bluuebunny/crossref_metadata_2025 huggingface dataset:
Large-scale flow generation in turbulent convection
Authors: Krishnamurti Ruby , Howard Louis N.
Published: May 2006

In a horizontal layer of fluid heated from below and cooled from above, cellular convection with horizontal length scale comparable to the layer depth occurs for small enough values of the Rayleigh number. As the Rayleigh number is increased, cellular flow disappears and is replaced by a random array of transient plumes. Upon further increase, these plumes drift in one direction near the bottom and in the opposite direction near the top of the layer with the axes of plumes tilted in such a way that horizontal momentum is transported upward via the Reynolds stress. With the onset of this large-scale flow, the largest scale of motion has increased from that comparable to the layer depth to a scale comparable to the layer width. The conditions for occurrence and determination of the direction of this large-scale circulation are described.
Phagocytosis of carbohydrate-modified phospholipid vesicles by macrophage.
Authors: Wu P S , Tin G W , Baldeschwieler J D
Published: May 2006

Modification of the surface of distearoyl phosphatidylcholine vesicles with synthetic glycolipids dramatically affects the rate of uptake of these vesicles by mouse peritoneal macrophage. The high rate of uptake of 6-aminomannose-modified vesicles is effectively inhibited by cytochalasin B and chloroquine but not by colchicine, indicating that the mechanisms of vesicle uptake is phagocytosis. Other modified vesicles appear to have some effect on the rate of uptake of 6-aminomannose-modified vesicles suggesting that the various vesicle types compete for the same initial binding sites. Analysis of 6-aminomannose-modified vesicles by gamma-ray perturbed angular correlation spectroscopy shows that the rotational correlation time of the encapsulated 111In3+ does not change when the vesicles associate with macrophage. This result is consistent with transmission electron microscopy, which indicates that the aminomannose-modified vesicles remain intact after phagocytosis as aggregates of fused and intact vesicles surrounded by a single bilayer membrane structure.
Vesicular stomatitis virus defective interfering particle containing a muted internal leader RNA gene.
Authors: Keene J D , Chien I M , Lazzarini R A
Published: May 2006

The RNA of a unique long defective interfering particle (DI-LT2) derived from the heat-resistant strain of vesicular stomatitis virus (VSV) contains 70 nucleotides at its 3' end that are complementary to the 5' end of the VSV RNA. Following this region of terminal complementarity, there is a precise copy of the 3' end of the nondefective VSV RNA. The sequence homology between the DI-LT2 RNA and the 3' end of VSV RNA extends for at least 60 bases and probably for most of the length of the DI-LT2 RNA. The DI-LT2 particle is capable of transcription in vitro but produces only a short RNA [defective interfering (DI) particle product], which is encoded by the extreme 3' terminus of the DI RNA. Neither leader RNA nor capped VSV mRNAs are synthesized by DI-LT2, although competent templates for these are present. These data suggest that the 3'-terminal initiation is a prerequisite of the production of competent transcripts and that the sequence coding for leader RNA is not, by itself, sufficient for initiation. We propose a model for the origin of this DI particle, involving specific termination and resumption of replication, which is similar to that described previously for another class of DI particle RNAs.
Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.
Authors: Levine R L , Oliver C N , Fulks R M , Stadtman E R
Published: May 2006

We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.
Predominance of core histones in formation of DNA--protein crosslinks in gamma-irradiated chromatin.
Authors: Mee L K , Adelstein S J
Published: May 2006

Chromatin and a subunit of chromatin containing a complex of DNA and the core histones--H2A, H2B, H3, and H4--have been prepared from cultured Chinese hamster cells. Comparison of the formation of radiation-induced DNA--protein crosslinks in whole chromatin with that in the DNA--core histone complex has demonstrated that the core histones are the specific proteins involved in crosslinking. gamma irradiation of the chromatin subunit in the presence of radical scavengers has shown the hydroxyl radical to be the most effective aqueous radical intermediate for the promotion of crosslinking and the solvated electron and superoxide radical to be essentially ineffective.
A cloned calmodulin structural gene probe is complementary to DNA sequences from diverse species.
Authors: Munjaal R P , Chandra T , Woo S L , Dedman J R , Means A R
Published: May 2006

Calmodulin mRNA has been partially purified from a total nucleic acid extract of the electroplax of Electrophorus electricus by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. A 9- to 10S fraction was determined to contain 39% calmodulin mRNA by translation in a reticulocyte lysate followed by immunoprecipitation with antibodies to calmodulin. Double-stranded cDNA was synthesized from the RNA fraction by using reverse transcriptase from avian myeloblastosis virus. The double-stranded cDNA was joined to pBR322 linearized by restriction endonuclease Pst I and used to transform Escherichia coli RRI. DNAs from 60 tetracycline-resistant cloned hybridized to [32P]cDNA synthesized from the partially purified calmodulin mRNA fraction. By direct DNA sequence analysis, one of these clones, pCM109, was shown to contain calmodulin-specific sequences corresponding to amino acid residues 93--148 of calmodulin or approximately 38% of the peptide-coding region of the calmodulin structural gene sequence. pCM109 was hybridized to DNA isolated from three vertebrate and one plant species by the procedure of Southern. Positive hybridization bands were noted regardless of the DNA source. These data suggest thaat calmodulin gene sequences are evolutionarily conserved, as has been shown to be the case for the primary amino acid sequence.
Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells.
Authors: Lin J J
Published: May 2006

Monospecific antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with rat skeletal myofibrils. After cloning 3 times on agarose, two stable clones were obtained and chosen for further characterization. The first clone, JLB1, produced an antibody that recognizing an antigen distributed in the M-line region and on either site of the Z line of myofibrils. The second clone, JLB7, produced an antibody reacting only with an antigen located at the M-line region of myofibrils. Both JLB1 and JLB7 antibodies decorate the typical intermediate filaments of a variety of cultured cells. Colcemid treatment of cells before reaction with both antibodies resulted in the coiling or capping (or both) of the fibers around the nucleus. Brief treatment of cells with cytochalasin B did not affect the integrity of the fibers stained by both antibodies whereas, under the same conditions, microfilament bundles visualized by another monoclonal antibody (JLA20) against actin were disassembled into many aggregates in the cytoplasm. Identical staining patterns of the intermediate filaments are obtained by double-label immunofluorescence microscopy of the same cell stained with these monoclonal antibodies and rabbit autoimmune serum (which has been shown to react with the components of the intermediate filaments). By using immunoprecipitation, protein bands at 210,000 and 95,000 daltons from chicken embryo fibroblasts were identified as the potential antigens recognized by JLB1 and JLB7 monoclonal antibodies, respectively. The widespread occurrence of these antigenic determinants in different cultured cells suggests the highly conservative property of these intermediate-filament components.
Stimulation of rheumatoid synovial cell collagenase and prostaglandin production by partially purified lymphocyte-activating factor (interleukin 1).
Authors: Mizel S B , Dayer J M , Krane S M , Mergenhagen S E
Published: May 2006

Human macrophages produce in culture a factor termed mononuclear cell factor (MCF) that increases the production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. A factor with similar biologic activity is also produced by the murine macrophage cell line P388D1. By using a sequential purification scheme involving ammonium sulfate fractionation; chromatography on DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose; and discontinuous polyacrylamide gel electrophoresis, the P388D1 cell-derived, synovial cell-stimulating factor was copurified with the lymphocyte-activating factor [LAF; interleukin 1 (IL 1)]. The specific activity of the partially purified LAF (IL 1) was approximately 15,000-fold higher than that of the LAF (IL 1) in the original P388D1 cell culture supernatant. On the basis of (i) the copurification of the P388D1 cell-derived LAF (IL 1) and synovial cell-stimulating factors; (ii) the similarity in cell of origin, molecular weight, and phenylglyoxal sensitivity of human MCF and murine LAF (IL 1); and (iii) the presence of LAF (IL 1) activity in preparations of partially purified human MCF, we have postulated that LAF (IL 1) may have effects on cell targets that are nonlymphoid in nature and also that human MCF may be similar to, or identical with human LAF (IL 1). The results of these studies have raised the possibility that LAF (IL 1) may play a role in macrophage-mediated activation of synovial cells and lymphocytes which are involved in the inflammatory responses associated with rheumatoid arthritis.
Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice.
Authors: Solanki V , Slaga T J
Published: May 2006

The binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDB) to intact living epidermal cells in monolayer culture was characterized. At 37 degrees C, the maximum specific [3H]PDB binding (binding displaceable by 30 microM unlabeled PDB) was attained in 15--20 min and was followed by a rapid decrease (down regulation) of radioactivity bound to the cells. The activity lost by the cells during this decrease was found in the incubation medium. Prior exposure of cells to phorbol 12-myristate 13-acetate (PMA; 12-O-tetradecanoylphorbol 13-acetate) but not to phorbol for 2 hr at 37 degrees C caused approximately 55% reduction in the number of measurable binding sites for [3H]PDB. The down regulation was temperature sensitive; there was no loss of radioactivity after 1 hr at 4 degrees C. The specific binding of [3H]PDB at 4 degrees C reached equilibrium in 15--20 min and was saturable and freely reversible. At equilibrium, epidermal cells contained 1.2 x 10(5) binding sites per cell, and binding sites had a KD of 10 nM. Specificity of binding was shown by the observation that the biologically active phorbol esters PMA and 12-deoxyphorbol 13-decanoate inhibited the binding, whereas the inactive parent compound phorbol and the nonphorbol tumor promoter anthralin did not have any effect. The abilities of these compounds to inhibit [3H]PDB binding directly correlates with their tumor promoting activities. Epidermal cells exposed to retinoic acid or fluocinolone acetonide for 24 hr had similar [3H]PDB binding characteristics as untreated cells suggesting that inhibition of tumor promotion induced by these compounds is not mediated through alterations in the phorbol ester binding sites.
Growth of a pure population of mouse mast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes.
Authors: Razin E , Cordon-Cardo C , Good R A
Published: May 2006

A pure population of mast cells was obtained after 14 days of culturing mouse bone marrow cells in the presence of medium derived from concanavalin A-stimulated mouse spleen cells. The cells were characterized as mast cells by their morphologic appearance and histologic staining, by their histamine content (450 ng per 10(6) cells) and by the demonstration of IgE receptors on their surface (150,000--440,000 receptor sites per cell). The histamine content and the number of IgE receptors remained constant for at least 7 wk of culture. These mast cells could be passively sensitized to mice hybridoma IgE. They then released 43% of their histamine content upon incubation with anti-mouse hybridoma IgE.
Nuclear uptake of 1,25-dihydroxy[3H]cholecalciferol in dispersed fibroblasts cultured from normal human skin.
Authors: Eil C , Marx S J
Published: May 2006

Because of the relative inaccessibility of known calciferol target tissues (i.e., intestine and bone), we examined fibroblasts derived from normal human skin and grown in tissue culture as a means of evaluating the interaction of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its effector system. When dispersed, intact cells were used, nuclear uptake of 1,25-dihydroxy[23,24(n)3-H]cholecalciferol [1,25(OH)2[3H]D3) was temperature-dependent, optimal at 45 min at 37 degrees C, and saturable. In competition experiments with other calciferols, the 1,25(OH)2[3H]D3 uptake showed specificity indistinguishable from that reported for 1,25(OH)2D3 receptors from calciferol target tissues. Analysis of 1,25(OH)2[3H]D3 nuclear uptake in fibroblast strains from six normal adults (four male, two female) yielded an average binding capacity (R0) of 10,600 +/- 2,000 (SEM) nuclear sites per cell and an apparent dissociation contant (Kd) of 0.50 +/- 0.07 (SEM) x 10(-9) M. Donor sex, donor age, or anatomic site of origin of the cell line did not affect the characteristics of uptake. Similar nuclear uptake was demonstrable with cultured MCF-7 cells (derived from human breast cancer) when assayed in the same fashion. When hypertonic extracts of nuclei obtained from skin fibroblasts incubated with 1,25(OH)2[3H]D3 were subjected to centrifugation on sucrose gradients, a single peak of radioactivity sedimented at approximately 3 S; when excess 1,25(OH)2D3 was coincubated during the cellular uptake phase, this 3S peak was not observed. Molybdate was an essential buffer component for receptor stabilization during cell fractionation and sedimentation analysis. In summary, by using fibroblasts cultured from normal human skin, we have identified a process of nuclear uptake of 1,25(OH)2[3H]D3 with the affinity, saturability, and specificity characteristics of a steroid hormone--receptor interaction. This method should be useful in studying 1,25(OH)2D3 recept physiology in cells from normal persons as well as in cells from patients who have disorders in the responsiveness of calciferol target tissues.
Uptake and release of iron from human transferrin.
Authors: Huebers H , Josephson B , Huebers E , Csiba E , Finch C
Published: May 2006

Purified fractions of human apotransferrin, monoferric transferrins with iron on the acid-labile binding site and on the acid-stable binding site, and diferric transferrin have been prepared. The iron loading and unloading behavior of these preparations has been examined by isoelectric focusing. Iron release from the two monoferric transferrin preparations to human reticulocytes was of similar magnitude. In a mixture containing equal amounts of diferic and monoferric iron, approximately 4 times the amount of iron delivered by the monoferric species was delivered by the diferric species. Iron loading of transferrin in vitro showed a random distribution between monoferric and diferric transferrin. Among the monoferric transferrins, loading of the acid-labile binding sites was greater than that of the acid-stable binding sites. In vivo iron distribution in normal subjects, as evaluated by in vitro-added 50Fe, gave similar results. Absorption of a large dose of orally administered iron in iron-deficient subjects resulted in a somewhat greater amount of diferric transferrin at low saturation and a somewhat smaller amount of diferric transferrin at higher saturations than would have been anticipated by random loading. These data would indicate that in the human, iron loading of transferrin may be considered essentially random. Unloading from the two monoferric transferrin species is of similar magnitude but far less than that delivered by diferric transferrin.
Use of cultured lymphoblastoid cells for the study of abnormal enzymes: molecular abnormality of a phosphoglycerate kinase variant associated with hemolytic anemia.
Authors: Fujii H , Chen S H , Akatsuka J , Miwa S , Yoshida A
Published: May 2006

A phosphoglycerate kinase (PGKase: ATP:3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3; X chromosome-linked) variant, PGKase-Tokyo, is associated with enzyme deficiency, nonspherocytic hemolytic anemia, and neurological disturbances. Because a sufficient amount of the patient's erythrocytes was not available, the variant enzyme was purified to homogeneity from the cultured lymphoblastoid cells of the patient. The enzyme activity of the variant lymphoblastoid cells was about 16% of that of the normal lymphoblastoid cells. PGKase-Tokyo, compared to the normal enzyme, had a lower specific activity (31% of normal in the backward reaction and 15% of normal in the forward reaction), higher than normal Michaelis constants for ATP and 3-phosphoglycerate, a more acidic pH optimum, and increased thermal instability. Microscale peptide mapping analysis revealed that the structural abnormality of PGKase-Tokyo is a single amino acid substitution from valine to methionine at position 266. Thus, the use of the cultured lymphoblastoid cells is proven to be useful for the study of structural and functional abnormalities of mutant enzymes.
Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.
Authors: Wiggs J L , Gilman M Z , Chamberlin M J
Published: May 2006

Preparations of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme II) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) and lacks the normal sigma subunit [Jaehning, J. A., Wiggs, J. L. & Chamberlin, M. J. (1979) Proc. Natl. Acad. Sci. USA 76, 5470-5474]. By heparin-agarose chromatography we have obtained holoenzyme II fractions that have no detectable holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template we have tested. These fractions are far more active with B. subtilis DNA than with T7 DNA or other heterologous templates. This high degree of specificity has allowed identification of plasmids containing cloned fragments of B. subtilis DNA that bear strong promoter sites for holoenzyme II. These promoter sites are not used at all by B. subtilis RNA polymerase holoenzyme I. The specificity of holoenzyme II is dictated by a peptide of Mr 28,000 as judged by copurification of the peptide with specific holoenzyme II activity and by reconstitution of the holoenzyme II promoter specificity when the isolated peptide is added to B. subtilis core polymerase. Hence the 28,000 Mr peptide appears to be a sigma factor that determines a promoter specificity distinct from that of RNA polymerase holoenzyme I and all other known bacterial RNA polymerases.
Expression of human and mouse nonhistone chromosomal proteins in hybrid mouse erythroleukemia cells containing a single human chromosome.
Authors: Bode U , Deisseroth A , Hendrick D
Published: May 2006

The nonhistone chromosomal proteins of a series of hybrid mouse erythroleukemia cell lines containing human chromosome 16 were investigated by two-dimensional gel electrophoresis to determine if such cells contained nonhistone chromosomal proteins of both human and mouse origin. Comparison of the two-dimensional gel electrophoretograms of the nonhistone chromosomal proteins of mouse and human cell lines showed 400 and 280 chromosomal proteins, respectively, of which about 75% were electrophoretically identical. The two-dimensional gel electrophoretogram of a cloned hybrid mouse erythroleukemia cell line that retained a tetraploid complement of mouse chromosomes and human chromosome 16 (as the only human chromosome) displayed a nonhistone chromosomal protein of pI 6.2 and Mr 65,000. This protein, which comigrates with a nonhistone chromosomal protein present in the human cell line used to produce this hybrid cell and which is also present in two additional human cells lines studied, could not be detected in the mouse erythroleukemia parent before fusion. This polypeptide also was shown by similar techniques to be associated with the presence of human chromosome 16 in four out of five other independently derived hybrid mouse erythroleukemia cell lines that contained a near tetraploid complement of mouse erythroleukemia chromosomes.
Interaction of alpha-actinin and vinculin with actin: opposite effects on filament network formation.
Authors: Jockusch B M , Isenberg G
Published: May 2006

The interaction of actin filaments with two actin-associated proteins, alpha-actinin and vinculin (Mr 130,000 protein), was studied in vitro with viscometry and light and electron microscopy. Vinculin, like alpha-actinin, binds to F-actin, and the two proteins were found to have different effects on the formation of filament networks: alpha-actinin crosslinks individual filaments in a manner strongly dependent on temperature and acts as a spacer, whereas vinculin forms actin bundles that display a paracrystalline substructure. In viscometric assays, alpha-actinin mimics the effect of actin gelation factors, whereas vinculin acts as a gelation inhibitor. These findings imply complementary functions of these proteins in the regulation of cellular mobility.
Structural analysis of an electrophoretically cryptic alcohol dehydrogenase variant from an Australian population of Drosophila melanogaster.
Authors: Chambers G K , Laver W G , Campbell S , Gibson J B
Published: May 2006

The existence of geographically widespread clines in genetic polymorphisms is persuasive evidence that the distribution of such genetic variance is determined by natural selection. However, when comparing clines it is important to be certain that identical structural genes are involved. We report a structural difference (proline-214 to serine) between the product of AdhF and an electrophoretically cryptic heat-stable variant isolated from an Australian natural population, ADH-FCh.D. ("fast" Chateau Douglas). The biochemical properties of this new variant must be taken into consideration when attempting to formulate a causal explanation of the maintenance of the three identified Adh alleles. Our data also show that the products of an AdhF and an AdhS allele in Drosophila melanogaster in an Australian population are identical over 70% of their amino acid sequences with their North American counterparts.
Dissection of distinct human immunoregulatory T-cell subsets by a monoclonal antibody recognizing a cell surface antigen with wide tissue distribution.
Authors: Irigoyen O H , Rizzolo P V , Thomas Y , Hemler M E , Shen H H , Friedman S M , Strominger J L , Chess L
Published: May 2006

A monoclonal antibody, PVR-11, was obtained after hybridization of X63Ag8.653 murine myeloma cells with spleen cells from a mouse immunized with human lymphocytes. It recognizes a 175,000- to 185,000-dalton surface antigen present on approximately 80% of normal human peripheral T lymphocytes, 50% of non-T non-B cells, and less than 10% of B cells as determined by complement-dependent microcytotoxicity. It is also present on various leukemia T cells, on some but not all T lymphoblastoid cell lines, and on a small fraction of some B lymphoblastoid cell lines. Some B-cell chronic lymphocytic leukemia cells also express the PVR-11 antigen. Functional analysis of normal human T lymphocytes demonstrated that the PVR-11-depleted T-cell subset contains the precursors of both cytotoxic and suppressor cells but lacks helper cells. On the other hand, cytotoxic effector T cells express the PVR-11 antigen. These results demonstrate that antigenic determinants with relatively wide tissue distribution can dissect functionally distinct human immunoregulatory T-cell subsets.
Changes in serum iron levels due to infection with hepatitis B virus
Authors: Blumberg Baruch S. , Lustbader Edward D. , Whitford Pamela L.
Published: May 2006

We found in two previous studies (Down syndrome patients and end-stage kidney patients receiving renal dialysis) that total serum iron is higher on average in carriers of the hepatitis B virus than in those who are not. The elevation of the serum iron is independent of elevations of serum L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) (SGPT), an indicator of liver cell damage. We have followed for 10 yr a large number of patients with end-stage renal disease receiving renal dialysis. In this paper we describe studies of serum iron and SGPT levels in patients (
i
) 1 mo before infection, (
ii
) after infection but within the month of infection, and (
iii
) 6-12 mo after infection. Comparisons of serum iron levels were made between those infected who retained the virus (carriers) and those who rejected the infection (transients). There were no differences between these groups before infection. Serum iron remained high in the carrier group and dropped in the transients. However, not all of the carriers retained high levels, although this was the case in general. Individual changes in the pre- and postconversion period were then considered. All carriers who had a preconversion decline in iron had an increase after infection, whereas this occurred in only some of the transients. Those carriers who had a decline after infection had raised levels before infection, and the decline was generally less than the increase. Consideration of the SGPT and the iron levels together led to the same conclusion as the previous studies, that elevation of iron may be independent of rise in SGPT. Several hypotheses were derived from these findings. Individuals who are carriers in general have higher iron levels and, therefore, are more likely to become infected with bacteria; this may contribute to increased morbidity and mortality. From experimental evidence, iron is required for the growth of tumor cells. Carriers with elevated iron levels may be more likely to develop detectable cancer of the liver than those who do not.
Changes in the promoter range of RNA polymerase resulting from bacteriophage T4-induced modification of core enzyme.
Authors: Goldfarb A
Published: May 2006

Primary transcripts made in vitro on bacteriophage T4 DNA by RNA polymerase isolated from normal or T4-infected Escherichia coli were compared by gel electrophoresis. Bacteriophage-modified RNA polymerase fails to initiate transcription at certain promoters recognized by unmodified enzyme. In the T4tRNA gene region, only one of the two promoters is active with the modified RNA polymerase. Reconstitution of separated RNA polymerase components demonstrates that this change in promoter site selection results from the modification of core enzyme and not sigma factor.
Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei.
Authors: Liu L F , Miller K G
Published: May 2006

Two type I DNA topoisomerases have been purified to homogeneity from the nuclei of HeLa cells. One topoisomerase has a peptide molecular weight of 100,000 and the other, a molecular weight of 67,000. Several lines of evidence indicate that these two topoisomerases are closely related, (a) Both exhibit similar enzymatic activities on DNA. (b) The chromatographic properties of the two topoisomerases during purification are similar. (c) Mild proteolysis of the purified molecular weight 100,000 topoisomerase in vitro generates a group of protein bands of molecular weight approximately 67,000, and these bands retain topoisomerase activity. (d) The peptides formed by partial proteolysis of the 67,000 topoisomerase in the presence of NaDodSO4 form a subset of those produced from the 100,000 enzyme. The 100,000 topoisomerase is the major type I enzyme in the cell. The 67,000 topoisomerase, which may be identical to the previously identified "nicking-closing" enzyme [Champoux, J. J. & Dulbecco, R. (1972) Proc. Natl. Acad. Sci. USA 69, 143-146], is probably formed by proteolysis of the 100,000 enzyme.
DNA double-strand breaks induced in normal human cells during the repair of ultraviolet light damage.
Authors: Bradley M O , Taylor V I
Published: May 2006

DNA double-strand breaks (DSBs) are formed in normal human IMR-90 cells during repair incubation after 100 and 300 J.m-2 of ultraviolet light. By contrast, no DSBs are formed after exposure to ultraviolet light in human XPA cells (from a patient with xeroderma pigmentosum complementation group A), which are unable to excise pyrimidine dimers. The DSBs are not due to immediate cell death, because all the cells excluded trypan blue at the time of assay and because XPA cells, which are much more sensitive to ultraviolet light than are IMR-90 cells, did not form DSBs after exposure to ultraviolet light. The DSBs do not appear to be due to either DNA synthesis or cellular single-strand endonucleases. We suggest that repair-induced DSBs would be potent lesions that might lead to cytotoxicity, chromosome aberrations, deletion mutations, and perhaps cellular transformation.
Statistical analysis of the Ames Salmonella/microsome test.
Authors: Margolin B H , Kaplan N , Zeiger E
Published: May 2006

A family of statistical models for analysis of Ames Salmonella/microsome test data is constructed that considers mutation and toxicity as competing risks and allows hyper-Poisson variability. These models have a parameter than can be employed as a mutagenic index because it approximates the slope at zero dose of a dose-response curve adjusted for toxicity. A second parameter quantifies plate-to-plate variability and lends itself to the study of factors affecting internal test reproducibility. The detection of aberrant plate counts is also addressed. This methodology is illustrated with data from a Salmonella test.
Murine binding protein of the fourth component of complement: structural polymorphism and its linkage to the major histocompatibility complex.
Authors: Kaidoh T , Natsuume-Sakai S , Takahashi M
Published: May 2006

The binding protein of the fourth component of complement (C4-BP) is a regulatory protein of the complement system with specific affinity for the fourth component. This paper describes a structural polymorphism of murine C4-BP and its linkage to the major histocompatibility complex of the mouse (H-2). After isoelectric focusing of whole mouse plasma in low-endosmosis agarose, C4-BP was demonstrated as a single precipitin band by overlaying monospecific antiserum on the agarose gel. Two C4-BP patterns were distinguished among many strains of mice on the basis of isoelectric point--C4-BP a type, which has a pH range of 6.5-7.0 (exemplified by B10.BR and B10.AKM), and C4-BP b type, which has a pH range of 6.3-6.6 (exemplified by B10 and B10.M). Genetic crosses between two strains bearing distinct C4-BP types demonstrate a C4-BP pattern representative of both types. A linkage study was carried out in which progeny of two backcross combinations--[(B10 X B10.BR)F1 X B10.BR] and [(B10.AKM X B10)F1 X B10.AKM]--were phenotyped for C4-BP type and serum fourth-component level. Results were obtained suggesting that C4-BP patterns are inherited by a single codominant locus (C4-Bp) linked to the H-2 complex. The recombination frequency between the C4-Bp locus and the S region was 0.017. By phenotyping appropriate intra-H-2 recombinants of three different backgrounds (B10, A, and HT), this locus was assigned to the right of the H-2D region.
Immunochemical delineation of an oncofetal antigen on normal and simian virus 40-transformed human fetal melanocytes.
Authors: Morgan A C , Galloway D R , Jensen F C , Giovanella B C , Reisfeld R A
Published: May 2006

Human melanoma cells of uveal origin shed 94,000- and 240,000-dalton glycoproteins in common with most melanoma cell lines of dermal origin. Normal human melanocytes derived from fetal uvea shed a 90,000-dalton glycoprotein that was found to be immunologically identical with the 94,000-dalton glycoprotein of melanoma cells. Expression of this 90,000-dalton molecule was confined to fetal cells of ectodermal origin. After simian virus 40 (SV40) transformation of human fetal melanocytes, there was an apparent increase in molecular size of this component to 94,000 daltons. In contrast, the 240,000-dalton glycoprotein was not synthesized or shed by uninfected or SV40-transformed fetal melanocytes. These data suggest that the 94,000-dalton glycoprotein is an oncofetal antigen of ectodermal origin.
Indole alkaloids: dihydroteleocidin B, teleocidin, and lyngbyatoxin A as members of a new class of tumor promoters.
Authors: Fujiki H , Mori M , Nakayasu M , Terada M , Sugimura T , Moore R E
Published: May 2006

Dihydroteleocidin B, which is a derivative of teleocidin from Streptomyces, showed potent tumor-promoting activity in vivo when painted on mouse skin. Although the chemical structure of dihydroteleocidin B is entirely different from those of phorbol esters, the tumor-promoting activity of dihydroteleocidin B was found to be comparable to that of 12-O-tetradecanoylphorbol 13-acetate (TPA) in vivo. Teleocidin from Streptomyces and lyngbyatoxin A and debromoaplysiatoxin from the marine blue-green alga Lyngbya majuscula induced ornithine decarboxylase activity when painted on mouse skin, their effects being similar to those of dihyroteleocidin B and TPA. 13-cis-Retinoic acid inhibited this ornithine decarboxylase induction when painted on the skin 1 hr before these natural products. These three compounds produced adhesion of human promyelocytic leukemia cells (HL-60) to the flasks and inhibited differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. The in vitro biological potencies of teleocidin and lyngbyatoxin A were almost as great as those of dihydroteleocidin B and TPA, but that of debromoaplysiatoxin was much weaker.
Solubilization of human platelet alpha-adrenergic receptors: evidence that agonist occupancy of the receptor stabilizes receptor--effector interactions.
Authors: Smith S K , Limbird L E
Published: May 2006

The alpha-adrenergic receptors of human platelet membranes can be directly identified by both a radiolabeled agonist, [3H]epinephrine, and a radiolabeled antagonist, [3H]yohimbine. Digitonin solubilizes a binding component from the membrane that is indistinguishable from the alpha-receptor identified in the native platelet membrane as assessed by (i) order of potency of agonists and antagonists and (ii) affinity of the receptor for [3H]-yohimbine and competing antagonists. However, the solubilized receptor demonstrates a reduced affinity for agonists and a loss of the ability of guanine nucleotides to modulate receptor affinity for agonists. Prelabeling of human platelet membranes with [3H]-epinephrine results in a guanine nucleotide-sensitive agonist-receptor complex that sediments more rapidly in sucrose gradients than do unoccupied or antagonist-occupied receptors. Thus, agonist occupancy of the alpha-receptor prior to membrane solubilization may promote or stabilize receptor interaction with effector components in the membrane, one of which may be the GTP regulatory protein responsible for modulation of receptor affinity.
DNA polymerase accuracy and spontaneous mutation rates: frequencies of purine.purine, purine.pyrimidine, and pyrimidine.pyrimidine mismatches during DNA replication.
Authors: Fersht A R , Knill-Jones J W
Published: May 2006

DNA from the am16 mutant of bacteriophage phi X174 may be replicated in vitro and expressed in vivo to give five classes of revertants. Each class may be specifically induced by the appropriate biasing of the concentrations of deoxynucleoside triphosphates in a predictable manner. The frequency of each reversion follows a kinetic rate equation relating it to the concentrations of the triphosphates involved in the substitution. The reversions corresponding to TAG leads to GAG, AAG, CAG, TGG, and TCG are calculated to occur with frequencies of 5 X 10(-7), 4 X 10(-7), 4 X 10(-7), approximately 2 X 10(-7), and approximately 5 X 10(-9), respectively, at the concentration of triphosphates found in vivo. The frequencies are in the range found for the reversion of the phage in vivo and so are consistent with errors in nucleotide selection by DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) III being largely responsible for the rate of spontaneous mutation in vivo. The relative frequency of mispairing leading to misincorporation is: purine.purine approximately purine.pyrimidine much greater than pyrimidine.pyrimidine, confirming predictions from model-building studies that transversions arise through purine.purine mismatches.
Induction of high serum levels of retroviral env gene products (gp70) in mice by bacterial lipopolysaccharide.
Authors: Hara I , Izui S , McConahey P J , Elder J H , Jensen F C , Dixon F J
Published: May 2006

In the present study, mice each given a single intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) responded with increased serum levels of the major envelope glycoprotein, gp70, of endogenous retrovirus. Concentrations of gp70 in their sera began to increase 4 hr after LPS injection, reached maximal 5- to 15-fold increases after 12--24 hr, and returned to the preinjection levels within 3 days. This response occurred only in the strains characterized by high base line levels of serum gp70 (greater than 10 micrograms/ml) such as NZB, NZB X NZW F1, BXSB, MRL, NZW, DBA/2, LG, 129(GIX+), and C57BL/6(GIX+). However, strains such as DBA/1, C3H/St, BALB/c, C57BL/6(GIX-), and 129(GIX-) with lower base line levels of serum gp70 (less than 5 micrograms/ml) made little or no response. This serum gp70 induced by LPS was structurally similar to the gp70 of NZB xenotropic virus that is dominantly expressed in sera from virtually all strains of mice. However, (i) the induced gp70 was virion-free; (ii) xenotropic virus was not isolatable from BXSB, MRL/1, or 129(GIX+) mice injected with LPS; and (iii) amounts of the major structural viral protein, p30, did not increase correspondingly in sera. All of these findings indicate that the increased expression of serum xenotropic viral gp70 in response to LPS did not result from activation of replication-competent xenotropic virus. In addition, the serum gp70 response to LPS was abolished by simultaneous inoculation of an inhibitor of protein synthesis, D-galactosamine. These results strongly suggest that LPS selectively stimulates synthesis of the env gene product, gp70, of NZB xenotropic virus but other viral gene products.
Intracellular disruption of rat heart lysosomes by leucine methyl ester: effects on protein degradation.
Authors: Reeves J P , Decker R S , Crie J S , Wildenthal K
Published: May 2006

Perfusion of rat hearts with Krebs--Henseleit medium containing 10 mM L-leucine methyl ester leads to swelling of lysosomes and loss of lysosomal integrity within 30-60 min. No morphological changes can be detected in the nuclei, mitochondria, sarcoplasmic reticulum, or Golgi complex as a result of the treatment with leucine methyl ester, and the hearts continue to beat normally during the treatment period. Homogenates of rat hearts perfused with the methyl ester exhibit a decrease in the sedimentability of cathepsin D activity compared to controls, thus providing additional evidence for a loss of lysosomal integrity. Swelling and disruption of the lysosomes presumably occurs because of the extensive accumulation of leucine within the organelles resulting from the intralysosomal hydrolysis of the freely permeating methyl ester. The lysosomal dysfunction that occurs with exposure to leucine methyl ester produces a 30% decrease in cardiac protein degradation. These results provide an estimate of the contribution of lysosomes to total protein degradation in the rat heart, and they also suggest that the enzymes released as a result of lysosomal disruption are relatively inactive in hydrolyzing cellular constituents under the perfusion conditions used here. The use of amino acid methyl esters to produce rapid, specific loss of lysosomal integrity in situ provides an approach to the study of lysosomal function in intact cells.
Specific transcriptional initiation in vitro on murine type C retrovirus promoters.
Authors: Ostrowski M C , Berard D , Hager G L
Published: May 2006

We have investigated the ability of molecularly cloned murine type C retroviral DNA to direct accurate initiation of RNA synthesis when added to cell-free extracts. Two different cloned proviruses were used. The first was derived from an integrated molecule of AKR murine leukemia virus and contains adjacent host information. The origin of the second was an unintegrated permuted copy of Harvey murine sarcoma virus. We found that the leukemia virus cloned provirus, as predicted by structural considerations, contained two functional RNA polymerase II promoters located in the U3 region present at either end of the molecule. These promoters initiate transcription at equal rates in vitro. We also found that the permuted sarcoma virus clone contained an RNA polymerase II promoter in the U3 region. Removal of viral sequences 49 bases upstream of the in vitro sarcoma virus initiation site by restriction cleavage results in loss of specific transcription, indicating a role for this information in in vitro promotion. The 5' ends of in vitro and in vivo viral RNA were compared by nuclease mapping techniques and found to be identical. Based on this evidence, we conclude that murine retroviral genomes contain sufficient information to initiate transcription independent of any host information in vitro and that these viral promoters are probably also active in vivo. In addition to the promoter in U3, Harvey murine sarcoma virus contains a second promoter in vitro that initiates near the 5' boundary of the transformation-specific (src) region of the virus. Initiation by this promoter was insensitive to low levels of alpha-amanitin, and the RNA transcript could be terminated to yield a 340-nucleotide product.
Direct immunoassay for detecting Escherichia coli colonies that contain polypeptides encoded by cloned DNA segments.
Authors: Kemp D J , Cowman A F
Published: May 2006

We describe a simple in situ immunoassay for screening Escherichia coli colonies, to detect those that express polypeptide antigens encoded by cloned DNA segments. In the colony immunoassay that we have developed, only one antibody molecule need bind to the polypeptide molecule. Hence short fragments of coding sequences should be detectable, and problems imposed by the conformation of polypeptides "fused" to protein fragments encoded by the vector should be minimized. The method should allow the use of monoclonal antibodies, which bind to only one determinant. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter papers. Antigens covalently bound to the CNBr paper were detected by reaction with antiserum, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. We have used the colony immunoassay to determine what proportion of ptrpED5-1 clones containing mouse mu chain cDNA segments express mu polypeptides. Surprisingly, about 50% of the clones were positive. Because only one in six clones would be expected to contain the insert in the correct orientation and reading frame to encode a fused polypeptide, it is likely that polypeptides that initiate within the insert can be detected. A similarly high proportion of pBR322 clones containing mu cDNA were positive, and about 25% of pBR322 clones containing mouse gamma 2a chain cDNA were positive to anti-gamma 2a serum. EAch of the positive mu clones reacted with affinity-purified anti-mu antibodies but not with normal serum, and clones containing irrelevant sequences were negative. The mu cDNA segment was inserted in the correct orientation in all six clones studied, and discrete mu polypeptides corresponding in size both to fused polypeptides and internally initiated polypeptides were detected by electrophoresis of protein extracts.
23,25-Dihydroxyvitamin D3: a natural precursor in the biosynthesis of 25-hydroxyvitamin D3-26,23-lactone.
Authors: Tanaka Y , DeLuca H F , Schnoes H K , Ikekawa N , Eguchi T
Published: May 2006

To elucidate the biosynthesis of 25-hydroxyvitamin D3-26,23-lactone, two known metabolites of 25-hydroxyvitamin D3--23,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3--were incubated individually with kidney homogenate prepared from vitamin D-supplemented chickens, a preparation known to produce the lactone from 25-hydroxyvitamin D3. The 25-hydroxyvitamin D3-26,23-lactone produced in vitro was then separated, purified, identified, and quantitated by consecutive straight-phase and reverse-phase high-performance liquid chromatography. 23,25-Dihydroxyvitamin D3 is a far better substrate for production of 25-hydroxyvitamin D3-26,23-lactone than is 25,26-dihydroxyvitamin D3. Production of lactone is highly selective for the natural 23(S)-hydroxy-23,25-dihydroxyvitamin D3 while both epimers of 25,26-dihydroxyvitamin D3 resulted in small amounts of product comigrating with natural lactone. It appears that 23(S),25-dihydroxyvitamin D3, but not 25,26-dihydroxyvitamin D3, is a natural precursor in the synthesis of 25-hydroxyvitamin D3-26,23-lactone; this result also implies that the configuration of the lactone at C-23 is S.
Single K+ channel currents of anomalous rectification in cultured rat myotubes.
Authors: Ohmori H , Yoshida S , Hagiwara S
Published: May 2006

The currents through single K+ channels of the anomalous (or inward) rectifier were recorded in tissue cultured rat myotubes by using the "gigohm seal" patch clamp technique developed by Sigworth and Neher. These unitary currents were detected as current fluctuations due to the blocking and unblocking of channels by Ba2+. The single-channel conductance was obtained from the slope of the linear relationship between unitary current amplitude and membrane potential. When the external solution contained 155 mM K+, the single-channel conductance was 10.4 +/- 2.6 pS (+/- SD; n = 6). This value was independent of the the concentration of blocking ions but increased with increasing external K+ concentration. The behavior of the unitary current agreed with that expected from the blocking kinetics of Ba2+ on the macroscopic K+ current of the anomalous rectifier. The density of the channel is likely to be small and may even be less than 1/micrometers 2.