bluuebunny/crossref_metadata_2025 huggingface dataset:
Two tumor antigens and their polypeptides in adenovirus type 12-infected and transformed cells
Authors: Shiroki Kazuko , Segawa Kaoru , Shimojo Hiroto
Published: May 2006

A tumor (T) antigen, designated T antigen g, was visualized as fine fluorescent granules in nuclei of adenovirus type 12 (Ad12)-infected cells by immunofluorescence with sera from rats bearing HY cell tumors (H sera). HY cells are rat cells incompletely transformed by the
Acc
I-H endonuclease fragment (0-4.7 map units) of Ad12 DNA. The antigen is different from the usually described T antigen, designated T antigen f, which is visualized as fluorescent flecks or filaments in both nucleus and cytoplasm of Ad12-infected cells when tested with narrowly reacting T sera. Extracts of [
35
S]methioninelabeled infected cells were immunoprecipitated with H sera, and the resultant precipitate was analyzed by the two-dimensional gel electrophoresis technique of O'Farrell. The autoradiogram showed the presence of a cluster of several polypeptides (
M
r
35,000-40,000, pI 5.0-5.5) that was absent in extracts of mock-infected cells. A similar autoradiogram of infected cells analyzed with narrowly reacting T sera showed the presence of a small polypeptide (
M
r
10,000, pI 6.4), that was absent in extracts of mock-infected cells. The results show that
M
r
35,000-40,000 polypeptides are components of T antigen g and a
M
r
10,000 polypeptide is a component of T antigen f. Ad12-transformed cells showed a similar result. T antigen g was present and T antigen f was absent in HY cells. Both T antigen g and T antigen f were present in CY cells, which are rat cells completely transformed by the
Eco
RI-C endonuclease fragment (0-16 map units) of Ad12 DNA. The possible functions of these proteins are discussed.
Pteridines as nonretinal regulators of light-dependent melatonin biosynthesis.
Authors: Cremer-Bartels G , Ebels I
Published: May 2006

6-Formylpterin was purified by paper chromatography, and bovine pineal hydroxyindole-O-methyltransferase (S-adenosyl-L-methionine:N-acetylserotonin O-methyl-transferase; EC 2.1.1.4) was purified by flat-bed gel electrofocusing. Fractions of the methyltransferase with isoelectric points at pH 5.0, 5.7, and 5.2 were tested in the presence of 6-formylpterin, and decreased activities were found. Because 6-formylpterin is one of the photolytic products of folic acid and decreased serum folic acid has been reported in light-exposed men, we suggest that pteridines may be involved in nonretinal effects of light on pineal melatonin biosynthesis. It may be that this metabolic pathway is an evolutionary relic of primitive light responses of pigmentation mechanism.
“Living electrode” as a long-lived photoconverter for biophotolysis of water
Authors: Ochiai Hideo , Shibata Hitoshi , Sawa Yoshihiro , Katoh Takashi
Published: May 2006

Living blue-green algae (
Mastigocladus laminosus
), immobilized on an SnO
2
optically transparent electrode with calcium alginate, functioned as an anodic photoelectrode on continuous illumination for periods of time adequate for use in a conventional electrochemical cell. This “living electrode” shows promise of use as a long-lived photoconverter of solar radiant energy to electric energy and as a suitable replacement for unstable chloroplast systems.
DNA sequence of regulatory region for integration gene of bacteriophage λ
Authors: Abraham Judith , Mascarenhas Desmond , Fischer Robert , Benedik Michael , Campbell Allan , Echols Harrison
Published: May 2006

The cII and cIII proteins specified by bacteriophage λ direct the lysogenic response to infection through the coordinate establishment of repression and integration of the viral DNA. The regulatory activity of cII/cIII involves positive regulation of two promoter sites: the
p
E
promoter, turning on expression of the cI protein that maintains lysogeny, and the
p
I
promoter, activating synthesis of the Int protein for integrative recombination. Regulation of the
p
I
promoter provides for differential expression of the Int protein with respect to the excision-specific Xis protein from the closely linked
int
and
xis
genes. We have determined the DNA sequence of the
p
I
promoter region for wild-type λ DNA and for two classes of mutations:
intc
mutations, which result in a high rate of Int synthesis in the absence of cII, and deletion mutations, some of which eliminate cII-activated expression of the
int
gene. We find a sequence with considerable homology (11 of 15 bases) to a “typical” (computer-generated) promoter sequence, adjacent to a region with striking homology (11 of 14 bases) to part of the
p
E
promoter region. This presumed
p
I
sequence overlaps the start of the
xis
gene and includes the site of two
intc
point mutations. A cII-insensitive
xis
+
deletion partially removes the proposed
p
I
sequence; a deletion that leaves the
p
I
sequence intact but terminates 21 bases upstream does not interfere with cII activation of the
int
gene. From our results and the analysis of the
p
E
region, we suggest that cII acts in the promoter -35 recognition region to facilitate binding by RNA polymerase at the -10 interaction region. Differential expression of the
int
and
xis
genes results because the
p
I
transcript lacks the initiation codon for Xis protein synthesis.
Subclasses of external adenosine receptors.
Authors: Londos C , Cooper D M , Wolff J
Published: May 2006

Cell surface adenosine receptors mediate either stimulation or inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], and the receptors that mediate these different responses can be discriminated with selected adenosine analogs. 5'-N-Ethylcarboxamide-adenosine is a more potent agonist at stimulatory receptors (Ra) than is N6-phenylisopropyladenosine, whereas the reverse potency order is seen with inhibitory receptors (Ri). The potency of adenosine is intermediate between the potencies of these two analogs. The relative potencies of adenosine receptor agonists are maintained in physiological responses in intact cells, such as steroidogenesis and inhibition of lipolysis. As with adrenergic receptors, subclasses of adenosine receptors differ functionally and pharmacologically.
Homologous pairing in genetic recombination: formation of D loops by combined action of recA protein and a helix-destabilizing protein.
Authors: Shibata T , DasGupta C , Cunningham R P , Radding C M
Published: May 2006

Escherichia coli single-strand binding protein (SSB) or phage T4 gene 32 protein reduced the amount of recA protein required to catalyze the formation of D loops from double-stranded DNA and homologous single-stranded fragments. Neither SSB nor gene 32 protein alone catalyzed the formation of D loops, and excessive amounts of either protein, amounts that were sufficient to saturate the single strands, inhibited the formation of D loops completely. Both the stimulatory activity and the inhibitory activity of SSB resisted boiling, which is consistent with the known thermal stability of SSB, whereas the gene 32 protein was inactivated by heating. The formation of D loops in the presence of both recA protein and SSB required homologous DNA and ATP. Spermidine aided the combined action of SSB and recA protein in forming D loops, but Mg2+ alone was sufficient as a counterion.
Sequences of the recA gene and protein.
Authors: Sancar A , Stachelek C , Konigsberg W , Rupp W D
Published: May 2006

We have determined the nucleotide sequence of the recA gene of Escherichia coli; this permits the formulation of the primary structure for the recA protein. This structure is consistent with the amino acid composition of the tryptic peptides obtained from the recA protein. The coding region of the recA gene has 1059 base pairs, which specify 352 amino acids. The recA protein has alanine and phenylalanine as its NH2- and COOH-terminal amino acids, respectively, and has the following amino acid composition: Cys3 Asp20 Asn15 Met9 Thr17 Ser20 Glu30 Gln13 Pro10 Gly35 Ala38 Val22 Ile27 Leu31 Tyr7 Phe10 His2Lys27 Trp2 Arg14. Of the three cysteine residues, only two can be alkylated under reducing and denaturing conditions. The molecular weight of the recA polypeptide is 37,842.
Insulin-treated 3T3-L1 adipocytes and cell-free extracts derived from them incorporate 32P into ribosomal protein S6.
Authors: Smith C J , Rubin C S , Rosen O M
Published: May 2006

Differentiated 3T3-L1 preadipocytes preincubated for 60 min with 32Pi incorporate 32P into ribosomal protein S6 within 5 min after exposure to 0.1-1.0 nM insulin. Undifferentiated 3T3-L1 cells, which possess only 3-5% of the high-affinity cell surface insulin receptors present on the differentiated cells, are less sensitive to this stimulation. Under the same conditions used for insulin, epidermal growth factor, antibody to the insulin receptor, and a combination of isoproterenol and 1-methyl-3-isobutylxanthine also promote 32P incorporation into S6 in the differentiated cells although less effectively than insulin. Cell-free extracts derived from cells treated for 5-10 min with either physiological concentrations of insulin or epidermal growth factor (0.1 microgram/ml) reflect intact cells and catalyze the incorporation of 32P from exogenous [gamma-32P]ATP into ribosomal protein S6.
Identification and molecular cloning of Moloney mouse sarcoma virus-specific sequences from uninfected mouse cells.
Authors: Jones M , Bosselman R A , van der Hoorn F A , Berns A , Fan H , Verma I M
Published: May 2006

When uninfected mouse cell DNA is cleaved with restriction endonuclease EcoRI, a DNA fragment of 14.0 kilobases can be identified by hybridization to cloned DNA containing sarcoma specific sequences of Moloney mouse sarcoma virus (M-MSVsrc). The cellular DNA fragment contains the entire M-MSVsrc specific sequences. The 14.0-kilobase EcoRI DNA fragment was cloned in bacteriophage lambda. The sequence organization of a recombinant clone, lambda . MTX-1, was analyzed by restriction endonuclease mapping, nuclease S1 mapping, and electron microscopy. The results indicate that lambda . MTX-1 contains an uninterrupted stretch of 1.0 kilobase similar to that found in the M-MSV genome.
Epidermal growth factor-induced down-regulation of receptor does not occur in HeLa cells grown in defined medium.
Authors: Wolfe R A , Wu R , Sato G H
Published: May 2006

125I-Labeled epidermal growth factor (EGF) binds to HeLa cells in culture under completely defined conditions in a specific and saturable manner. When the cultures are maintained in serum, all the reported phenomena of down-regulation occur. In contrast, in cultures maintained in serum-free hormone-supplemented medium, binding is maximal after 50 min and remains constant for at least 18 hr. EGF retains its mitogenic activity in this defined system; as little as 4 pM EGF elicits a 2-fold increase in cell number in 5 days. EGF appears to be in reversible equilibrium with a constant number of surface receptors, and gel filtration analysis indicates a decrease in the rate of degradation and release of bound 125I-labeled EGF. These results indicate that EGF can retain its mitogenic activity in the absence of concomitant down-regulation.
Prenatal diagnosis of sickle cell anemia by restriction and endonuclease analysis: HindIII polymorphisms in gamma-globin genes extend test applicability.
Authors: Phillips J A , Panny S R , Kazazian H H , Boehm C D , Scott A F , Smith K D
Published: May 2006

Polymorphism for a Hpa I restriction endonuclease site associated with about 60% of beta S genes in American Blacks allows exact prenatal diagnosis of sickle cell anemia by amniocentesis in 36% of couples at risk. In three families in whom exact diagnosis by Hpa I sites was impossible, we found analysis for the presence of polymorphic HindIII sites in the G gamma and A gamma intervening sequences would allow an exact prenatal diagnosis of sickle cell status in all three. In one of these families, the presence of an A gamma HindIII site in amniocyte DNA confirmed the diagnosis (sickle cell trait) made by synthetic studies using fetal erythrocytes obtained at fetoscopy. Studies of other Black families and individuals provide evidence for linkage disequilibrium in the G gamma-A gamma-delta-beta gene complex involving the four sites, G gamma HindIII, A gamma HindIII, beta S, and Hpa I, which span 33 kilobases (kb). Ten of 14 chromosomes bearing a beta S gene in a 7.6-kb Hpa I fragment contained a G gamma but not an A gamma HindIII site, whereas 16 of 16 chromosomes bearing a beta S gene in a 13-kb Hpa I fragment lacked both the G gamma and A gamma HindIII sites. Two-thirds of beta A-bearing chromosomes lacked both G gamma and A gamma sites, whereas one-third contained either the G gamma or both G gamma and A gamma sites. These data demonstrate that combined analysis of both Hpa I and HindIII polymorphisms and verification of their linkage phase should increase the fraction of couples for whom amniocentesis can provide an exact diagnosis of sickle cell status from 36% to greater than 80%.
T-T hybridoma product specifically suppresses tumor immunity.
Authors: Nelson K , Cory J , Hellström I , Hellström K E
Published: May 2006

Culture supernatants of spleen or thymus cells from BALB/c mice bearing transplanted, syngeneic, methylcholanthrene-induced sarcomas suppress T lymphocyte-mediated lysis of cells from the tumor borne by the donor of the spleen or thymus cells. On this basis, we hybridized thymus cells from mice bearing sarcoma MCA-1490 with cells from the T lymphoma BW5147. The hybrids (hybridomas) formed were tested for production of factors that could suppress T lymphocyte-mediated lysis of MCA-1490 cells. One hybridoma, and a clone derived from it, produced factors that suppressed the lysis of MCA-1490 cells in vitro. In addition, these factors enhanced the growth of MCA-1490 in immune mice and prevented the destruction of MCA-1490 cells by immune lymphocytes in tumor neutralization (Winn) assays. In vitro lysis of cells from another MCA-induced sarcoma by immune lymphocytes was not suppressed. The suppressor factors did not affect the proliferative response of BALB/c lymphocytes to mitogens or the generation of a cytotoxic response to C57BL/6 alloantigens. Neither did they inhibit the generation of primary or secondary cytotoxic responses to murine leukemia virus-related antigens present on a BALB/c lymphoma line, LSTRA. Although our findings suggest that these suppressor factors are specific for MCA-1490, their specificity for antigens restricted to this tumor needs further definition.
Evidence for structured variation in self-renewal capacity within long-term bone marrow cultures.
Authors: Mauch P , Greenberger J S , Botnick L , Hannon E , Hellman S
Published: May 2006

Bone marrow pluripotent stem cells (CFUs) demonstrate capacity for both proliferation and differentiation. The proliferative capacity of CFUs has been measured by serial transplantability and by the Rs, a measurement of CFU production in a single 14-day transfer. In the present study, the self-renewal capacity fo both adherent and nonadherent CFUs from long-term bone marrow cultures was measured. Culture conditions were established such that nonadherent cells were derived from the adherent cell layer. Both adherent and non-adherent cells produced spleen colonies, demonstrating that significant proliferative potential was present in both locations; however, at all times in culture, the CFUs within the adherent stromal cell layer had a significantly greater self-renewal capacity than did the nonadherent CFUs. During the initial establishment of the cultures, the self-renewal capacity of the adherent CFUs decreased as the total number of CFUs per flask increased. After 3 weeks in culture, the self-renewal potential of the adherent CFUs stabilized and was maintained. These results suggest two different mechanisms of stem cell proliferation. In order to increase the most primitive stem cell pool size, there was initial proliferation of early stem cells with a concomitant decrease in self renewal capacity. Once this pool was established, the self-renewal capacity of the adherent CFUs maintained for 13 weeks in culture suggests that CFU production and cell maintenance were achieved by clonal succession.
Monoclonal antibody against a 250,000-dalton glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neutralizing antigen.
Authors: Hoffman G J , Lazarowitz S G , Hayward S D
Published: May 2006

An antibody-secreting hybrid cell line was produced by fusion of mouse myeloma cells with splenocytes from mice immunized with virions of the B95-8 strain of Epstein-Barr Virus (EBV). The monoclonal IgG antibody was shown to have anti-EBV activity by the following criteria: (i) It reacted with the membranes and the cytoplasm of seven different EBV-producing lines, but with no nonproducing line. (ii) The individual cells identified by the murine antibody were shown to be the same cells identified by a human serum having anti-EBV activity. (iii) The antibody significantly reduced the infectivity of two independent strains of EBV (namely, P3HR1K and B95-8). The antigen being recognized was characterized by immunoprecipitations of radiolabeled EBV-producer cell lysates. A single glycoprotein with an estimated molecular weight of 250,000 was identified. It is concluded that neutralization of EBV can be achieved by an IgG-class monoclonal antibody directed against a single antigenic site on a 250,000-dalton glycoprotein, which is a constituent of the EBV virion.
Epstein-Barr virus (B95-8) DNA VII: molecular cloning and detailed mapping.
Authors: Dambaugh T , Beisel C , Hummel M , King W , Fennewald S , Cheung A , Heller M , Raab-Traub N , Kieff E
Published: May 2006

Two of the Sal I fragments and all of the internal BamHI fragments (with the exception of BamHI c, a 0.6 x 10(6) dalton fragment) of Epstein-Barr virus (EBV) DNA have been cloned in pBR322. The termini and other parts of the DNA (including the EcoRI fragment which contains BamHI c) have been cloned as EcoRI fragments in bacteriophage Charon 4A. The cloned DNAs have been used to derive a complete map of the BamHI fragments of EBV DNA and to align the BamHI, EcoRI, HindIII, and SalI cleavage sites in EBV DNA.
Identification of a proviral genome associated with avian myeloblastic leukemia.
Authors: Souza L M , Komaromy M C , Baluda M A
Published: May 2006

We have identified and isolated a presumptive leukemogenic provirus from myeloblasts of a chicken in which leukemia had been induced by avian myeloblastosis virus (AMV). Leukemic myeloblasts isolated from peripheral blood or from converted yolk sac cultures of various strains of chickens, regardless of the endogenous proviral content or AMV pseudotype used for infection, contain an EcoRI 2.2-megadalton (MDaI) and a HindIII 2.6-MDal proviral fragment. A proviral genome flanked by chicken DNA sequences on either side and containing both the EcoRI 2.2-MDal and the HindIII 2.6-MDal fragments was inserted by molecular recombination into lambda phage Charon 4A and then cloned. This presumptive AMV proviral genome has a mass of approximately 4.9 MDal and contains terminal redundancies with respect to 3' viral RNA sequences.
Recombinants between avian sarcoma virus genome and chicken helper factor gene of the host cell: cloning by transfection.
Authors: Carloni G , Kaczorek M , Hill M
Published: May 2006

Chicken cells of chicken helper factor-positive (chf+) phenotype were infected with a cloned (envE-free) Rous sarcoma virus, subgroup D, and examined for the presence of parent and recombinant proviruses by transfection in chicken and turkey cells, respectively. It was found that most parent virus DNA is integrated into the host cell genome during the first 18 hr after infection, and no significant integration occurs between 18 and 72 hr after infection. On the other hand, no recombinant virus DNA was detected at 18 hr, although both unintegrated and integrated (provirus) forms of this DNA occurred 72 hr after infection. Recombination proviruses were also found in chronically virus-infected chf+ cells but not in chf- cells lacking virus-related RNA. Our results show that recombinants between the exogenous virus and endogenous chf gene can be cloned from the DNA of the host cell by transfection and suggest that a second replicative cycle of the virus is required to generate such recombinants.
Independence of the positive inotropic effect of ouabain from the inhibition of the heart Na+/K+ pump.
Authors: Godfraind T , Ghysel-Burton J
Published: May 2006

Isolated left atria from guinea pigs were stimulated at 3.3 Hz and bathed at 30 degrees C in Tyrode's solution containing 6 mM KCl. After equilibration, this solution was replaced by a low-K solution or by Tyrode's solution containing ouabain or dihydroouabain. These treatments evoked an increase in the contractility of the atria. The time to peak increase was about 30 min, and the inotropic effect was sustained for at least 40 min. After 30 min, 42K was added to the bathing solution in order to estimate the activity of the Na+/K+ pump. A linear relationship was observed between the degree of inhibition of the Na+/K+ pump and the increase in systolic tension. The regression line was the same for low-K solutions and dihydroouabain but not for ouabain. For a given degree of inhibition of the pump, ouabain evoked a higher increase in contractility. These findings indicate that inhibition of the Na+/K+ pump can be the only mechanism responsible for the positive inotropic effect of dihydroouabain but cannot be the sole mechanism for that of ouabain.
Cleavage of the Escherichia coli lexA protein by the recA protease.
Authors: Little J W , Edmiston S H , Pacelli L Z , Mount D W
Published: May 2006

The recA and lexA proteins of EScherichia coli are involved in a complex regulatory circuit that allows the expression of a diverse set of functions after DNA damage or inhibition of DNA replication. Exponentially growing cells contain a low level of recA protein, and genetic evidence suggests that lexA protein is involved in its regulation, perhaps as a simple repressor. Recent models for recA derepression after DNA damage have suggested that an early event in this process is the proteolytic cleavage of lexA protein, leading to high-level expression of recA. We present several lines of evidence that the specific protease activity of the recA protein, previously described with the lambda repressor as substrate, is capable of cleaving the wild-type lexA+ protein. First, lexA protein can be cleaved in vitro under the same conditions as prevously described for lambda repressor cleavage in a reaction requring both recA protease and ATP or an analogue, adenosine 5'-[lambda-thio]-triphosphate. Second, lexA protein can be observed in vivo as a physical entity after infection with lambda lexA+ transducing phage of host strains containing ittle or no active protease, but not in strains containing high levels of active protease. Finally, infection of host cells containing active protease with a lambda lexA+ transducing phage does not lead to repression of recA, but does so in cells lacking active protease. In all of these conditions the mutant lexA3 protein is largely resistant to inactivation or cleavage; this resistance can explain the dominant phenotype of lexA3 over lexA+. We discuss models for recA derepression and re-establishment of repression which propose that modulation of the protease activity of recA protein regulates both of these transitions.
Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences.
Authors: Van Beveren C , Goddard J G , Berns A , Verma I M
Published: May 2006

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.
A beta-like DNA polymerase activity in the slime mold Dictyostelium discoideum.
Authors: Baril E F , Scheiner C , Pederson T
Published: May 2006

Two distinct DNA polymerases (DNA nucleotidyltransferases) have been partially purified from exponentially growing cells of the slime mold Dictyostelium discoideum. The major DNA polymerase activity, DNA polymerase A, has physical and enzymatic properties similar to DNA polymerase alpha from vertebrate cells. DNA polymerase B, representing less than 10% of the polymerase activity in homogenates, is similar to DNA polymerase beta from vertebrate cells in that its activity is not inhibited by N-ethylmaleimide, aphidicolin, or KCl at a concentration below 200 mM. Like DNA polymerase beta, the activity of Dictyostelium DNA polymerase B is sensitive to 2', 3'-dideoxythymidine 5'-triphosphate. However, it differs from vertebrate DNA polymerase beta in that it chromatographs as an acidic protein and has an apparent molecular weight of 70,000. Previous reports of the absence of a beta-like DNA polymerase in D. discoideum apparently resulted from inadequate extraction and assay conditions for the enzyme. It seems quite probable from the results of this study and those of other studies (e.g., yeast and Tetrahymena pyriformis) that lower eukaryotic organisms, as well as vertebrates and prokaryotes, contain more than one form of DNA polymerase.
A cholesteryl ester transfer complex in human plasma.
Authors: Fielding P E , Fielding C J
Published: May 2006

Immunoadsorption affinity chromatography has been used to define the structure of lipoproteins in human plasma containing lecithin:cholesterol acyltransferase (EC 2.3.1.43) (LCAT) and transfer protein (apo D). The whole of LCAT was absorbed by antibodies specific for apo D and for apo A-1, indicating that the enzyme is present in plasma exclusively as a complex with its cofactor (apo A-1) and product transfer protein (apo D). About 80% of apo D (but no LCAT) was removed by antibody to apo A-2, indicating the presence of most of apo D in the form of an enzyme-free complex will apo A-1 and apo A-2. After removal of LCAT with antibody to apo D, plasma was unreactive as a substrate with isolated LCAT, but substrate activity was generated by ultracentrifugal flotation with either intact or adsorbed plasma. The apparent stoichiometry of the complex with LCAT (LCAT:apo A-1:apo D) was 1.0:0.9:1.8; that of the complex containing apo A-1, apo A-2, and apo D was 3.9:2.2:1.0.
Covalent structure of human haptoglobin: a serine protease homolog.
Authors: Kurosky A , Barnett D R , Lee T H , Touchstone B , Hay R E , Arnott M S , Bowman B H , Fitch W M
Published: May 2006

The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions.
The distal nephron of rat kidney: a target site for glucagon.
Authors: Bailly C , Imbert-Teboul M , Chabardès D , Hus-Citharel A , Montégut M , Clique A , Morel F
Published: May 2006

Glucagon-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity was measured in nine different portions of the rat nephron. Each sample contained a single piece of tubule isolated by microdissection from collagenase-treated kidney tissue. As compared to basal activity, 1 microM porcine glucagon stimulated adenylate cyclase 60-fold in the medullary portion and 40-fold in the cortical portion of the thick ascending limb, 23-fold in the early distal convoluted tubule, 11-rold in the cortical collecting tubule, and 8-fold in the medullary collecting tubule. No stimulation was observed in proximaly tubules and thin segments of the loop of Henle. Half-maximal stimulations were obtained with about 10 nM glucagon in the responsive nephron portions.
Hormonal control of human colon carcinoma cell growth in serum-free medium.
Authors: Murakami H , Masui H
Published: May 2006

A human colon carcinoma cell line, HC84S, was established in serum-supplemented medium from a colon tumor line T84 transplanted in nude mice. These cells also grew in a serum-free, synthetic medium supplement with insulin, glucagon, epidermal growth factor, transferrin, hydrocortisone, triiodothyronine, selenium, and ascorbic acid. HC84S cells grew 3 times faster in this medium than in serum-containing medium and formed gland-like structures closely resembling the original tumor morphologically. In serum-containing medium, the cells grew as a monolayer and did not form such structures. Primary cultures from transplantable human colon tumor lines maintained in nude mice and a primary tumor from a patient were established directly in this hormone-supplemented medium in collagen-treated plastic dishes without fibroblast overgrowth. The hormone-supplemented medium may be generally useful for the establishment of human colon carcinoma cell lines.
Transformation of Streptococcus pneumoniae with S. pneumoniae-lambda phage hybrid DNA: induction of deletions.
Authors: Claverys J P , Lefevre J C , Sicard A M
Published: May 2006

The genetic fate of a fragment of Streptococcus pneumoniae DNA cloned into a derivative of the Escherichia coli bacteriphage lambda has been studied in pneumococcal transformation. Transforming activity of this hybrid DNA is 8 times higher than standard S. pneumoniae DNA. Hybrid DNA is mutagenic for the recipient bacteria. Mutations are induced at a rate of 2 per 1000 transformation events. Most of these mutations are deletions adjacent to the cloned pneumococcal fragment, starting at or near its extremities and extending outside. The length of these deletions, estimated by genetic analysis or by gel electrophoresis of DNA fragments generated by restriction endonucleases, is quite variable, ranging from 150 base pairs to more than 1800 base pairs. Insertion of lambda DNA bas been detected in two large deletions by using DNAxDNA hybridization as a probe. This suggests that nonhomologous regions adjacent to the cloned fragment may be illegitimately integrated by the tranformation process. During the genetic analysis of these induced mutations we have observed that not only these deletions but also spontaneous deletions drastically increase recombination rates when present on donor DNA in transformation of neighboring markers. Such an effect is interpreted as partial exclusion of deletions from synapsis between donor and recipient DNA.
Prokaryotic histone-like protein interacting with RNA polymerase.
Authors: Lathe R , Buc H , Lecocq J P , Bautz E K
Published: May 2006

firA mutation of Escherichia coli can render RNA synthesis thermosensitive and confer abnormal sensitivity to rifampicin, an antibiotic that specifically inhibits the activity of RNA polymerase. We previously described the cloning of a chromosomal HindIII fragment containing the firA gene, and we now present strong evidence that the product of this gene is a 17,000-dalton polypeptide which, by various criteria, closely resembles the eukaryotic histones. This protein forms the largest of a unique set of three abundant histone-like proteins (HLP) found in E. coli and is hence referred to as HLPI. We discuss possible routes by which these proteins might affect transcription.
Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene.
Authors: Orkin S H , Kolodner R , Michelson A , Husson R
Published: May 2006

Restriction endonuclease mapping permitted identification of a form of beta 0-thalassemia in which a partial deletion of the beta-globin structural gene occurred [Orkin, S. H., Old, J. M., Weatherall, D. J. & Nathan, D. G. (1979) Proc. Natil. Acad. Sci. USA 76, 2400-2404]. To analyze its structure more directly, this abnormal human gene has now been cloned in bacteriophage lambda gtWES. Restriction mapping of the cloned gene and of a normal beta-globin gene contained in the phage H beta G1 confirmed previous findings regarding the presence of a deletion toward the 3' end of the gene but could not establish its position more accurately. Electron microscopy of the hybrid of the beta-thalassemia gene with globin RNA (R-loop analysis) provided unequivocal evidence for a deletion with the beta-globin structural gene. Hybridization of restriction fragments of the mutant gene with homologous fragments of H beta G1 (heteroduplex analysis) revealed a continuous, internal deletion of about 0.6 kilobase of DNA in the mutant gene and permitted its localization within the beta-globin gene region. This deletion removed the terminal third of the large intervening sequence within the beta-globin gene, the entire 3' coding block (extending from codon 105 to the end of the gene), and approximately 150 base pairs of DNA past the end of the normal globin gene.
Transformation of mammalian cells with an amplifiable dominant-acting gene.
Authors: Wigler M , Perucho M , Kurtz D , Dana S , Pellicer A , Axel R , Silverstein S
Published: May 2006

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments.
Intracisternal A-particle genes: identification in the genome of Mus musculus and comparison of multiple isolates from a mouse gene library.
Authors: Lueders K K , Kuff E L
Published: May 2006

The genome of Mus musculus contains multiple copies (500 -1000) of DNA sequences related to the 35S RNA of intracisternal type A particles (IAPs). Using labeled IAP RNA as a probe in blot-hybridization experiments, we have identified a characteristic electrophoretic pattern of reactive fragments generated by restriction endonuclease cleavage of mouse DNA. From the genomic blots, we deduced a composite restriction map for a 6.5- to 7-kilobase (kb) DNA region containing sequences homologous to the IAP RNA. Units of this type appeared to be interspersed without obvious regularity in nonhomologous flanking regions. A 5.2-kb segment of this unit was inserted directly into plasmid pBR322 from HindIII/EcoRI digest of mouse DNA. The fragment was cloned and then labeled by nick-translation and used to scan a mouse embryo gene library (average 16-kb inserts in lambda Charon 4A); 1% of the library samples hybridized, confirming the extensive reiteration of IAP genetic units. Among six different library isolates containing 6.5- to 7-kb IAP units, some restriction sites were highly conserved whereas others varied in both occurrence and position. Despite this variation, heteroduplexes between the individual isolates showed continuous IAP homology regions of 7 kb. No flanking region homologies were seen in this limited sample. Some evidence suggests that mouse DNA may contain other dispersed sequence elements related to but smaller than the genetic unit defined above.
Surface component of primate thymus-derived lymphocytes related to a heavy chain variable region.
Authors: Marchalonis J J , Warr G W , Rodwell J D , Karush F
Published: May 2006

In a study designed to determine whether T cells of man and higher primates express a surface component related to the variable region of immunoglobulin heavy chain (VH), chickens were immunized with the purified VH fragment of a monoclonal Waldenström macroglobulin. The antibody preparation reacted with a mu chain determinant contained in the Fd fragment and with individual determinants characteristic of the orginal Waldenström protein. As estimated by immunofluorescence analysis, a subpopulation of normal human peripheral T cells (approximately 30%) bound the anti-VH antibody. B-Cell lymphoma lines grown in vitro, as well as some T-cell leukemia lines of the cotton-topped marmoset (Sagiunus oedipus), also bound the anti-VH antibody. The VH-bearing component of the T-cell line 70-N-2 was labeled biosynthetically by incorporation of [3H]leucine and was precipitated specifically by anti-VH antibody. This component was characterized by an apparent mass of 70,000 daltons as assessed by polyacrylamide gel electrophoresis under reducing conditions in buffers containing sodium dodecyl sulfate. These data provide direct support for the hypothesis that some T cells express and synthesize a component related to immunoglobulin heavy chains.
Inhibitory effect of alpha-fetoprotein on the binding of myasthenia gravis antibody to acetylcholine receptor.
Authors: Brenner T , Beyth Y , Abramsky O
Published: May 2006

The binding of myasthenia gravis antibody acetylcholine receptor (AcChoR) as measured in vitro by Radioimmunoassay with 125I-labeled alpha-bungarotoxin (alpha-BuTx), can be blocked by amniotic fluid, maternal serum, and umbilical cord serum. This inhibitory effect is due to alpha-fetoprotein present in high concentrations in amniotic fluid and serum, as shown by: (i) selective removal of several components from amniotic fluid and serum; (ii) selective addition of different components present in amniotic fluid and serum, including alpha-fetoprotein, to be radioimmunoassay; (iii) correlation between the inhibitory effect of both amniotic fluid and serum and between the amounts of alpha-fetoprotein they contain; (iv) blocking of the alpha-fetoprotein in vitro suggests a similar effect in vivo in pregnant women with myasthenia gravis. This effect may explain in part the variability in the development of neonatal myasthenia gravis in the babies, due to transplacental transfer of maternal anti-AcChoR antibody, only after delivery and only in the minority of the cases. It also may explain the appearnace of remissions in females with myasthenia gravis during the second and third trimesters of pregnancy. Similar phenomena observed during pregnancy in other autoimmune and immunopathogenic diseases also might be attributed to activity of alpha-fetoprotein.
Development of chemotactic responsiveness in myeloid precursor cells: studies with a human leukemia cell line.
Authors: Fontana J A , Wright D G , Schiffman E , Corcoran B A , Deisseroth A B
Published: May 2006

We have studied the events that occur during the development of chemotaxis in HL60, a promyelocytic leukemia cell line that acquires the features of mature neutrophils when exposed to dimethylformamide (DMF). Chemotactic function first appears between 48 and 96 hr of DMF induction and is associated not only with the coincidental development of deformability, spontaneous motility, greatly increased binding of fMet-Leu-Phe, and orientation but also with decreasing cell size and pleomorphism of nuclei. Surface adhesiveness develops earlier (36-48 hr) and is coincident with a 10-fold increase in protein synthesis not seen in other DMF-inducible cell lines. This burst of protein synthesis precedes the expression of chemotactic function. These studies show that the HL60 cell line can provide a useful model for delineating control mechanisms responsible for the development of complex cellular functions present in differentiated myeloid cells in humans.
Poly(ADP-ribose) synthesis, a marker of granulocyte differentiation.
Authors: Ikai K , Ueda K , Fukushima M , Nakamura T , Hayaishi O
Published: May 2006

By using an indirect immunofluorescence technique, the distribution of poly(ADP-ribose) synthesis in human blood cells was investigated. The antibody used was reactive with poly(ADP-ribose) larger than trimers. The specific immunofluorescence of poly(ADP-ribose) synthesized in situ from NAD+ was observed in nuclei of lymphocytes and monocytes in normal peripheral blood. No immunofluorescence, however, was detected in granulocytes and erythrocytes. In agreement with this finding, no incorporation of radioactivity from [adenine-14C]NAD+ into acid-insoluble material was detectable in nuclei isolated from granulocytes. In normal bone marrow, the immunofluorescence of poly(ADP-ribose) was not observed in myelocytes or in their descendants but was observed in nuclei of lymphocytes and erythroblasts. Myelocytes and mature granulocytes in peripheral blood as well as in bone marrow of patients with chronic myelocytic leukemia were totally negative in the polymer-specific immunofluorescence. In marked contrast, prominent fluorescence was observed in nuclei of myeloblasts that appeared in peripheral blood as well as in bone marrow of patients with acute myeloblastic leukemia. Myeloblasts appearing in peripheral blood of patients in blastic crisis of chronic myelocytic leukemia also showed the nuclear immunofluorescence. These results suggest that the capacity for synthesizing poly(ADP-ribose) serves as a marker of differentiation of granulocytes, and its immunohistochemical analysis may be useful for differential diagnosis of leukemias, especially in blastic crisis.
Acetylcholine receptor site density affects the rising phase of miniature endplate currents.
Authors: Land B R , Salpeter E E , Salpeter M M
Published: May 2006

The relationship between acetylcholine receptor (AcChoR) site density (sigma) and the rising phase of the miniature endplate current was determined in esterase-inactivated lizard intercostal neuromuscular junctions. The currents were recorded by using a voltage clamp. The receptor site density was determined by electron microscope autoradiography after labeling with 125I-labeled alpha-bungarotoxin in normal endplates and in those partially inactivated with nonradioactive alpha-bungarotoxin. We found that as sigma is decreased the rise time in increased and the amplitude is decreased. These results are compatible with a previously stated "saturating disk" model, which suggests that a quantum of acetylcholine (AcCho) acts on a small postsynaptic area at saturating concentration. We conclude that in the normal neuromuscular junction the most likely number of AcCho molecules needed to open an ion channel is 2, and that the 20--80% rise time of < 100 musec is influenced both by the sigma-dependent factors such as diffusion and binding of AcCho to AcChoR and by the sigma-independent time delays such as the conformation change time to open the ion channels. From our data we calculate the lower limits to the forward rate constant of AcCho binding to AcChoR greater than or equal to 3 X 10(7) M-1 sec-1 and the diffusion constant for AcCho in the cleft greater than or equal to 4 X 10(-6) cm2 sec-1.
Positive cooperativity in a (dissected) lectin-membrane glycoprotein binding event.
Authors: Ketis N V , Girdlestone J , Grant C W
Published: May 2006

A transmembrane glycoprotein, glycophorin, was assembled into large liposomes that may be handled like intact cells. Under appropriate conditions, lectin binding to these simple model cells accurately mimics the positive cooperative behavior commonly reported for binding of various lectins to real cells. Hence we suggest that the simple observation of cooperativity in such a cell-surface recognition event does not necessarily imply an involvement of complex cellular machinery; rather, it may simply reflect interaction of a multivalent ligand with conformationally deformable headgroups.
Permissive effect of the extracellular matrix on cell proliferation in vitro.
Authors: Gospodarowicz D , Delgado D , Vlodavsky I
Published: May 2006

Corneal endothelial cells maintained in tissue culture retain the ability to synthesize and secrete an extracellular matrix (ECM) along their basal cell surface. Treatment of confluent cultures with 0.5% Triton X-100 results in the removal of the cell monolayer, thereby exposing the ECM, which adheres strongly to the tissue culture dish. Dishes coated with ECM were used to study the permissive effect of such a substrate on cell proliferation. The proliferation of bovine granulosa and adrenal cortex cells maintained on plastic tissue culture dishes was compared to that on dishes coated with ECM. Neither cell type, even when exposed to optimal serum concentration, replicated when seeded at low cell density on plastic. In contrast, when seeded on ECM they proliferated actively. None of the cultures maintained on ECM required fibroblast growth factor in order to reach confluence, although when maintained on plastic they were totally dependent on fibroblast growth factor for proliferation. Because cells maintained on plastic do not respond to factors present in serum or plasma, although they do so respond when maintained on ECM, it is likely that the close contact of the cells with the ECM restores their sensitivity to agents present in serum and plasma.
Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts.
Authors: Birchmeier C , Kreis T E , Eppenberger H M , Winterhalter K H , Birchmeier W
Published: May 2006

The distributions of both fibronectin (LETS, CSP) fibers and focal contacts to the substratum, as viewed by fluorescence and reflection contrast microscopy, respectively, have been compared in freshly plated WI-38 human fibroblasts. Most frequently, the actual focal attachment plaques did not contain fibronectin fluorescence and, furthermore, fibronectin spots and fibers often alternated with focal contacts. Overlap, however, was observed between focal contacts and the endings of actin-containing stress fibers [see also Wehland, J., Osborn, M. & Weber, K. (1979) J. Cell Sci. 37, 257-273]. Thus, the fibroblast attachment membrane might best be described as a corrugated sheet that undulates between alternating microfilaments and fibronectin fibers, at the points of closest and farthest distance to the substratum, respectively.