bluuebunny/crossref_metadata_2025 huggingface dataset:
Methyl-directed repair of frameshift mutations in heteroduplex DNA.
Authors: Dohet C , Wagner R , Radman M
Published: May 2006

DNA heteroduplexes with single unpaired bases of the four different kinds were prepared by annealing separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli. Genetic analysis of the progeny phages obtained from transfected bacteria indicates that the E. coli mismatch repair system can recognize and repair heteroduplexes with single unpaired bases--i.e., frameshift/wild-type heteroduplexes. The repair of a particular strand of the heteroduplex is inhibited by full methylation of the adenines in the GATC sequences of that strand. Thus, it appears that the E. coli mismatch repair system can act on newly synthesized DNA strands to remove replication errors involving the insertion or deletion of a single base.
Monoclonal antibodies to HLA-DP-transfected mouse L cells.
Authors: Heyes J , Austin P , Bodmer J , Bodmer W , Madrigal A , Mazzilli M C , Trowsdale J
Published: May 2006

Mouse L cells transfected with human HLA-DP (DPw4) alpha and beta genes were used to make monoclonal antibodies in C3H mice. A polymorphic antibody, DP11.1, was obtained, as well as several monomorphic antibodies. In ELISAs, DP11.1 bound to DPw4 cells and, more weakly, to DPw2, but not DPw1, -3, -5, or -6, using HLA homozygous cells. It also bound to L-cell transfectants expressing either DPw2 or DPw4 products. From B lymphoblastoid cell lysates labeled with [35S]methionine, the antibody immunoprecipitated alpha and beta chains of a similar size to those precipitated by a well-characterized DP monoclonal antibody, B7/21.2. Immunoblotting indicated that the DP11.1 antibody was directed against the alpha chain. This result confirms partial sequence data that showed that the DP alpha chain, as well as DP beta, is polymorphic, and that DPw2 and -4 alpha chains are very similar, if not identical.
Brain-specific hyaluronate-binding protein: an immunohistological study with monoclonal antibodies of human and bovine central nervous system.
Authors: Bignami A , Dahl D
Published: May 2006

Hyaluronectin is a protein isolated from acid extracts of human brain by affinity chromatography on immobilized hyaluronate. With polyclonal antibodies, it was immunohistologically localized in the rat at the nodes of Ranvier of central and peripheral myelinated fibers and in mesenchymal tissues. Compared to adult rat, hyaluronectin-immunoreactive material was more abundant in embryonal rat brain and mesenchyma. We report a different localization in human and bovine tissues with monoclonal antibodies reacting with human hyaluronectin by NaDodSO4/PAGE and immunoblotting but not staining rat tissues by immunohistology. In human and calf the antigen reacting with hyaluronectin monoclonal antibodies was brain specific, while several peripheral tissues were stained by the polyclonal antibodies. In human and bovine central nervous system monoclonal antibodies stained white matter and tissues formed predominantly by glial fibers (e.g., subependymal glia). In white matter hyaluronectin-immunoreactive material formed a delicate mesh surrounding individual myelinated fibers, a pattern compatible with the distribution of fine astroglial processes in this location. Gray matter did not stain with monoclonal antibodies, the granular layer of the cerebellum excepted. The findings suggest that human hyaluronectin is heterogeneous and comprises at least two fractions. The main fraction is a brain-specific protein, probably produced by white matter astrocytes. Another fraction cross-reacting with rat is more abundant in embryonal tissues, including mesenchyma and brain.
Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution.
Authors: Brenner B , Eisenberg E
Published: May 2006

Crossbridge models of muscle contraction based on biochemical studies predict that there may be a relationship between the rate-limiting step in the actomyosin ATPase cycle in vitro and the rate of force development in vivo. In the present study, we measured the rate of force redevelopment in skinned rabbit muscle fibers following unloaded isotonic shortening and a rapid restretch. For comparison, ATPase activity was measured under identical conditions, using myosin subfragment-1 chemically crosslinked to actin. We found that the time course of force redevelopment is well fitted by a single exponential function, implying that force redevelopment is a first-order process, described by a single rate constant. The magnitude of this rate constant is in close agreement with the rate constant necessary to simulate the experimental force-velocity relation on the basis of a crossbridge model of the type proposed by A. F. Huxley in 1957. In addition, the observed close correlation between the rate constant for force redevelopment and the maximal actin-activated actomyosin ATPase rate under a variety of conditions suggests that the step that determines the rate of force generation in the crossbridge cycle may be the physiological equivalent of the rate-limiting step in the actomyosin ATPase cycle in solution.
The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase.
Authors: Honess R W , Bodemer W , Cameron K R , Niller H H , Fleckenstein B , Randall R E
Published: May 2006

Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.
Pantothenate is required in Neurospora crassa for assembly of subunit peptides of cytochrome c oxidase and ATPase/ATP synthase.
Authors: Brambl R , Plesofsky-Vig N
Published: May 2006

One polypeptide subunit of cytochrome c oxidase (EC 1.9.3.1) and two subunits of the ATPase/ATP synthase (EC 3.6.1.34) in mitochondria of Neurospora crassa are covalently modified with a derivative of pantothenic acid. In asexual spores of a pantothenate auxotroph of Neurospora, deprivation of pantothenic acid blocked the increase of the specific activities of cytochrome c oxidase and the ATPase above the basal activities in the dormant spores. Under cellular panthothenate deprivation, all the subunit peptides of these two enzymes apparently were synthesized and accumulated in the mitochondria, but these subunits were not assembled into normal complexes, and 55Fe-labeled heme a was incorporated into immunoprecipitable cytochrome c oxidase to a very low extent. In pantothenate-supplemented cells, the pantothenate derivative apparently is attached to the free unassembled subunits and appears not to be present in the assembled enzymes. It is likely that cellular deprivation of pantothenate, resulting in failure to modify the three subunit peptides, causes an interruption of the assembly pathway of cytochrome c oxidase and the ATPase/ATP synthase.
Redox pathways in electron-transfer proteins: correlations between reactivities, solvent exposure, and unpaired-spin-density distributions.
Authors: Tollin G , Hanson L K , Caffrey M , Meyer T E , Cusanovich M A
Published: May 2006

The relative reactivities toward reduction by free flavin semiquinones of cytochromes (c-type cytochromes, cytochrome b5, c'-type cytochromes) iron-sulfur proteins (high-redox-potential ferredoxins, rubredoxins, low-redox-potential ferredoxins), and blue copper proteins (plastocyanin, azurins) are shown to correlate with calculations of the solvent exposure of the various prosthetic groups. In the case of the c-type cytochromes, one of the major centers of exposure is the sulfur atom of the thioether bridge that covalently links heme ring C to the protein. Charge-iterative extended Hückel calculations on a heme c model indicate that both porphyrin pi and Fe(III)d pi orbitals can delocalize onto the bridging sulfur atom. Unpaired spin densities are comparable to those obtained for individual aromatic porphyrin ring carbon atoms. Thus, the exposed sulfur of ring C may act to facilitate electron transfer.
Rat transferrin gene expression: tissue-specific regulation by iron deficiency.
Authors: Idzerda R L , Huebers H , Finch C A , McKnight G S
Published: May 2006

Rats raised on a low-iron diet were used as a model system for investigating the regulation of transferrin gene expression by iron deficiency. We quantitated transferrin mRNA in a variety of tissues from normal and iron-deficient rats and found that the level of transferrin mRNA in normal rat liver was about 6500 molecules per cell, while the level in iron-deficient animals was 2.4-fold higher. The increase of transferrin mRNA in iron deficiency was the result of a specific induction of transferrin gene transcriptional activity as measured in isolated nuclei. This increase in transferrin gene expression resulted in a corresponding increase in serum total-iron-binding capacity. Of the other tissues examined, moderate amounts of transferrin mRNA were found in brain (83 molecules per cell) and testis (114 molecules per cell), and low levels were measured in spleen and kidney. The transferrin mRNA content of brain, testis, spleen, and kidney remained unchanged in iron deficiency. The small intestine had no detectable transferrin mRNA in either normal or iron-deficient rats; however, transferrin protein was present, and its level was 2-fold higher in the iron-deficient group. We hypothesize that intestinal transferrin is synthesized in the liver and is delivered to the gut via the bile. Consistent with this idea, bile transferrin content was found to be elevated in iron deficiency and appeared to be sufficient to account for intestinal transferrin levels. In addition, treatment of plasma transferrin with bile caused an acidic shift in its isoelectric-focusing behavior so that it comigrated with intestinal transferrin.
Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.
Authors: Itoh H , Kozasa T , Nagata S , Nakamura S , Katada T , Ui M , Iwai S , Ohtsuka E , Kawasaki H , Suzuki K
Published: May 2006

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.
Adipocyte P2 gene: developmental expression and homology of 5'-flanking sequences among fat cell-specific genes.
Authors: Hunt C R , Ro J H , Dobson D E , Min H Y , Spiegelman B M
Published: May 2006

We have isolated the mouse gene encoding adipocyte P2, aP2, the differentiation-dependent adipocyte protein homologous to myelin P2. The aP2 gene is present in a single copy in the mouse and is present in single or few copies in species from human to Drosophila. The entire gene spans 4 kilobases and consists of four exons encoding 25, 57, 34, and 16 amino acids; the overall exon structure is similar to the gene encoding liver fatty acid binding protein. A plasmid vector was constructed containing the entire aP2 gene with flanking sequences, modified by linker insertion. When this gene is stably introduced into 3T3-F442A cells, it is expressed only upon adipose differentiation, with a time course of induction very similar to that of the endogenous aP2 gene. We have compared the DNA sequence of the 5'-flanking region of the aP2 gene to the promoter regions of two other genes activated during adipocyte differentiation, glycerol-3-phosphate dehydrogenase and adipsin, and find a 13-base region of homology (Formula: see text) present in multiple copies in the 5'-flanking region of each gene. An adjacent 15-base sequence is present only in glycerol-3-phosphate dehydrogenase and aP2 genes. Both of these elements share homology with putative viral enhancer core sequences. These results indicate that the aP2 gene contains sequence information necessary for differentiation-dependent expression in fat cells; common elements shared by adipocyte-specific genes may play a role in this process.
Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene.
Authors: Brotcorne-Lannoye A , Maenhaut-Michel G
Published: May 2006

Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously.
Control of hemopoiesis in mice by sensitized L3T4+ Lyt2-lymphocytes during infection with bacillus Calmette-Guérin.
Authors: Marchal G , Milon G
Published: May 2006

When injected intravenously with bacillus Calmette-Guérin (BCG; 10(7) viable units), C57BL/6 mice rapidly develop a transient anemia associated with an increased number of granulocytes and monocytes, whereas C3H/He mice do not. Because these two features are lacking in C57BL/6 nude mice we postulated that T lymphocytes can regulate hemopoiesis during infection. To assess further the role in hemopoiesis of T lymphocytes present in bone marrow of C57BL/6 and C3H/He mice, the frequency of BCG-specific T lymphocytes and their surface marker phenotype were determined by limiting dilution analysis and use of monoclonal antibodies. The number of BCG-specific T lymphocytes was estimated to be 50- to 100-fold higher in bone marrow of C57BL/6 than in that of C3H/He mice. Although L3T4+ Lyt2-and L3T4- Lyt2+ BCG-specific T lymphocytes were generated in mice of both strains, in C57BL/6 mice L3T4+ cells were induced preferentially from day 1 through day 5 after infection in correlation with hemopoietic changes. The relation between T-cell immune response and hemopoietic changes was substantiated by results obtained after in vivo treatment with monoclonal antibodies. Selective depletion of L3T4+ T cells by in vivo injection of anti-L3T4 monoclonal antibodies (GK 1-5) inhibited the development of the anemia and the related increased production of phagocytes in C57BL/6 mice receiving BCG.
T-cell receptor variable region gene usage in T-cell populations.
Authors: Garman R D , Ko J L , Vulpe C D , Raulet D H
Published: May 2006

We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens.
Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (tau)
Authors: Wood J G , Mirra S S , Pollock N J , Binder L I
Published: May 2006

The relationship of the neurofibrillary tangle, found in Alzheimer disease and aged brains, to normal or abnormal cytoskeletal proteins remains elusive. Although immunohistochemical studies have yielded disparate results, most antigenic determinants localized to neurofibrillary tangles are cytoskeletal constituents normally present in neuronal perikarya or dendrites. We report light and electron microscopic immunolabeling of neurofibrillary tangles by a monoclonal antibody to the microtubule-associated protein tau (tau). Dephosphorylation of tissue slices not only increased the number of tau-positive tangles but also produced marked positive immunoreactivity of neuritic plaques. The localization of tau, an axonal protein, to neurofibrillary tangles in the perikaryon in particular suggests that abnormal synthesis, modification, or aggregation of tau may induce aberrant cytoskeletal--cell organelle interactions, subsequent interference with axonal flow, and resultant tangle formation.
Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.
Authors: Kosik K S , Joachim C L , Selkoe D J
Published: May 2006

The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.
Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro.
Authors: Roberts A B , Sporn M B , Assoian R K , Smith J M , Roche N S , Wakefield L M , Heine U I , Liotta L A , Falanga V , Kehrl J H
Published: May 2006

Transforming growth factor type beta (TGF-beta), when injected subcutaneously in newborn mice, causes formation of granulation tissue (induction of angiogenesis and activation of fibroblasts to produce collagen) at the site of injection. These effects occur within 2-3 days at dose levels than 1 microgram. Parallel in vitro studies show that TGF-beta causes marked increase of either proline or leucine incorporation into collagen in either an NRK rat fibroblast cell line or early passage human dermal fibroblasts. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) do not cause these same in vivo and in vitro effects; in both rat and human fibroblast cultures, EGF antagonizes the effects of TGF-beta on collagen formation. We have obtained further data to support a role for TGF-beta as an intrinsic mediator of collagen formation: conditioned media obtained from activated human tonsillar T lymphocytes contain greatly elevated levels of TGF-beta compared to media obtained from unactivated lymphocytes. These activated media markedly stimulate proline incorporation into collagen in NRK cells; this effect is blocked by a specific antibody to TGF-beta. The data are all compatible with the hypothesis that TGF-beta is an important mediator of tissue repair.
nu 1, a Mr 43,000 component of postsynaptic membranes, is a protein kinase.
Authors: Gordon A S , Milfay D
Published: May 2006

Acetylcholine receptor-enriched membranes from the electric organ of Torpedo californica show a major band at Mr 43,000 on NaDodSO4/polyacrylamide gels. This band is composed of three polypeptides: nu 1, nu 2, and nu 3. Polypeptide nu 1 has been found to be localized exclusively at the innervated face of the electrocyte and at the neuromuscular junction in rat muscle. We show here that monoclonal antibody to nu 1 precipitates a radioactive Mr 43,000 polypeptide from detergent-solubilized extracts of Torpedo membranes covalently labeled with periodate-oxidized [alpha-32P]ATP. The monoclonal antibody also precipitates protein kinase activity from neutralized pH 11 extracts of the acetylcholine receptor-rich membranes. These data suggest that nu 1 is a postsynaptic membrane protein kinase.
Characterization of leukotriene A4 synthase from murine mast cells: evidence for its identity to arachidonate 5-lipoxygenase.
Authors: Shimizu T , Izumi T , Seyama Y , Tadokoro K , Rådmark O , Samuelsson B
Published: May 2006

Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was identified by demonstration of two epimers of 6-transleukotriene B4, methanol trapping, as well as further transformation to leukotriene B4 by leukotriene A4 hydrolase. Leukotriene A4 synthase stereospecifically eliminated the D-hydrogen at C-10 (pro-R) in the synthesis of leukotriene A4 when incubated with [10D-3H;3-(14)C]5-HPETE. The purified enzyme also exhibited 5-lipoxygenase activity toward arachidonic acid and 8-lipoxygenase activity towards 8,11,14-cisicosatrienoic acid. All of these activities required Ca2+ and ATP for their maximal velocities. The effects of heat treatment and of several lipoxygenase inhibitors on these enzyme activities as well as coelution in various chromatographic systems strongly suggest that lipoxygenase and leukotriene A4 synthase activities reside in the same enzyme molecule.
Both ATP and the electrochemical potential are required for optimal assembly of pro-OmpA into Escherichia coli inner membrane vesicles.
Authors: Geller B L , Movva N R , Wickner W
Published: May 2006

Pro-OmpA is processed to OmpA by isolated inverted plasma membrane vesicles from Escherichia coli. In the presence of ATP and a membrane potential, 58% (+/- 13%) of the OmpA is sequestered in the vesicles. We sought to determine which of these two metabolic energy sources is used for protein translocation. The plasma membrane F1F0-ATPase is the central enzyme that interconverts the energy of membrane electrochemical potential and ATP. To separate the effects of these two forms of energy in vitro, the ATPase was inactivated, either by "stripping" the F1 from the membranes with low salt and EDTA or by using membrane vesicles derived from a strain without the atp operon. In each case, optimal translocation and processing of pro-OmpA required both a membrane potential and ATP. We conclude that ATP and membrane potential are separate requirements for bacterial protein export.
Electron-transport-driven proton pumps display nonhyperbolic kinetics: Simulation of the steady-state kinetics of cytochrome
c
oxidase
Authors: Brzezinski Peter , Malmström Bo G.
Published: May 2006

A reaction cycle for electron-transportdriven proton pumps is proposed. It includes two distinct conformational states of the pump protein in which the primary electron acceptor has different reduction potentials. This has as an unavoidable consequence that the steady-state rate equation for the catalytic reaction driving the pump is nonhyperbolic. The model can be used to simulate experimental results for the kinetics of cytochrome oxidase (EC 1.9.3.1) in a wide range of experimental conditions (ionic strength, pH, temperature). It is thus not necessary to invoke more than one binding site for cytochrome
c
to account for the biphasic response of the oxidase activity to the concentration of this substrate.
Selective regulation of trypsin gene expression by calcium and by glucose starvation in a rat exocrine pancreas cell line.
Authors: Stratowa C , Rutter W J
Published: May 2006

Treatment of the rat pancreatic acinar cell line AR4-2J with the calcium ionophore A23187 selectively increases, within a few hours, the steady-state level of trypsin mRNA. Addition of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate potentiates the calcium-induced increase. The mRNA level of the other tested exocrine pancreatic genes decreases. These results were confirmed by DNA transfection experiments, using the 5' flanking region of the trypsin and chymotrypsin genes linked to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. In calcium-induced cells transfected with the trypsin constructs, an increase in CAT activity was observed, whereas the chymotrypsin constructs revealed a decreased CAT activity. Glucose starvation of AR4-2J cells similarly elicited a selective increase in trypsin mRNA. This selective regulation of trypsin may reflect its role as the key activator of the other zymogen species.
Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes.
Authors: Tondravi M M , Yao M C
Published: May 2006

The ribosomal RNA genes (rDNA) of Tetrahymena thermophila macronucleus exist as free linear 21-kilobase molecules that contain replication origins and telomeres. A mutation in this gene confers resistance to the antibiotic paromomycin. We have isolated rDNA from such a mutant (strain p2f), microinjected it into the macronucleus of a sensitive strain, and obtained drug-resistant cells at a frequency of 1-3%. The transformed cells have a distinct and stable phenotype. The rDNA of the transformants contains the expected sequences of the mutant rDNA as determined by oligonucleotide hybridization. rDNA from a different inbred line (C3-368), which contains heteromorphic restriction sites, has also been used for injection, and the results confirm the fact that the injected rDNA is indeed present in the transformants. Injection of rDNA from the C3 strains also increases the transformation frequency 5- to 10-fold and leads to the total replacement of the resident rDNA of the B-inbred strains. This is presumably due to the replication dominance of rDNA from the C3 strains over that of the B strains. Using this method, we have also been able to transform developing cells, at similar frequencies, by microinjecting into the macronuclear anlagen.
A recognition site on synthetic helical oligonucleotides for monoclonal anti-native DNA autoantibody.
Authors: Stollar B D , Zon G , Pastor R W
Published: May 2006

The binding site in native DNA for a murine monoclonal anti-DNA autoantibody was investigated by measurements of competitive binding of a series of synthetic helical oligonucleotides. The antibody bound to a (dG-dC)3 or (dG-dC)4 core in the center of a base-paired octadecanucleotide. Reactions of analogues containing modifications or substitutions at specific sites indicated that the antibody bound to portions of cytosine and guanine in the major groove, a limited region of the backbone, and the 2-amino group of one guanine in the minor groove. For these interactions to occur, the antibody combining site would straddle the backbone of one of the helical strands of DNA.
Activation of human peripheral blood mononuclear cells by anti-T3: killing of tumor target cells coated with anti-target-anti-T3 conjugates.
Authors: Jung G , Honsik C J , Reisfeld R A , Müller-Eberhard H J
Published: May 2006

Cytotoxic T-cell clones have been shown to be capable of killing unrelated target cells in the presence of heteroconjugates consisting of OKT3 (monoclonal antibody against cell surface antigen T3) and targeting antibody. In contrast, human peripheral blood T cells were not known to have this ability, although these cells have been shown to undergo proliferation in response to OKT3 and accessory signals. Here we report that OKT3-stimulated human peripheral blood mononuclear cells effectively killed human melanoma or Raji cells coated with anti-target-cell--OKT3 conjugates. The extent of target-cell killing by stimulated effector cells was much greater than that observed with unstimulated cells, in spite of down-regulation of the T3 structure on the OKT3-treated cells. Target-cell lysis occurred at physiological concentrations of effector cells and was inhibited by monomeric OKT3, indicating that killing was T-cell-mediated.
Role of hydrogen peroxide and hydroxyl radical formation in the killing of Ehrlich tumor cells by anticancer quinones.
Authors: Doroshow J H
Published: May 2006

The cytotoxicity of the clinically important antineoplastic quinones doxorubicin, mitomycin C, and diaziridinylbenzoquinone for the Ehrlich ascites carcinoma was significantly reduced or abolished by the antioxidant enzymes catalase and superoxide dismutase, the hydroxyl radical scavengers dimethyl sulfoxide, diethylurea, and thiourea, and the iron chelators deferoxamine, 2,2-bipyridine, and diethylenetriaminepentaacetic acid. However, tumor cell killing by 5-iminodaunorubicin, a doxorubicin analog with a modified quinone function that prohibits oxidation-reduction cycling, was not ameliorated by any of the free radical scavengers tested. Furthermore, treatment of intact tumor cells with doxorubicin, mitomycin C, and diaziridinylbenzoquinone but not 5-iminodaunorubicin generated the hydroxyl radical, or a related chemical oxidant, in vitro in a process that required hydrogen peroxide, iron, and intact tumor cells. These results suggest that drug-induced hydrogen peroxide and hydroxyl radical production may play a role in the antineoplastic action of redox active anticancer quinones.
Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1.
Authors: Bevilacqua M P , Pober J S , Majeau G R , Fiers W , Cotran R S , Gimbrone M A
Published: May 2006

Human recombinant tumor necrosis factor (rTNF) was found to act directly on cultured human vascular endothelium to induce a tissue factor-like procoagulant activity (PCA). After a 4-hr incubation in rTNF (100 units/ml), serially passaged endothelial cells isolated from umbilical veins, saphenous veins, iliac arteries, and thoracic aortae demonstrated a dramatic increase (4- to 15-fold, 21 experiments) in total cellular PCA as measured with a one-stage clotting assay. rTNF-induced PCA was also expressed at the surface of intact viable endothelial monolayers. Induction of PCA by rTNF was concentration dependent (maximum, 500 units/ml), time dependent, reversible, and blocked by cycloheximide and actinomycin D, and it occurred without detectable endothelial cell damage. Actions of rTNF were compared with those of natural human interleukin 1 (IL-1) derived from stimulated monocytes and two distinct species of recombinant IL-1, each of which also induced endothelial PCA. The use of recombinant polypeptides and specific neutralizing antisera established the distinct natures of the mediators. The kinetics of the endothelial PCA responses to TNF and IL-1 were similar, demonstrating a rapid rise to peak activity at approximately equal to 4 hr, and a decline toward basal levels by 24 hr. This characteristic decline in PCA after prolonged incubation with TNF or IL-1 was accompanied by selective endothelial hyporesponsiveness to the initially stimulating monokine. Interestingly, the effects of TNF and IL-1 were found to be additive even at apparent maximal doses of the individual monokines. Endothelial-directed actions of TNF, alone or in combination with other monokines, may be important in the initiation of coagulation and inflammatory responses in vivo.
Defective RNA splicing in the absence of adenovirus-associated RNAI.
Authors: Svensson C , Akusjärvi G
Published: May 2006

We have analyzed late gene expression in 293 cells infected with an adenovirus type 5 mutant dl331, which is defective in production of the low molecular weight virus-associated (VA) RNAI. The results show that several steps in late gene expression are affected. In addition to the previously characterized defect in late mRNA translation, mutant infected cells also show an aberrant selection of RNA splice sites and a substantially reduced L2, L3, and L5 mRNA accumulation. Normal or even slightly elevated amounts of mRNA from region L1 are produced. However, the L1 pre-mRNA is spliced only to generate the mRNA encoding the Mr 52,000-55,000 polypeptide and no detectable mRNA for polypeptide IIIa. Cotransfection of a plasmid encoding VA RNAI complemented the splicing defect in trans, suggesting that the abnormalities are due to the absence of VA RNAI, rather than to a cis-acting change in the nuclear precursor RNA. In a HeLa cell variant, which allows late protein synthesis also in the absence of VA RNAI, because of a lack of eukaryotic initiation factor 2 alpha kinase expression, a normal repertoire of late mRNA was produced. We conclude that a soluble factor, most likely a late viral protein, controls differential RNA splicing and late mRNA accumulation during an adenovirus infection.
Junctional plasma membrane domains isolated from aggregating Dictyostelium discoideum amebae.
Authors: Ingalls H M , Goodloe-Holland C M , Luna E J
Published: May 2006

Regions of plasma membrane involved in Dictyostelium discoideum intercellular adhesion resist solubilization with the nonionic detergent Triton X-100. Electron microscopy shows that these regions of the plasma membrane adhere to each other, forming many bi- and multilamellar structures. NaDodSO4/polyacrylamide gels of these regions contain major polypeptides at 225 kDa (residual myosin), 105 kDa, 88 kDa, 84 kDa, 47 kDa (residual actin), and 34 kDa. These membranes contain a subset of the total plasma membrane proteins, as analyzed by labeling of electrophoretically fractionated and blotted membrane proteins with radioiodinated Con A and by electrophoresis of membrane proteins from surface-labeled cells. Antibodies specific for gp80, a glycoprotein implicated in intercellular adhesion, intensely stain the 88-kDa and 84-kDa bands. Since these membrane regions resist Triton extraction, they appear to be stabilized by protein-protein interactions. Such stabilizing interactions may involve multivalent linkages with adjacent cells, or associations with intracellular actin and myosin, or both. Since these membranes appear to represent regions of intercellular contact, we call them "contact regions."
Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.
Authors: Grundke-Iqbal I , Iqbal K , Tung Y C , Quinlan M , Wisniewski H M , Binder L I
Published: May 2006

A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.
Biochemical and pharmacological characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist.
Authors: Chang R S , Lotti V J
Published: May 2006

3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl)-1H-indole-2-carboxamide (L-364,718) interacted in a competitive manner with rat pancreatic cholecystokinin (CCK) receptors as determined by Scatchard analysis of the specific binding of 125I-labeled CCK. The affinity of L-364,718 for both pancreatic (IC50, 81 pM) and gallbladder (IC50, 45 pM) CCK receptors in radioligand binding assays greatly exceeded that of other reported nonpeptide CCK antagonists and was similar to that of CCK itself. In vitro functional studies utilizing CCK-induced contractions of the isolated guinea pig ileum and colon further demonstrated that L-364,718 acts as a competitive CCK antagonist, which lacks agonist activity and has a similar high affinity in these tissues (pA2, 9.9). L-364,718 exhibited a very high selectivity for peripheral CCK receptors relative to brain CCK, gastrin, and various other peptide and nonpeptide receptors in both in vitro radioligand and isolated tissue assays. In vivo, low intravenous doses of L-364,718 (0.1 mg/kg) markedly antagonized the contractions of the guinea pig gallbladder produced by intravenous administration of CCK for at least 2 hr. Administered orally, L-364,718 (ED50, 0.04 mg/kg) was highly effective as an antagonist of CCK-induced inhibition of gastric emptying in mice. The biochemical and pharmacological properties of L-364,718--namely, very high affinity and selectivity for peripheral CCK receptors, long-lasting in vivo efficacy, and oral bioavailability--makes this compound a powerful tool for investigating the physiological and pharmacological actions of CCK, and possibly its role in gastrointestinal disorders.
Inhibition of gamma-glutamyl transpeptidase and induction of glutathionuria by gamma-glutamyl amino acids.
Authors: Anderson M E , Meister A
Published: May 2006

Treatment of mice with various gamma-glutamyl amino acids leads to marked urinary excretion of glutathione and other gamma-glutamyl compounds. There is good correlation between the affinity of gamma-glutamyl transpeptidase for various gamma-glutamyl amino acids and the extent of glutathionuria. The findings indicate that the administered gamma-glutamyl compounds effectively compete with glutathione (exported from kidney cells and present in the glomerular filtrate) for the enzyme. The administration of certain gamma-glutamyl amino acids appears to be a specific and nontoxic procedure for in vivo inhibition of gamma-glutamyl transpeptidase that may be useful in experimental work on glutathione metabolism and function and also for treatment of certain toxicities and for modulation of the metabolism of endogenous glutathione conjugates.
Mutations affecting the signal sequence alter synthesis and secretion of yeast invertase.
Authors: Perlman D , Raney P , Halvorson H O
Published: May 2006

Insertion mutations previously constructed within the proximal region of the yeast invertase signal sequence did not interfere with secretion or glycosylation of the enzyme. We now describe deletion mutations within the same signal sequence. Large deletions truncating the hydrophobic core of the signal peptide prevented both secretion and glycosylation of the enzyme and increased the intracellular concentration of nonglycosylated invertase. This increase was coupled with the appearance of a new invertase polypeptide, 2 kilodaltons larger than cytoplasmic invertase. The new polypeptide was consistent in size with uncleaved (signal peptide intact) pre-secretory invertase previously identified by using in vitro translation (apparent molecular mass, 62 kilodaltons). The data on enzyme activity indicate that invertase whose secretion is aborted by large deletion mutations augments the normal pool of cytoplasmic invertase found in sucrose-fermenting yeast cells.
Molecular cloning of partial cDNAs for rabbit liver apolipoprotein B and the regulation of its mRNA levels by dietary cholesterol.
Authors: Kroon P A , DeMartino J A , Thompson G M , Chao Y S
Published: May 2006

Apolipoprotein B (apoB) is the major protein of plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Here we report the molecular cloning of cDNAs for rabbit liver apoB, by use of the expression vector lambda gt11, and the use of these cDNAs to study the regulation of apoB mRNA levels by dietary cholesterol. The beta-galactosidase-apoB fusion proteins expressed by recombinant clones were identified with guinea pig anti-rabbit LDL antibodies. The cloned cDNAs hybridized to an 18-kilobase mRNA that was present in liver and intestine. Slot blot analysis showed that this mRNA was not present in other tissues studied, with the possible exception of kidney. When rabbits are fed a high-cholesterol diet, they develop severe hypercholesterolemia. Most of the excess cholesterol is contained in beta-VLDL, a cholesteryl ester-rich lipoprotein that contains apoB and apoE. We addressed the question of whether increased apoB mRNA levels, and by inference increased apoB synthetic rates, are responsible for the accumulation of beta-VLDL. A comparison of apoB mRNA levels showed that cholesterol-fed rabbits had lower liver apoB mRNA levels than control rabbits. We suggest that the accumulation of plasma beta-VLDL in cholesterol-fed rabbits is not due to an increased production of beta-VLDL but solely due to a suppression of hepatic LDL receptors.
Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.
Authors: Sai S , Tanaka T , Kosher R A , Tanzer M L
Published: May 2006

A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals.
In vitro regulation of cartilage matrix assembly by a Mr 54,000 collagen-binding protein.
Authors: Chandrasekhar S , Laurie G W , Cannon F B , Martin G R , Kleinman H K
Published: May 2006

In cartilage, type II collagen is present as thin, short, randomly oriented fibrils. In vitro, however, type II collagen forms fibrils of large diameter, indicating that additional factors may be involved in the regulation of collagen fibril formation. We have examined extracts of a cartilage-producing tumor for the presence of collagen-binding proteins. In addition to fibronectin and link protein, a Mr 54,000 protein was found to bind to collagen fibrils as well as to native and denatured type II collagen. Immunological studies using antibody against the protein indicate that it is a cartilage matrix protein, not present in bone or in several other tissues. In vitro studies show that the Mr 54,000 protein in combination with cartilage proteoglycan decreases the rate of type II fibril formation and causes the fibrils to be of small diameter (24 +/- 8 nm). These studies indicate that complexes between collagen and proteoglycans mediated by this protein may regulate the assembly of cartilage matrix.
Topoisomerase I identified by scleroderma 70 antisera: enrichment of topoisomerase I at the centromere in mouse mitotic cells before anaphase.
Authors: Maul G G , French B T , van Venrooij W J , Jimenez S A
Published: May 2006

Antibodies derived from scleroderma patients positive for the extractable 70-kDa antigen were shown to react with topoisomerase I. Topoisomerase I was identified by molecular size and by antibody inhibition of the topoisomerase I-specific relaxation of supercoiled plasmid DNA. By using in situ localization by indirect fluorescence, we found topoisomerase I preferentially located in the centromeric regions of the mouse G2-phase cells and chromosomes, while the distribution in human cells is much more dispersed. Moreover, comparison of a published consensus sequence for topoisomerase I binding with mouse satellite DNA revealed a high degree of homology. The localization of topoisomerase I in the centromeres of mouse cells in the later part of the cell cycle and prior to anaphase suggests functional involvement in mitosis.