr before analysis reversed this phenomenon. Putrescine treatment alone did not affect pyruvate utilization. Pyruvate utilization in methylglyoxal bis(guanylhydrazone)-treated cells was depressed to a greater extent than that in α-difluoromethylornithine-treated cells.
Receptor for transferrin may be a "target" structure for natural killer cells.
Authors: Vodinelich L , Sutherland R , Schneider C , Newman R , Greaves M
Published: May 2006

Human transferrin receptors detected by monoclonal antibody OKT9 appear to be well expressed on cell types known to provide sensitive targets for natural killer (NK) cells. The possibility that transferrin receptors are recognized by NK effector cells has been investigated by three series of experiments: (i) analysis of the correlation between sensitivity to natural killing and the proportion of transferrin receptor-positive cells in different cell lines, (ii) study of the relationship between levels of transferrin receptor expression in cell lines and their capacity to competitively inhibit recognition and killing of the target cell K562 by NK cells, and (iii) comparison of affinity-purified soluble proteolytic fragments of the transferrin receptor and HLA-A, -B molecules for their ability to inhibit the natural killing effect. The data indicate that the transferrin receptor can provide a "target" structure for NK cells. Because transferrin receptors are ubiquitously expressed on normal and malignant proliferating cells, these observations have interesting implications for the possible function of NK cells in vivo.
Evolutionary aspects of immunoglobulin heavy chain variable region (VH) gene subgroups.
Authors: Rechavi G , Ram D , Glazer L , Zakut R , Givol D
Published: May 2006

We isolated and determined the sequences of two human germ-line heavy chain variable region (VH) genes and compared them with mouse VH genes. The results show that the human VHI subgroup is evolutionarily related to the mouse VHII subgroup. Evolutionary preservation of homologies in VH genes of the same subgroup includes not only the coding region but also intron size and homology in noncoding regions. This suggests that a VH gene subgroup constitutes a multigene family that undergoes concerted evolution. The homology between genes of the same subgroup in different species is greater than that between genes of different subgroups within a species. One of the VHII genes contains, in complementarity-determining region 2 (CDR2), a 13-base-pair previously shown to be in CDR2 of a VHIII gene and in a heavy chain diversity region gene, DH [Wu, T. T. & Kabat, E. A. (1982) Proc. Natl. Acad. Sci. USA 79, 5031-5032], suggesting the insertion of diversity region gene sequences into the VH gene. One of the human VH genes is a pseudogene because of a terminator, which, together with our previous results, shows that the VH gene repertoire contains 40% pseudogenes. In one of the VH genes, direct and inverted repeats at both 5' and 3' ends of the gene suggest a potential transposable element that encompasses the entire VH gene. It is possible that such a structure may facilitate saltatory replication and rapid expansion of VH gene families.