bluuebunny/crossref_metadata_2025 huggingface dataset:
Friend murine leukemia virus-immortalized myeloid cells are converted into tumorigenic cell lines by Abelson leukemia virus.
Authors: Oliff A , Agranovsky O , McKinney M D , Murty V V , Bauchwitz R
Published: May 2006

Friend murine leukemia virus (Fr-MuLV) is a replication-competent murine retrovirus that induces acute nonlymphocytic leukemias in NFS/n mice. Fr-MuLV disease is divided into two stages based on the ability of the leukemia cells to grow in culture and transplant into syngeneic mice. Hematopoietic cells taken from the early stage of disease after Fr-MuLV infection grow as immortal myeloid cell lines in the presence of WEHI-3 cell-conditioned medium (CM) or interleukin 3. These growth factor-dependent cell lines do not grow in culture in the absence of CM and do not form tumors in syngeneic animals. If these Fr-MuLV-infected cells are superinfected with Abelson murine leukemia virus (Ab-MuLV), they lose their dependence on WEHI-3 CM and proliferate in culture in the absence of exogenous growth factors. Concomitant with the loss of growth factor dependence in culture, the Ab-MuLV-infected cell lines become tumorigenic in syngeneic mice. This secondary level of transformation is Ab-MuLV specific. Fr-MuLV-immortalized myeloid cell lines superinfected with Harvey murine sarcoma virus (Ha-MuSV) or amphotropic virus remain dependent on WEHI-3 CM for growth in vitro and are not tumorigenic in vivo. Neither Ab-MuLV- nor Ha-MuSV-infected normal mouse myeloid cell cultures produce growth factor-independent or tumorigenic cell lines. We conclude that at least two genetic events are needed to convert a murine myeloid precursor into a tumorigenic cell line. The first event occurs in Fr-MuLV-infected mice, generating cells that are growth factor dependent but immortal in vitro. The second event, which can be accomplished by Ab-MuLV infection, converts these immortal myeloid precursors into growth factor-independent and tumorigenic cells.
Human T-lymphocyte response in vitro to synthetic peptides of herpes simplex virus glycoprotein D.
Authors: DeFreitas E C , Dietzschold B , Koprowski H
Published: May 2006

Immunization of mice with a synthetic peptide that corresponds to a murine antibody-defined immunodominant domain of herpes simplex virus (HSV) glycoprotein D (gD) induced neutralizing antibodies against HSV types 1 and 2 and protected animals against a lethal challenge with HSV type 2 (Dietzschold, B., Eisenberg, R., Ponce de Leon, M., Golub, E., Hudecz, F., Varicchio, A. & Cohen, G. (1984) J. Virol. 52, 431-435). We report here that human peripheral blood T cells from HSV-seropositive and -seronegative adult donors are activated by this synthetic peptide in vitro. Interleukin-2-dependent T-cell lines established from these cultures respond specifically to peptides containing residues 1-23 of HSV gD and to a panel of overlapping peptides within this domain. The T-cell proliferative response was maximal when the majority of interleukin-2-propagated T cells were of the helper phenotype and the peptides were at least 16 amino acids long. Peptides of 8 or 12 amino acids from the carboxyl terminus were nonstimulatory. Peptide-activated T-cell lines from sero-negative donors less than 11 years old could be established in vitro, but most cells were of the suppressor/cytotoxic phenotype and demonstrated no antigen-specificity when tested with the panel of synthetic peptides.
Is an acetylcholine transport system responsible for nonquantal release of acetylcholine at the rodent myoneural junction?
Authors: Edwards C , Dolezal V , Tucek S , Zemková H , Vyskocil F
Published: May 2006

Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the endplate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 microM), quinacrine (0.1 microM), and tetraphenylborate (1 microM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated in inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 microM) and by 42% in the presence of quinacrine (0.1-1 microM). The AcCho released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.
Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution.
Authors: Mastrangelo I A , Hough P V , Wilson V G , Wall J S , Hainfeld J F , Tegtmeyer P
Published: May 2006

Large tumor (T) antigen and its bound multimeric states are positioned by scanning transmission electron microscopy (STEM) within a few base pairs at control sequences of the simian virus 40 DNA origin of replication region. Proximal and distal edge positions for each multimer group match the end positions of previously mapped fragments protected from DNase cleavage. Since chance correspondence is shown to be extremely unlikely, STEM mass measurements, obtained concurrently with STEM map positions, indicate that the DNase fragments arise from bound monomers, dimers, trimers, and tetramers in binding region II and monomers, dimers, and trimers in binding region I. Simultaneous binding of seven monomer-equivalent masses is observed, three in region I and four in region II, with an ordered and interpretable mass distribution in the plane of the foil. Although this observation does not prove that the six G-A-G-G-C and one T-A-G-G-C sequences, similarly distributed, function as recognition sequences for T-antigen monomer, it provides strong support for such a model. The stable existence in solution of low-and intermediate-mass structures, observed at lower T-antigen concentrations, suggests a role as assembly intermediates.
Deletion of the terminus region (340 kilobase pairs of DNA) from the chromosome of Escherichia coli.
Authors: Henson J M , Kuempel P L
Published: May 2006

A strain of Escherichia coli with a 7-minute (340 kilobase pairs of DNA) deletion of the terminus region of the chromosome was isolated. This deletion was probably an IS10-promoted event and its extent was characterized by both genetic and DNA hybridization analyses. The most dramatic property of strains harboring this deletion was the absence of the sites that inhibit clockwise- and counterclockwise-traveling replication forks. These strains also grew slowly, produced many nonviable cells, were filamentous, and appeared to have an induced SOS system.
Perpetual production of hair cells and maturational changes in hair cell ultrastructure accompany postembryonic growth in an amphibian ear.
Authors: Corwin J T
Published: May 2006

Sensory hair cells are produced in the ears of birds and mammals only during early development, so that a programmed termination of hair cell proliferation leaves adult birds and mammals susceptible to irreversible deafness and balance disorders. This study reports that this is not an inherent feature of hair cells and is not shared through all the vertebrate classes. In toads (Bufo marinus) hair cells accumulate throughout life, increasing in the sacculus from approximately 400 cells at metamorphosis to more than 1600 in adulthood. In both embryonic and postembryonic ears new hair cells have been identified by scanning electron microscopy and through uptake of radioactively labeled thymidine. In the otic vesicle of postneurulation embryos there is a single primordial sensory epithelium that contains approximately 100 hair cells. Scanning electron microscopy has demonstrated that these newly formed hair cells all have stereocilia bundles that are shorter than 1.5 micron, whereas the majority of hair cells in postembryonic ears have stereocilia bundles that are at least 3 micron long. In the postembryonic sacculus the proliferation of hair cells never appears to cease, since newly produced hair cells identified by their short stereocilia have been found in a distinct peripheral growth zone at the edge of the sensory epithelium even in specimens from the oldest 1% of natural populations. This peripheral growth zone is also the site of the most frequent labeling of newly synthesized DNA in hair cells. It appears that the postembryonic enlargement of the toad sacculus occurs primarily through appositional addition of new hair cells at its edge, with few hair cells added within the existing structure of the epithelium.
Is there a single pathway for the folding of a polypeptide chain?
Authors: Harrison S C , Durbin R
Published: May 2006

We argue that folding of the compact domains of proteins can occur with adequate rapidity in the absence of a unique directed mechanism, provided that native-like local structure dominates the folding process. We further suggest that the evolution of amino acid sequences should favor multiple paths to the folded state. Existing physicochemical and mutational data are not inconsistent with a many-pathway model. The analogy of a jigsaw puzzle, with multiple routes to a unique solution, appears to be particularly apt.
Purification and crystallization of the calcium binding protein of sarcoplasmic reticulum from skeletal muscle.
Authors: Maurer A , Tanaka M , Ozawa T , Fleischer S
Published: May 2006

The calcium binding protein of skeletal muscle sarcoplasmic reticulum (also referred to as calsequestrin) was purified by release from the compartment of the vesicles with the detergent octaethyleneglycol mono-n-dodecyl ether (C12E8) and by subsequent precipitation of the calcium binding protein with specific divalent cations. The isolated protein exhibited a single band on NaDodSO4/PAGE and bound 903 nmol of Ca2+ per mg of protein. The calcium binding protein could be crystallized in the presence of Ca2+, Mg2+, Sr2+, or combinations of these three cations used in a narrow concentration range. Needle-shaped crystals of up to 500 X 50 micron were obtained. The removal of the divalent cations resulted in solubilization of the crystals. The spacings and angles of the crystals were obtained by electron microscopy using three different methods of sample preparation. By freeze-drying and negative staining electron microscopy, the spacings along axes a and b were determined to be 10-11 nm each, and the angle between the two axes was 90 degrees. By thin section electron microscopy, the spacing along axis a was 11 nm, along axis c was 15-16 nm, and the angle between the two axes was 75 degrees. This study reports (i) a simple and rapid method for purification of the calcium binding protein; (ii) conditions to crystallize the protein using Ca2+, Mg2+, Sr2+, or combinations of the three; and (iii) some preliminary characteristics of the crystals. The crystalline nature was characterized by electron microscopy and x-ray diffraction. The larger crystals diffracted beyond 3-A Bragg spacing.
Type beta transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval.
Authors: Shipley G D , Tucker R F , Moses H L
Published: May 2006

Type beta transforming growth factor/growth inhibitor (TGF-beta/GI) is demonstrated to be a potent stimulator of DNA synthesis in AKR-2B mouse embryo cells with a prolonged (greater than 24 hr) prereplicative phase when compared with other growth factors (epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor) that induce DNA synthesis 12-14 hr after stimulation. In addition, TGF-beta/GI inhibits the early peak of DNA synthesis produced by EGF and insulin before the later stimulatory effects of TGF-beta/GI become manifest. TGF-beta/GI induces a marked morphologic transformation in these cells prior to their entry into S phase. Like the other growth factors, TGF-beta/GI stimulates an early increase in the rate of protein synthesis in AKR-2B cells and its stimulatory effect on DNA synthesis is enhanced by insulin. The data show that this molecule is a growth factor for certain mesenchymal cells in monolayer culture but only after a prereplicative phase that is significantly longer than that of other growth factors.
Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay.
Authors: Sawadogo M , Roeder R G
Published: May 2006

We describe a new assay system that allows a rapid, direct, and quantitative detection of promoter-dependent in vitro transcription by RNA polymerase II. The template used is a hybrid plasmid containing the adenovirus major late promoter linked to a synthetic 400-base-pair DNA fragment that lacks cytidine residues on the transcribed strand--i.e., generates a transcript with no guanosine residues. In vitro transcriptions are carried out in the absence of GTP or, if the reactions contain GTP, in the presence of RNase T1 and the chain terminator 3'-0-methyl-GTP. Under these conditions the only RNAs that can accumulate, whether from a circular or linearized DNA template, are the 400-nucleotide RNase T1-resistant transcripts resulting from accurate initiation at the major late promoter. Thus, specific transcription can be directly monitored by conventional RNA quantitation methods. Using this fast assay, we show that three basic transcription factors, TFIIB, TFIID, and TFIIE, are absolutely required, in addition to the RNA polymerase II, for specific transcription initiation from the adenovirus major late promoter. Units of activity can be defined for each of these individual components. The applicability of this kind of assay to other systems is discussed.
Liver mRNA probes disclose two cytochrome P-450 genes duplicated in tandem with the complement C4 loci of the mouse H-2S region.
Authors: Amor M , Tosi M , Duponchel C , Steinmetz M , Meo T
Published: May 2006

A search for uncharacterized genes of the S region of the murine H-2 major histocompatibility complex was undertaken; a series of cosmid clones previously aligned by overlap hybridizations were used as radiolabeled probes. Sequences hybridizing with liver poly(A)+ RNA were found within a cosmid covering a region 3' to the C4-Slp gene (the gene encoding the hemolytically inactive isoform of the fourth component of serum complement). Radiolabeled, short cDNA complementary to liver poly(A)+ RNA was used to establish the transcriptional polarity of the newly detected gene and to define fragments containing its 3' end. DNA sequence analyses and comparisons with porcine peptides established that the gene encodes the enzyme steroid 21-hydroxylase (EC 1.14.99.10), a cytochrome P-450 often referred to as P-450(C21), whose major site of expression is the adrenal gland. Two copies of the P-450(C21) gene, very similar yet distinguishable by restriction endonuclease analysis, were found individually associated with C4 and C4-Slp, genes that encode isoforms of mouse fourth component of complement. One of the P-450(C21) genes is coamplified with C4-Slp in H-2w7, a haplotype carrying a rare elongation of the S region. Comparisons with other members of the P-450 gene family show that the P-450(C21) genes encode peptides of extraordinary evolutionary conservation. The detection of a liver transcript of P-450(C21) raises the issue of the specific metabolic role of this enzyme in this organ and may have implications for the interpretation of human congenital adrenal hyperplasia.
Metabolism of retinoic acid and retinol during differentiation of F9 embryonal carcinoma cells.
Authors: Williams J B , Napoli J L
Published: May 2006

Retinol and retinoic acid dose-response curves were obtained for promotion of the differentiation of F9 murine embryonal carcinoma cells with an enzyme-linked immunoadsorbent assay for laminin, a product of differentiated F9 cells. Retinoic acid produced a half-maximum response at 1.3 nM and a maximum response at about 30 nM; retinol was 1/175th as potent. Maximum differentiation required 48 hr of continuous exposure to retinoic acid, whereas retinol required 72 hr of exposure. The half-time of retinoic acid conversion into polar metabolites was 3.5 hr; metabolism was accelerated by pretreating F9 cells with retinoic acid. An inhibitor of oxidative metabolism, ketoconazole, decreased the rate of retinoic acid metabolism and decreased the concentration of retinoic acid required to produce a half-maximum response. Unchanged retinoic acid was the sole compound isolated from nuclei of F9 cells incubated with retinoic acid. Retinol had a half-life approximately 5-fold longer than retinoic acid, attained greater cell concentrations, and was converted into retinoic acid by F9 cells. These data indicate that retinoic acid itself directs the differentiation of F9 cells and may mediate differentiation induced by retinol.
Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome.
Authors: Jenkins F J , Casadaban M J , Roizman B
Published: May 2006

An earlier technique for insertional mutagenesis of large viral genomes involved the insertion of the thymidine kinase (TK) gene at a specific target site, cotransfection of the fragment carrying the insertion with the intact viral genome, and selection of the progeny for viral recombinants expressing the TK gene. The inserted TK gene could then be replaced by cotransfection of the recombinant DNA with fragments carrying a foreign sequence or a deletion in the target sequence. To enable the probing of larger target domains and facilitate insertional mutagenesis, we extended this technique by insertion of a 2.2-kilobase-pair (kbp) herpes simplex virus 1 (HSV-1) chimeric alpha TK gene into the 7.5-kbp mini-Mu-phage (alpha TK-mini-Mu) and lysogenized Escherichia coli with the helper Mu phage and the alpha TK-mini-Mu. Induction of phage multiplication of the lysogenized E. coli after transformation with plasmids carrying HSV-1 DNA and subsequent infection of E. coli RecA+ lysogenized with Mu phage yielded plasmid populations carrying randomly inserted alpha TK-mini-Mu DNA. Application of this procedure for insertional mutagenesis of the BamHI B fragment, which spans the junction between the unique and reiterated sequences of the L component of viral DNA, yielded two types of recombinants. Viral recombinant designated RBMu1 contained the intact alpha TK-mini-Mu inserted into unique sequences of BamHI B fragment. In recombinant RBMu2, the alpha TK-mini-Mu was inserted at or in the repeated sequences, but approximately equal to 14 kbp comprising most of the internal reiterations of the viral genome in the prototype arrangement were deleted.
Differentiation of normal marrow and HL60 cells induced by antithymocyte globulin.
Authors: Hunter R F , Mold N G , Mitchell R B , Huang A T
Published: May 2006

Antithymocyte globulin (ATG) therapy is an important treatment alternative for patients with acquired aplastic anemia. The mechanism by which it exerts its effects on hematopoiesis is unknown. In this report, we describe the ability of horse ATG to induce growth and differentiation of normal bone marrow. A single cell suspension of normal human bone marrow was cultured in methylcellulose medium and examined for the growth and maturation after incubation with ATG (10 micrograms/ml). After 3-4 days of culture, spherical colonies containing mature myeloid elements were found in cultures containing ATG but not in cultures containing medium or preimmunization horse IgG. The addition of 10% colony-stimulating factor increased growth by 40%. The number of spherical colonies is not dependent on the presence of macrophages or T lymphocytes. This property of ATG may be relevant to the mechanism behind the hematologic recovery in some patients with acquired aplastic anemia. We also describe the ability of ATG to induce terminal differentiation in the HL60 leukemic cell line. ATG binds to HL60 cells and at concentrations between 10 and 100 micrograms/ml, 50% of the cells become mature granulocytes, acquire the ability to reduce nitroblue tetrazolium, and lose their proliferative capacity in the clonogenic assay. These new observations of ATG-induced differentiation of normal marrow myeloid elements and terminal differentiation of the HL60 cell line point to different avenues for future search of differentiation-inducing agents.
Phosphatidylcholine produced in rat synaptosomes by N-methylation is enriched in polyunsaturated fatty acids.
Authors: Tacconi M , Wurtman R J
Published: May 2006

Rat brain synaptosomes contain an enzyme, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), that catalyzes the methylation of phosphatidylethanolamine to form its mono-, di-, and trimethyl (phosphatidylcholine) derivatives. Synaptosomal phosphatidylethanolamine is much richer in polyunsaturated fatty acids (43.4%) than is synaptosomal phosphatidylcholine (4.6%). It thus seemed possible that the phosphatidylcholine derived via the N-methylation of phosphatidylethanolamine might also be especially enriched in polyunsaturated fatty acids. To test this hypothesis, we examined the incorporation of [3H]methyl groups into various molecular species of phosphatidylcholine, by incubating rat synaptosomes for 10, 30, or 90 min in a medium containing S-adenosyl[methyl-3H]methionine. Phosphatidylcholine was extracted and separated from other lipids by TLC, after which its molecular species were isolated by argentation TLC (which distinguishes among the phospholipid molecules by the degree of unsaturation of their fatty acid moieties.) We found that approximately 65% of the [3H]methyl incorporated into phosphatidylcholine during the incubation period was present in the fraction associated with pentaene or hexaene fatty acids; an additional 30% was present in the tetraene fraction, while the remaining phosphatidylcholine radioactivity was distributed between the dienes and monoenes. Similar distributions were observed among synaptosomes incubated for 10 or 30 min; however, after 90 min the phosphatidyl[3H]choline contained proportionately less of the tetraenes. These observations indicate that neuronal phosphatidylcholine molecules formed via N-methylation are especially richer in polyunsaturated fatty acids, and they raise the possibility that these molecules constitute a distinct pool with particular physiologic functions.
Unusual apparently constitutive interferons and antagonists in human placental blood.
Authors: Duc-Goiran P , Robert-Galliot B , Lopez J , Chany C
Published: May 2006

We have detected seemingly uninduced interferons (IFNs) in 29/37 human placental samples obtained during caesarian sections at different periods of pregnancy, mostly around the 37th week. The amounts were usually low and did not enable us to correlate our findings with any physiological or pathological conditions. Occasionally the presence of IFN was masked by a lectin-like antagonist. Therefore, in a number of cases, substantially higher amounts of IFN were found after purification by affinity chromatography using concanavalin A, Cibacron blue, or antiserum to IFN-alpha, each coupled to Sepharose. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of IFN-alpha and IFN-beta with molecular masses between 15 and 80 kilodaltons. Some of the high molecular weight components were neutralized either only by monospecific antiserum to IFN-alpha or, to the same extent, by antiserum to IFN-alpha or to IFN-beta, reminiscent of those previously reported after viral induction in the human amniotic membrane. We postulate that both IFNs and antagonist play a physiological role during fetal development.
Malignant transformation of Bloom syndrome B-lymphoblastoid cell lines by carcinogens.
Authors: Shiraishi Y , Yosida T H , Sandberg A A
Published: May 2006

Three types of Bloom syndrome B-lymphoblastoid cell lines, as well as one derived from a normal person, treated with 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine (0.3 micrograms/ml for 24 hr), were studied for tumorigenicity in nude mice, colony formation in soft agar, cytogenetic changes, and immunoglobulin markers. When normal and Bloom syndrome cells with normal sister chromatid exchange (SCE) levels and karyotypes (type I) were treated with carcinogens, no significant changes occurred in the immunoglobulin profile and karyotype, only rare colony formation was seen, and no tumors were produced. In contrast, when Bloom syndrome cells with high SCE levels (type II with normal karyotype and type III with an abnormal karyotype) were treated with carcinogens, tumors were produced in 22 of 53 nude mice injected; a high rate of colony formation in soft agar was seen; the cells exhibited virtual loss of immunoglobulin markers; and structural changes in chromosomes 1, 2, 3, 4, 5, 7, 11, 14, and 15 were found in the tumors in addition to the original chromosome abnormalities present in the injected cells. It appears that Bloom syndrome B-lymphoblastoid cell lines with high levels of SCE are highly susceptible to the action of carcinogens, as evidenced by tumor formation in nude mice and colony formation in agar. Apparently, the carcinogens were capable of transforming only those cells that had a critical level of SCE (approximately 140 per cell) and not those with only mildly increased levels (approximately 13 per cell).
Selective alkylation of carcinogenic 9-anthryloxirane at the N-3 position of adenine in DNA.
Authors: Yang N C , Chang C W
Published: May 2006

Carcinogenic 9-anthryloxirane binds covalently to calf thymus DNA and poly(dA-dT). Application of the technique for DNA sequence determination shows that acid cleavage of the modified DNA frees approximately half of the anthryl groups from the DNA. HPLC analysis indicates that an adenine adduct and the glycol derived from 9-anthryloxirane are the major acid-labile products. Spectroscopic analyses establish that the adenine adduct is the N-3 adduct of 9-anthryloxirane to adenine. Similar analyses of modified poly(dA-dT) indicate that the binding of 9-anthryloxirane takes place selectively at the N-3 position of adenine. The significance of this finding is briefly discussed.
Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair.
Authors: Karran P
Published: May 2006

Mex+ human lymphoma cell lines contain O6-methylguanine-DNA methyltransferase, a DNA repair enzyme that undergoes suicide inactivation on interaction with its substrate. The cells are therefore competent to remove the alkylation lesion O6-methylguanine from their DNA. However, several repair-deficient lymphoma cell lines (Mex-) are also known. It is shown here that Mex+ cells can be converted temporarily to a Mex- phenotype by growth in nontoxic concentrations of free O6-methylguanine. The depletion of methyltransferase activity is not a result of O6-methylguanine incorporation into DNA and subsequent demethylation by the enzyme. It is proposed that O6-methylguanine is mistakenly incorporated into tRNA molecules by means of a post-transcriptional ribosyl transfer reaction. The demethylation of such bases in tRNA has been demonstrated by using bacterial and human DNA repair enzymes. The existence of such a subversive repair of a methylated base in tRNA raises the possibility of competition between DNA and RNA for cellular DNA repair enzymes. Furthermore, it is proposed that the known aberrant methylation of tRNA in certain transformed cells, together with subversive tRNA repair, could account for the Mex- phenotype.
Structural similarities between the mammalian beta-adrenergic and reovirus type 3 receptors.
Authors: Co M S , Gaulton G N , Tominaga A , Homcy C J , Fields B N , Greene M I
Published: May 2006

The mechanism by which viruses bind to and infect specific tissues to cause disease has only recently begun to be understood. The mammalian reoviruses provide an especially attractive model for studying the details of cell surface recognition. The cell and tissue tropism of reovirus is determined by a portion of the viral hemagglutinin termed the neutralization domain. We have reported previously on the generation of both monoclonal and polyclonal anti-idiotypic antibodies that mimic the viral hemagglutinin in the specificity of binding to the reovirus receptor. By using these anti-idiotypic antibodies as specific probes, we have successfully isolated the mammalian reovirus receptor from neuronal and lymphoid cells. In the present study, we report that the reovirus receptor is structurally similar to the mammalian beta-adrenergic receptor. This conclusion is based on the following observations: (i) purified beta-adrenergic receptor is immunoprecipitable by anti-reovirus receptor antibody; (ii) purified reovirus receptor obtained from murine thymoma cells and beta-adrenergic receptor obtained from calf lung exhibit identical molecular masses and isoelectric points; (iii) trypsin digests of purified reovirus and beta-adrenergic receptors display indistinguishable fragment patterns; (iv) purified reovirus receptor binds the beta-antagonist [125I]iodohydroxybenzylpindolol and this binding is blocked by the beta-agonist isoproterenol.
Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin.
Authors: Smith F R , Ackers G K
Published: May 2006

Tetrameric human hemoglobin can assume ten molecular forms that differ in the number and configuration of ligands bound at the four heme sites. For each of these species we have determined the cooperative free energy--i.e., the deviation in free energy of ligation from that which would obtain for the same sites binding as independent alpha and beta subunits. These cooperative free energies were resolved from measurements on the dissociation into dimers of tetramers in which each subunit is either unligated (Fe2+ deoxy) or "ligated" by conversion into the cyanomet form (Fe3+ CN). The results indicate that each hemoglobin tetramer acts as a three-level molecular switch. During the course of ligation, the total cooperative free energy (6 kcal/mol over all four binding steps) is expended in two transitions that are synchronized with particular ligation steps. Whether a cooperative energy transition occurs or not depends upon how the ligation step changes both the number and configuration of ligated subunits. The hemoglobin tetramer is thus a "combinatorial switch." The finding of three distinct free energy levels for the ten ligation states suggests the existence of three major structural forms of the hemoglobin tetramer.
Variable stoichiometry in active ion transport: theoretical analysis of physiological consequences.
Authors: Johnson E A , Tanford C , Reynolds J A
Published: May 2006

Active ion transport systems with fixed stoichiometry are subject to a thermodynamic limit on the ion concentration gradients that they can generate and maintain, and their net rates of transport must inevitably decrease as this limit is approached. The capability to vary stoichiometry might thus be physiologically advantageous: a shift to lower stoichiometry (fewer ions pumped per reaction cycle) at increasing thermodynamic load could increase the limit on the supportable concentration gradient and could accelerate the rate of transport under high-load conditions. Here we present a theoretical and numerical analysis of this possibility, using the sarcoplasmic reticulum ATP-driven Ca pump as the example. It is easy to introduce alternate pathways into the reaction cycle for this system to shift the stoichiometry (Ca2+/ATP) from the normal value of 2:1 to 1:1, but it cannot be done without simultaneous generation of a pathway for uncoupled leak of Ca2+ across the membrane. This counteracts the advantageous effect of the change in transport stoichiometry and a physiologically useful rate acceleration cannot be obtained. This result is likely to be generally applicable to most active transport systems.
P
-element distribution in Eurasian populations of
Drosophila melanogaster
: A genetic and molecular analysis
Authors: Anxolabéhère Dominique , Nouaud Danielle , Périquet Georges , Tchen Paul
Published: May 2006

Genetic and molecular investigations were carried out with Eurasian
Drosophila melanogaster
populations on the
P-M
system of hybrid dysgenesis. In 27 strains sampled from France to Middle Asia, a clear gradient exists between Western Europe, in which most modern strains are of the
Q
type, and eastern areas, in which
M
-cytotype strains predominate. Molecular analysis on individual flies was performed with two complementary probes of the cloned 2.9-kilobase
P
element. The results provide evidence for a gradually decreasing frequency of
P
elements from west to east, but the presence of
P
-homologous sequences has been ascertained in all of the wild
M
-cytotype populations analyzed. Moreover, some active
P
elements with
GD
sterility potential were revealed in the majority of
M
-cytotype populations when tested with a highly sensitive reference line. The gradual change in distribution of the polymorphic
P
family in Eurasia is discussed in relation to the structure of the elements together with the theories of
P-M
evolution and is interpreted as the present invasion of Eurasian populations by these elements.
A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion.
Authors: Baird A , Mormède P , Ying S Y , Wehrenberg W B , Ueno N , Ling N , Guillemin R
Published: May 2006

The addition of fibroblast growth factor (FGF) to primary cultures of rat anterior pituitary cells modifies their response to thyrotropin-releasing factor in a dose-dependent manner. While the pituitary response to the other releasing factors (corticotropin-releasing factor, growth hormone-releasing factor, and gonadotropin-releasing factor) is not altered, FGF increases both the sensitivity of the cells to thyrotropin-releasing factor and the amounts of prolactin and thyrotropin released. A minimum of 24 hr of preincubation with FGF is required to modify the pituitary response, and maximal effects were observed with 48 and 72 hr of preincubation. The effective doses of FGF are similar to those described for its mitogenic activity (i.e., 1-100 pM), but inhibition of cell growth with 5-fluorodeoxyuridine does not modify the effect of FGF on thyrotropin and prolactin release. These results suggest a novel paracrine, if not autocrine, role of pituitary FGF in the homeostatic mechanisms that regulate the secretion of prolactin and thyrotropin. They also suggest that the biological significance of the presence of FGF in various tissues may not be directly related to its in vitro mitogenic activity.
Outlining social physics for modern societies—locating culture, economics, and politics: The Enlightenment reconsidered
Authors: Iberall A. S.
Published: May 2006

A groundwork is laid for a formulation of the modern human social system as a field continuum. As in a simple material physical field, the independent implied relationships of materials or processes in flux have to be based on local conservations of mass, energy, and momentum. In complex fields, the transport fluctuations of momentum are transformed into action modes (e.g., [unk]
pdq
= Σ

H
i

=
H
, a characteristic quantum of action over a characteristic cycle time). In complex living systems, a fourth local conservation of population number, the demographic variable, has to be added as a renormalized variable. Modern man, settled in place via agriculture, urbanized, and engaged largely in trade and war, invents a fifth local conservation—value-in-trade, the economic variable. The potentials that drive these five fluxes are also enumerated. Among the more evident external and internal physical-chemical potentials, the driving potentials include a sheaf of internal potential-like components that represent the command-control system emergent as politics.
In toto
, culture represents the social solvent with the main processes of economics and politics being driven by a social pressure.
Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide.
Authors: Okamura T , John M E , Zuber M X , Simpson E R , Waterman M R
Published: May 2006

Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services--namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11 beta-hydroxylase cytochrome P-450.
Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle.
Authors: Scott J D , Fischer E H , Takio K , Demaille J G , Krebs E G
Published: May 2006

The amino acid sequence of rabbit skeletal muscle heat-stable inhibitor of the cAMP-dependent protein kinase has been determined by microsequencing techniques. Proof of the structure involved a series of nonoverlapping tryptic fragments for primary identification of 86% of the amino acids. Complementary fragments generated by cleavage with chymotrypsin, Staphylococcus aureus V8 proteinase, and mast cell proteinase II contributed to proof of the structure. The inhibitor is a single polypeptide chain of 75 residues and has a molecular weight of 7829. It lacks tryptophan, proline, and sulfur-containing amino acids. The amino terminus of the inhibitor is blocked by an unidentified group. The amino-terminal region of the molecule contains the kinase inhibitory domain, and synthetic peptides based on the sequence of residues 11-30 are potent competitive inhibitors of the cAMP-dependent protein kinase [Scott, J. D., Fischer, E. H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383]. Residues 14-22 show considerable homology to the "hinge-regions" of the regulatory subunits of the cAMP-dependent protein kinase. The remainder of the molecule shows no similarity to the known amino acid sequence of any protein.
Chromosome localization of the gene for human terminal deoxynucleotidyltransferase to region 10q23-q25.
Authors: Isobe M , Huebner K , Erikson J , Peterson R C , Bollum F J , Chang L M , Croce C M
Published: May 2006

Complementary DNA clones representing the 3' half, the 5' half, and the entire coding region of the human terminal deoxynucleotidyltransferase gene (TdT; DNA nucleotidylexotransferase, nucleosidetriphosphate: DNA deoxynucleotidylexotransferase, EC 2.7.7.31) were used to screen a panel of mouse X human somatic cell hybrid DNAs to determine the chromosomal location of the human TdT gene. The results of the Southern transfer analysis of hybrid DNAs indicate that the gene for TdT is located on human chromosome 10. The in situ hybridization technique was then used to further localize the gene for TdT to region q23-q25 of human chromosome 10.
Control of yeast alpha-specific genes: evidence for two blocks to expression in MATa/MAT alpha diploids.
Authors: Ammerer G , Sprague G F , Bender A
Published: May 2006

In yeast alpha cells, the product encoded by the MAT alpha 1 gene of the mating-type locus is required for transcription of at least two genes, STE3 and MF alpha 1. To learn whether the lack of the MAT alpha 1 product in a and a/alpha cells is sufficient to explain the failure to express STE3 and MF alpha 1 in these cells, we have provided MAT alpha 1 product via a hybrid gene that circumvents the normal regulation of the MAT alpha 1 gene. We find by RNA blot analysis that provision of MAT alpha 1 protein permits production of STE3 and MF alpha 1 mRNA in a cells but not in a/alpha cells. These data suggest the existence of an additional regulatory mechanism that prevents expression of alpha-specific genes in a/alpha cells, even when MAT alpha 1 product is present. This regulatory mechanism appears to control expression of STE3 and MF alpha 1 at the transcriptional level rather than at the posttranscriptional level, because we show that MF alpha 1 mRNA supplied from a constitutive promoter is translated and processed in a/alpha cells to yield functional alpha-factor pheromone. This result shows further that a/alpha cells possess all the machinery necessary for pheromone maturation, even though these cells do not normally carry out these reactions.
Mini-F plasmid-induced SOS signal in Escherichia coli is RecBC dependent.
Authors: Bailone A , Sommer S , Devoret R
Published: May 2006

Dispensable replicons such as F plasmid [95 kilobases (kb)] or its mini-derivatives such as mini-F (9.3 kb) or lambda mini-F efficiently induced cellular SOS genes such as sfiA (sulA) when they were damaged by UV irradiation and then introduced into a recipient bacterium. To generate an SOS signal, UV light-damaged mini-F or mini-F conditional mutants deficient in replication required that the bacterial RecBC enzyme retained some activity different from the nuclease activity that was dispensable. In contrast, UV light-damaged F plasmid produced an SOS signal independently of the activity of the RecBC enzyme and of the expression of the mini-F, -H, and -G proteins. Our findings are consistent with a picture in which the SOS signal is constituted by stretches of single-stranded DNA on a replicon. Moreover, our present data combined with other data previously published lead to the hypothesis that the SOS signal induced by mini-F plasmid is located in trans on the host chromosome, whereas the one generated by UV light-damaged F plasmid is in cis on the transferred DNA.
Changes in cell volume measured with an electrophysiologic technique.
Authors: Reuss L
Published: May 2006

Epithelial cells of the gallbladder of Necturus maculosus were loaded with tetramethylammonium (Me4N+) by transient exposure of the apical (lumen-facing) surface to a solution of high Me4N+ concentration containing also the polyene antibiotic nystatin. Upon removal of nystatin, in the continued presence of Me4N+, spontaneous restoration of the native ionic permeability of the apical cell membrane was observed. At this time, external Me4N+ was removed; intracellular [Me4N+] measured with ion-sensitive microelectrodes was 2-15 mM and remained unchanged for several hours. Changes in cell volume were estimated from the changes in intracellular [Me4N+] produced by alterations in the osmolality of the mucosal bathing solution. Assuming that such changes are caused entirely by water fluxes across the apical membrane, the minimum value of its hydraulic permeability coefficient (Lp) was 1-3 X 10(-3) cm.sec-1.(osmoles/kg)-1, suggesting that an osmolality difference across the apical membrane as small as 1-3 milliosmoles/kg could explain the average rate of transepithelial water transport. These results agree with optical measurements [Persson, B. O. & Spring, K. R. (1982) J. Gen. Physiol. 79, 481-505]. The effective thickness of the apical unstirred layer was estimated from the time courses of both the apical membrane voltage and the response of an extracellular K+-sensitive microelectrode to an increase in [K+] in the mucosal bath. Since changes in concentration of the osmotically active solute at the membrane surface were thus shown to be significantly delayed by diffusion, the Lp value, calculated assuming a step-change in osmolality, is an underestimate.
Transcription of the human hsp70 gene is induced by serum stimulation.
Authors: Wu B J , Morimoto R I
Published: May 2006

We have examined the expression of the heat shock protein (hsp70) gene in human cells. The transcription of the hsp70 gene and accumulation of cytoplasmic hsp70 mRNA is induced by serum stimulation. Populations of HeLa cells and human embryonic kidney cells (cell line 293) were serum starved. Upon serum stimulation, the level of hsp70 mRNA transiently increases between 12 and 18 hr to a 10-fold higher level. The increased levels of hsp70 mRNA can be accounted for by a 10- to 15-fold increase in the rate of transcription of the hsp70 gene. When cells were serum-stimulated in the presence of an inhibitor of DNA synthesis, 1-beta-D-arabinofuranosylcytosine (araC), the levels of hsp70 mRNA were induced to only 20% of the maximal level detected in the absence of the inhibitor. This suggests that the expression of the hsp70 gene is coupled to DNA synthesis. The cloned human hsp70 gene contains regulatory sequences that confer serum-stimulated transcriptional control. The endogenous hsp70 gene and the transfected chimeric gene containing sequences upstream of the hsp70 gene fused to bacterial chloramphenicol acetyltransferase are both temporally expressed in stable transfectants of cell line 293 cells. The endogenous hsp70 mRNA and the chimeric mRNA reach maximum levels 12-18 hr after serum stimulation.
Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium.
Authors: Widdicombe J H , Welsh M J , Finkbeiner W E
Published: May 2006

The tracheal mucosa from a 12-year-old girl was digested with collagenase 4 hr after her death from cystic fibrosis. Forty million viable cells were obtained. The cells, plated at 10(6) per cm2 onto four Nuclepore filters coated with human placental collagen, formed confluent monolayers after 1 day. Their ultrastructure was similar to that of normal human cells. They were studied in conventional Ussing chambers or with intracellular microelectrodes on days 5-7 after plating. The monolayers displayed resistance of 380 +/- 50 omega X cm2 and short-circuit current (Isc) of 1.8 +/- 0.4 microA X cm-2. This resistance is similar to that obtained for dog or normal human monolayers. The Isc is less than normal human (approximately 3 microA X cm-2) or dog (approximately 10 microA X cm-2) cells. The cystic fibrosis cells resembled normal monolayers in that serosal ouabain and mucosal amiloride inhibited Isc, while mucosal ouabain or serosal amiloride had no effect. They differed from normal human or dog cells in that Isc was not inhibited by bumetanide and the stimulation of Isc by isoproterenol or prostaglandin E2 was greatly reduced or abolished. Addition of isoproterenol depolarized apical membrane potential (psi a) and decreased fractional resistance (fR) in normal human and dog but had no effect on psi a or fR in cystic fibrosis cells. Reduction of mucosal chloride from 120 to 5 mM by replacement with gluconate increased fR of dog and normal human monolayers and depolarized psi a by 22 (dog) or 30 (human) mV. In cystic fibrosis monolayers, chloride replacement hyperpolarized psi a by 2 mV and had little effect on fR. These results suggest that the primary defect in cystic fibrosis is reduced apical membrane chloride conductance.