bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies to the hemagglutinin Sa antigenic site of a/pr/8/34 influenza virus distinguish biologic mutants of swine influenza virus.
Authors: Kilbourne E D , Gerhard W , Whitaker C W
Published: May 2006

The dimorphic L and H hemagglutinin mutants of A/NJ/11/76(H1N1) (swine) influenza virus differ pleiotropically in their replication and virulence characteristics and in their antigenicity. L mutants replicate less well in chicken embryos and Madin-Darby canine kidney cells and are more infective for swine than are H mutants. L and H mutants are not antigenically distinguishable in cross-neutralization tests with homotypic antisera, but they can be identified with certain heterotypic heterogeneous antisera. The present studies demonstrate that two monoclonal antibodies (Sa-5 and Sa-13) to the Sa antigenic site of the hemagglutinin of A/PR/8/34H1N1 influenza virus react with mutants and viral reassortants containing the H hemagglutinin in radioimmunoassay, neutralization, and hemagglutination-inhibition tests but to a lesser degree or not at all with L mutants and reassortants. Conversely, monoclonal antibody (9C8) to the L mutant does not react with H mutants. L to H and H to L revertants, whether or not selected with monoclonal antibody, demonstrate concomitant change in biological and antigenic phenotype. Reactivity of H mutants with Sa monoclonal antibodies localizes the mutational site to a position on the hemagglutinin near the receptor binding site--a position in which single amino acid changes could readily influence both antigenic and biologic activity.
Comparative localization of acetylcholinesterase and pseudocholinesterase during morphogenesis of the chicken brain.
Authors: Layer P G
Published: May 2006

The histochemical localization of specific acetylcholinesterase (AcChoEase) and nonspecific cholinesterase (BtChoEase) is described during the early morphogenesis of the whole chicken head with main emphasis on the visual system. It is found that: (i) Expression of AcChoEase is an early differentiation event in the entire brain. Its pattern of first appearance on the external part of the neuroepithelium correlates with the general spatio-temporal pattern of differentiation. AcChoEase thus represents an early differentiation marker. (ii) The late pattern of AcChoEase (at E18), reflecting at least partially the distribution of synaptic AcChoEase shows no direct correlation to the distributions found at early stages when synapses are not yet formed. This argues for a nonsynaptic function of the early appearing AcChoEase. (iii) BtChoEase in early nervous tissue is diffusely localized on the ventricular side of the neural tube. At later stages it becomes concentrated on the ependymal layer as well as along fibers reaching from this inner layer outwards. Minor activities appear in specific external layers of tectum and retina. (iv) During the course of differentiation the enzymes express pronounced graded distributions within the areas where they are detectable. (v) Mesenchymal and epidermal BtChoEase is abundant in the entire head. Prominent amounts of activity are expressed on the rostral epidermis, along the eye cup next to the sclera, and on the rostro-dorsal surrounding of the optic nerves. The results are discussed in the light of possible morphogenetic functions of these enzymes.
At least six forms of extremely homologous cytochromes P-450 in rat liver are encoded at two closely linked genetic loci.
Authors: Rampersaud A , Walz F G
Published: May 2006

A subpopulation of phenobarbital-induced cytochrome P-450 in rat liver has been shown to consist of four closely related forms of the enzyme that appeared to be strain-related. In the present study, polypeptides composing this family were analyzed by two-dimensional electrophoresis of hepatic microsomes from 64 individual phenobarbital-treated rats. The animals surveyed included both sexes from four inbred and five outbred strains/colonies and F1 progenies from 10 crosses. Two new members of this polypeptide family were identified on the basis of their unique electrophoretic behavior and peptide maps. Eight phenotypes were observed that consisted of two to four member polypeptides. The six closely related cytochromes P-450 were found to be encoded at two genetic loci with at least four alleles at the P-450b locus and at least two alleles at the P-450e locus. Most colonies of outbred strains were characterized by polymorphism at one or both of these loci, and in no case did they contain unique alleles. Analyses of parents and their F1 progenies indicated that the P-450b and P-450e loci are closely linked on the same autosome and are expressed codominantly. Furthermore, the products of these loci appear to be coordinately regulated. The extreme homology between P-450b and P-450e genes, their high degree of polymorphism, and their close linkage suggest that they are subject to the same genetic mechanisms that maintain these features in other multigene families.
Biological significance of carbohydrate chains on monoclonal antibodies.
Authors: Nose M , Wigzell H
Published: May 2006

We have prepared monoclonal hapten-specific mouse IgG2b antibodies depleted of asparagine-linked carbohydrate chains by treating the hybridoma cells with tunicamycin. The carbohydrate-deficient antibodies behaved in an identical manner to the normal antibodies with regard to fine antigen-binding reactivity (a Fab fragment feature) and protein A binding capacity [a feature requiring integrity at the CH2 and CH3 domain-interaction regions in the constant region of the heavy chain (CH)]. However, they lost the ability to activate complement, to bind to Fc receptors on macrophages, and to induce antibody-dependent cellular cytotoxicity. Furthermore, antigen-antibody complexes produced from such carbohydrate-deficient antibodies failed to be eliminated rapidly from the circulation. We conclude that removal of carbohydrate chains from IgG molecules may have a profound and highly select impact on the biological activity to these antibodies.
Mechanisms of hydroxylation by cytochrome P-450: metabolism of monohalobenzenes by phenobarbital-induced microsomes.
Authors: Burka L T , Plucinski T M , Macdonald T L
Published: May 2006

The monohydroxylation of halobenzenes by phenobarbital-induced rat liver microsomes was studied. The p-halophenol was found to be the major metabolite from all four halobenzenes; o-halophenol formation decreased as the halogen atom size increased. Vmax for total hydroxylation (ortho and para products) correlated well with the sigma + Hammett constant with a negative rho value. This implies a positively charged intermediate in the rate-determining step. Vmax for either ortho or para hydroxylation alone did not correlate with a Hammett constant, implying that the product-determining step occurs after the rate-determining step. Rate-determining formation of a radical cation intermediate is postulated to explain this data.
Metabolism of tobacco-specific N-nitrosamines by cultured human tissues.
Authors: Castonguay A , Stoner G D , Schut H A , Hecht S S
Published: May 2006

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are present in cigarette smoke and snuff and are carcinogens in laboratory animals. In tobacco smokers, the buccal mucosa, trachea, esophagus, bronchi, and peripheral lung are exposed to smoke containing significant amounts of these N-nitrosamines. The results of the present study demonstrate that explants of these tissues as well as of the urinary bladder have the capacity to metabolize NNN and NNK by alpha-carbon hydroxylation. This metabolic pathway yields alkyldiazohydroxides, which are reactive and DNA-damaging electrophiles. The extent of alpha-carbon hydroxylation of NNN and NNK in human tissues was only 1/10th to 1/100th of that in animal tissues. Although the levels of alpha-carbon hydroxylation of NNN among different tissues of the same individual were similar, a 10-fold variation among individuals was observed. Reduction of the NNK carbonyl group was a major metabolic pathway observed with all human explants and may occur in the surface epithelia of the respiratory tract of smokers. These results provide further evidence that tobacco-specific N-nitrosamines could play a role in cancers related to the smoking and chewing of tobacco.
Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex.
Authors: Levin E G
Published: May 2006

Conditioned medium from cultures of human umbilical vein endothelial cells was analyzed for the presence of tissue plasminogen activator (tPA) and urokinase. Immunoprecipitation studies using metabolically labeled conditioned medium and anti-tPA IgG revealed a single band on autoradiographs corresponding to a Mr of 100,000. No bands were observed after immunoprecipitation with anti-urokinase IgG. The Mr 100,000 tPA was found to be inactive and did not bind to fibrin clots. However, exposure of this tPA to 1% NaDodSO4 resulted in the appearance of plasminogen activator activity with no apparent change in its Mr. Treatment with 10 mM diisopropylfluorophosphate prior to NaDodSO4 activation did not inhibit the NaDodSO4-induced appearance of plasminogen activator activity, indicating that the active site was not available for diisopropylfluorophosphate binding. The possibility that the properties of this Mr 100,000 tPA reflected a tPA-inhibitor complex was examined. Attempts to dissociate such a complex by denaturation, reduction, or extremes of temperature were not successful. However, after treatment of conditioned medium with 1 M hydroxylamine in the presence of 0.1% NaDodSO4, the Mr of the anti-tPA immunoprecipitable material declined by 40,000 to Mr 60,000, a Mr consistent with that of other human tPAs. Hydroxylamine has been shown previously to dissociate covalently coupled serine protease-inhibitor complexes. Furthermore, incubation of purified human melanoma cell tPA with conditioned medium resulted in an increase in its Mr by 40,000 with a concomitant decline in tPA activity. The data suggest that the latent tPA present in the conditioned medium of endothelial cells is composed of a Mr 60,000 tPA associated with an inhibitor.
Affinity labeling and partial purification of nerve growth factor receptors from rat pheochromocytoma and human melanoma cells.
Authors: Grob P M , Berlot C H , Bothwell M A
Published: May 2006

A cell surface glycoprotein receptor for nerve growth factor (NGF) has been identified by covalent crosslinking to 125I-labeled NGF (125I-NGF). Either ethyldimethylisopropyl-aminocarbodiimide or hydroxysuccinimidyl-p-azidobenzoate causes highly specific crosslinking of 125I-NGF to a similar receptor species on rat pheochromocytoma PC12 cells and on human melanoma A875 cells. The NGF-receptor complex migrates as a broad band in NaDodSO4/polyacrylamide gel electrophoresis with an apparent Mr of approximately equal to 100,000. Because the NaDodSO4-denatured complex apparently contains a single Mr 13,000 NGF chain, the apparent molecular weight of the receptor itself is 87,000. Inhibition of protein glycosylation by tunicamycin generates smaller 125I-NGF-receptor complexes. The mobility of the smallest of these in NaDodSO4 gel electrophoresis corresponds to a Mr of 90,000 for the complex and, hence, 77,000 for the carbohydrate-deficient receptor. A second NGF-receptor complex with a Mr of 225,000 also is obtained from A875 cells but only occasionally from PC12 cells. Tunicamycin treatment decreases the molecular weight of these species by 10,000-15,000. Substantial purification of the Mr 100,000 NGF-receptor complex was achieved by lectin affinity chromatography on columns of wheat germ agglutinin linked to Affi-Gel 15. The specific absorption of NGF-receptor complexes to these columns indicates that the receptor is a glycoprotein that contains N-acetyl-D-glucosamine.
Comparative risk analysis of technological hazards (a review).
Authors: Kates R W , Kasperson J X
Published: May 2006

Hazards are threats to people and what they value and risks are measures of hazards. Comparative analyses of the risks and hazards of technology can be dated to Starr's 1969 paper [Starr, C. (1969) Science 165, 1232-1238] but are rooted in recent trends in the evolution of technology, the identification of hazard, the perception of risk, and the activities of society. These trends have spawned an interdisciplinary quasi profession with new terminology, methodology, and literature. A review of 54 English-language monographs and book-length collections, published between 1970 and 1983, identified seven recurring themes: (i) overviews of the field of risk assessment, (ii) efforts to estimate and quantify risk, (iii) discussions of risk acceptability, (iv) perception, (v) analyses of regulation, (vi) case studies of specific technological hazards, and (vii) agenda for research. Within this field, science occupies a unique niche, for many technological hazards transcend the realm of ordinary experience and require expert study. Scientists can make unique contributions to each area of hazard management but their primary contribution is the practice of basic science. Beyond that, science needs to further risk assessment by understanding the more subtle processes of hazard creation and by establishing conventions for estimating risk and for presenting and handling uncertainty. Scientists can enlighten the discussion of tolerable risk by setting risks into comparative contexts, by studying the process of evaluation, and by participating as knowledgeable individuals, but they cannot decide the issue. Science can inform the hazard management process by broadening the range of alternative control actions and modes of implementation and by devising methods to evaluate their effectiveness.
Kinetic characterization and regulation of phosphoenolpyruvate-dependent methyl alpha-D-glucopyranoside transport by Salmonella typhimurium membrane vesicles.
Authors: Liu K D , Roseman S
Published: May 2006

Membrane vesicles from Salmonella typhimurium SB3507 were used to study the kinetics of methyl alpha-D-glucopyranoside (MeGlc) transport by the phosphoenolpyruvate: glycose phosphotransferase system (PTS). During the first minute of phosphoenolpyruvate-dependent MeGlc transport, two distinct rates were observed; an initial rapid rate, V1 (Vmax, 7.4-8.4 nmol X mg-1 X min-1; Km, 8.2-11.2 X 10(-6)M), followed by a second slower rate, V2 (Vmax, 4-4.6 nmol X mg-1 X min-1; Km, 3.4-6.4 X 10(-6) M). The change in rate occurred when the intravesicular MeGlc phosphate concentration was 0.2 mM or less, depending on the external MeGlc concentration. The rate-limiting component in MeGlc transport was found to be enzyme II-BGlc, not phosphoenolpyruvate uptake or the PTS proteins enzyme I, HPr, and IIIGlc. The change from V1 to V2 thus suggests that the PTS is regulated in intact vesicles. However, this regulation was completely relieved by permeabilizing the vesicles with toluene. That is, the toluene-treated vesicles showed only V1 for MeGlc phosphorylation. Evidence was obtained to show that pyruvate and its metabolic products generated by the vesicles exerted no effect on the rate of MeGlc transport. Furthermore, the result from a dual-label experiment excluded exchange transphosphorylation as the mechanism for regulating MeGlc transport by the vesicles. Possible mechanisms for regulation of the PTS are discussed.
Translational diffusion in phospholipid monolayers measured by fluorescence microphotolysis.
Authors: Peters R , Beck K
Published: May 2006

A method is described that eliminates surface flow in monolayers at the air-water interface and makes possible diffusion measurements by fluorescence microphotolysis ("photobleaching"). In contrast to previous studies that did not account for surface flow, lipid probe diffusion has been found to be similar in densely packed monolayers and in related bilayers. Furthermore, it seems that lipid diffusion is based on the same molecular mechanism in monolayers, bilayers, and potentially also cell membranes. In monolayers of L-alpha-dilauroylphosphatidylcholine (Lau2-PtdCho) the translational diffusion coefficient D of the fluorescent lipid probe N-4-nitrobenzo-2-oxa-1,3 diazole egg phosphatidylethanolamine decreased from 110 microns2/s at a surface pressure II = 1 mN/m to 15 microns2/s at II = 38 mN/m (T = 21-22 degrees C). Data could be fitted by the "free volume model." In monolayers of L-alpha-dipalmitoylphosphatidylcholine (Pam2-PtdCho) D decreased by greater than 3 orders of magnitude upon increasing II at constant temperature, thus indicating a fluid-to-crystalline phase transition. In Lau2-PtdCho/Pam2-PtdCho monolayers phase separation has been visualized in the fluorescence microscope and the effect on D measured. These results suggest that monolayers are a promising model system for studying the molecular mobility of lipids and other cell membrane components.
Site-specific recombination of yeast 2-micron DNA in vitro.
Authors: Vetter D , Andrews B J , Roberts-Beatty L , Sadowski P D
Published: May 2006

Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two 599-base-pair precise inverted repeats across which a site-specific inversion event occurs in vivo. This inversion is promoted by a plasmid-encoded function called "FLP." We have cloned the FLP gene of 2-micron DNA under control of a strong yeast promoter and transformed yeast cells with a plasmid containing the cloned FLP gene. Cell-free extracts from such a transformant promote highly efficient inversion of 2-micron DNA in vitro. The reaction requires a cation and works efficiently on supercoiled, relaxed circular, or linear DNA. The FLP activity bears certain similarities to the cre protein, a site-specific recombinase encoded by bacteriophage P1.
Amplified C lambda and c-abl genes are on the same marker chromosome in K562 leukemia cells.
Authors: Selden J R , Emanuel B S , Wang E , Cannizzaro L , Palumbo A , Erikson J , Nowell P C , Rovera G , Croce C M
Published: May 2006

The human leukemia cell line K562, derived from a patient with Philadelphia chromosome-positive chronic myelogenous leukemia, contains amplified c-abl oncogenes and unrearranged C lambda genes. Using in situ hybridization techniques, we have determined that the amplified c-abl and C lambda DNA sequences of K562 cells are both located on the same abnormal acrocentric marker chromosome, which may represent an altered Philadelphia chromosome.
Sequences essential for transposition at the termini of IS50.
Authors: Sasakawa C , Carle G F , Berg D E
Published: May 2006

The DNA sequences found repeated in opposite orientation at the ends of insertion (IS) elements are thought to contain sites at which transposase proteins act during transposition. Many elements have repeats of at least 15 base pairs (bp). Those of IS50 are quite short, however: just 8 of the first 9 bp. Functional tests had indicated that one end of IS50 is more effective in transposition than the other end and suggested that at least one of the recognition sites of IS50 extends beyond the common 8/9 bp. To determine the lengths of recognition sites of IS50 we mutagenized IS50 in vitro and tested the transposition proficiency of the resulting mutants. Our results show that the recognition sites at each end of IS50 are about 19 bp long. These findings suggest models for the evolution of IS elements from simpler immobile gene complexes.
Fibrinogen Baltimore II: congenital hypodysfibrinogenemia with delayed release of fibrinopeptide B and decreased rate of fibrinogen synthesis.
Authors: Ebert R F , Bell W R
Published: May 2006

A congenital hypodysfibrinogenemia, fibrinogen Baltimore II, was found in a young asymptomatic Caucasian female. Prothrombin, partial thromboplastin, and euglobulin lysis times were normal, as were platelet function and coagulation factor assays. Subnormal plasma fibrinogen levels were found using chronometric, rate-independent, and immunologic assay methods. Kinetic analysis of fibrinopeptide release revealed a delay in the thrombin-catalyzed release of fibrinopeptide B from the abnormal protein. Proteolysis of fibrinopeptide A by thrombin or Arvin, fibrin monomer polymerization, and fibrin polymer ligation occurred at normal rates. Catabolism of radiolabeled autologous and homologous fibrinogen was also normal, but the fibrinogen synthetic rate was less than half the normal value. Comparison of the coagulation characteristics of fibrinogen Baltimore II with those of other abnormal fibrinogens indicates that it represents a unique example of hypodysfibrinogenemia.
Preferential masking by the receptor of immunoreactive sites on the alpha subunit of human choriogonadotropin.
Authors: Milius R P , Midgley A R , Birken S
Published: May 2006

125I-Labeled human choriogonadotropin (125I-hCG) bound to rat ovarian receptor was solubilized in Triton X-100. By using increasing concentrations of nine different antisera specific for the individual subunits of human choriogonadotropin (hCG), free 125I-hCG or 125I-hCG-receptor complex was precipitated by double-antibody technique. The ability of any antiserum to bind to the hormone-specific beta subunit was not affected by hCG binding to receptor, suggesting that this subunit is not directly involved with the receptor in the final state of the hormone-receptor complex. In contrast, every antiserum specific for the alpha subunit was dramatically inhibited in binding to the solubilized 125I-hCG-receptor complex. These results suggest that the alpha subunit directly interacts with the receptor, thereby masking immunoreactive sites normally available on the free hormone. Because a number of reports describe binding activity of high concentrations of immunopurified beta subunits of hCG, we propose a two-step model for the binding of hCG to receptor and postulate separate and distinct roles for the subunits. We propose that the binding of hCG to the receptor involves a specific low-affinity initial interaction of the beta subunit with the receptor that activates a second site for the high-affinity binding of alpha subunit and stabilization of the hormone-receptor complex.
Both hypoxanthine and adenosine stimulate DNA synthesis independently in serum-starved L cells treated with platelet protein.
Authors: Klenow H , Flodgaard H
Published: May 2006

The effect of human platelet factors and purine derivatives on DNA synthesis has been investigated in mouse fibroblast-like L cells whose growth was arrested by serum starvation. When such cells were exposed to diluted platelet extract (e.g., 35 micrograms of protein per ml), a stimulatory effect on net DNA synthesis was observed. This effect was almost abolished by dialysis of the extract. The stimulation was, however, recovered by supplementing the diluted and dialyzed extract with hypoxanthine or adenosine. Similar phenomena were observed in pulse-labeling experiments performed with [3H]thymidine. In this case, however, there was a marginal stimulatory effect of adenosine or hypoxanthine alone. When the cells were treated with saturating concentrations of pure platelet-derived growth factor (PDGF), a stimulatory effect on pulse labeling was again obtained by the simultaneous presence of hypoxanthine or adenosine. In serum-starved cells of a mutant line of L cells deficient in hypoxanthine phosphoribosyltransferase, there was, however, no stimulatory effect on pulse labeling by hypoxanthine when it was added alone or together with either PDGF or diluted dialyzed platelet extract. It is suggested that the stimulation of DNA synthesis by the purine derivatives in the presence of a certain type of platelet proteins, probably involving PDGF, may be explained by their function as precursors for a purine ribonucleotide pool that is specifically related to DNA synthesis. Treatment of serum-starved L cells with high concentrations of dialyzed platelet extract (e.g., 240 micrograms of protein per ml) showed that platelets contain an additional type of factor that may substitute for the requirement of adenosine or hypoxanthine for DNA synthesis to take place. It is suggested that the effect of this type of factor may be to activate the catabolic activity of the purine salvage pathway.
Cloning of cDNA sequences of human adenosine deaminase.
Authors: Wiginton D A , Adrian G S , Friedman R L , Suttle D P , Hutton J J
Published: May 2006

Cloned cDNA sequences of human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been isolated from a cDNA library constructed in bacteriophage lambda gt10. The cDNA for the library was prepared from poly(A)+ RNA isolated from a human T-lymphoblast cell line, CCRF-CEM. The library was initially screened by differential plaque hybridization to labeled cDNA prepared from human T- and B-lymphoblast cell lines with a 21-fold difference in levels of translatable ADA mRNA. Two recombinants containing cloned cDNA sequences for ADA were identified by hybridization-selected translation. Both recombinants contained approximately 1,600 base pairs of inserted human DNA. Restriction maps of the two inserts were not identical. One contained approximately 40 base pairs of additional DNA toward the center of the cDNA. The cloned cDNA specifically hybridized to five fragments generated by HindIII digestion of human genomic DNA. It also hybridized to human lymphoblast RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast cell lines. ADA mRNA levels correlate closely with levels of ADA catalytic activity and ADA protein in cell lines containing structurally normal ADA. A leukemic T-lymphoblast line produced 6 to 9 times as much ADA protein and ADA mRNA as transformed B-lymphoblast lines. Two mutant B-lymphoblast lines from patients with hereditary ADA deficiency contained unstable ADA protein but had 3 to 4 times the normal level of ADA mRNA.
Early sporulation gene spo0F: nucleotide sequence and analysis of gene product.
Authors: Shimotsu H , Kawamura F , Kobayashi Y , Saito H
Published: May 2006

We have determined the sequence of a 1,162-base-pair DNA fragment containing a spo0F gene which is required for an early stage of sporulation in Bacillus subtilis. The sequence has only one long open reading frame consisting of 173 codons, which has been confirmed to be the spo0F cistron by DNA-mediated transformation and in vitro transcription. In UV-irradiated "maxicells" containing pUBSF13, the plasmid that carries cloned spo0F gene, we have observed the synthesis of a 20-kilodalton polypeptide that is absent from cells carrying a vector plasmid pUB110. The molecular weight of this protein is in agreement with the calculated molecular weight of the spo0F gene product (Mr, 19,065). The putative promoter sequences of spo0F gene were 5' T-A-T-A-A-T 3' at -10 and 5' T-T-G-A-T-T 3' at -35. An octamer sequence, 5' A-A-A-G-G-A-G-G 3', situated 8 base pairs prior to the initiation codon was found to be perfectly complementary with the 3' end of 16S ribosomal RNA. This result offers additional evidence for the proposal by Rabinowitz's group that an extensive mRNA-rRNA interaction is a requirement for efficient translation by B. subtilis ribosomes.
Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay.
Authors: Arendes J , Kim K C , Sugino A
Published: May 2006

Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-microns plasmid DNA templates. A crude extract from a S. cerevisiae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42 degrees C in vitro. This heat-inactivated extract was fully complemented by the addition of either wild-type or cdc8-1 single-stranded DNA binding protein (SSB). restoration by SSB of the activity of the mutant cell extract allowed replication like that of a wild-type crude extract, as shown by bidirectional DNA synthesis from the in vivo origin. The DNA binding protein specifically stimulates the reaction catalyzed by yeast DNA polymerase I, a true DNA replicase, using the hybrid of phi X174 single-stranded DNA and a restriction endonuclease fragment as a template. It also increases processivity of DNA polymerase I at least 10-fold. Escherichia coli SSB, but not T4 gene 32 protein, can substitute for yeast SSB. Both restoration of DNA synthesis in the heated mutant cell extract and stimulation of the DNA polymerase I reaction by SSB from cdc8-1 cells are inactivated at nonpermissive temperatures, suggesting that yeast SSB is the CDC8 gene product.
Localization of different photosystems in separate regions of chloroplast membranes
Authors: Anderson Jan M. , Melis Anastasios
Published: May 2006

The stoichiometric amounts and the photoactivity kinetics of photosystem I (PSI) and of the α and β components of photosystem II (PSII
α
and PSII
β
) were compared in spinach chloroplast membrane (thylakoid) fractions derived from appressed and nonappressed regions. Stroma-exposed thylakoid fractions from the nonappressed regions were isolated by differential centrifugation following a mechanical press treatment of the chloroplasts. Thylakoid vesicles derived mainly from the appressed membranes of grana were isolated by the aqueous polymer two-phase partition method. Stroma-exposed thylakoids were found to have a chlorophyll
a
/chlorophyll
b
ratio of 6.0 and a PSII
β
/PSI reaction center ratio of 0.3. Kinetic analysis of system II photoactivity revealed the absence of PSII
α
from stroma-exposed thylakoids. The photoactivity of system I in stroma-exposed thylakoids showed a single kinetic component identical to that of unfractionated chloroplasts, suggesting that PSI does not receive excitation energy from the PSII-chlorophyll
ab
light-harvesting complex. Thus, stroma-exposed thylakoids are significantly enriched in both PSI and PSII
β
. Inside-out vesicles from the appressed membranes of grana-partition regions had a chlorophyll
a
/chlorophyll
b
ratio of 2.0 and a PSII/PSI reaction center ratio of 10.0. The photoactivity of system II showed the membranes of the grana-partition regions to be significantly enriched in PSII
α
. We conclude that PSII
α
is exclusively located in the membranes of the grana partitions while PSII
β
and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles derived from grana partitions and the kinetic homogeneity of the PSI complex suggest total exclusion of P700 as a functional component in the membrane of the grana-partition region.
Calcium/calmodulin-dependent phosphorylation of vimentin in rat sertoli cells.
Authors: Spruill W A , Zysk J R , Tres L L , Kierszenbaum A L
Published: May 2006

Ca2+-dependent protein phosphorylation and the role of calmodulin in this process was investigated in subcellular fractions of primary cultures of rat Sertoli cells. Significant Ca2+/calmodulin-dependent protein phosphorylation in Sertoli cells was restricted to the cytosol fraction. The calmodulin dependence of these effects was confirmed by using the calmodulin inhibitor trifluoperazine. One of the Ca2+/calmodulin-dependent phosphoproteins was identified as the intermediate filament protein vimentin, based on the following criteria: (i) migration pattern in two-dimensional polyacrylamide gels, (ii) Ca2+/calmodulin-dependent phosphorylation of a 58-kilodalton protein present in detergent-insoluble intermediate filament protein extract of Sertoli cells, and (iii) peptide mapping of the phosphoprotein. These data support a role for Ca2+/calmodulin-dependent protein phosphorylation in the modulation of Sertoli cell cytoskeletal components.
Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.
Authors: Liu P K , Chang C C , Trosko J E , Dube D K , Martin G M , Loeb L A
Published: May 2006

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.
Polyploid nuclei in human artery wall smooth muscle cells.
Authors: Barrett T B , Sampson P , Owens G K , Schwartz S M , Benditt E P
Published: May 2006

Although polyploid nuclei have long been known to be present in many adult human tissues, the ploidy of smooth muscle cells in human artery wall has never been determined. We measured DNA content in individual smooth muscle cell nuclei of artery wall specimens by two means: Feulgen microdensitometry and flow microfluorimetry. A significant percentage of nuclei were polyploid; most of these were tetraploid, although higher levels were also found. The frequency of polyploidy varied with age from less than 1% at birth to a mean of 7% in adult aortic, carotid, and iliac vessels. Atherosclerotic plaques had a lower tetraploid content than the underlying media, whereas normal intima was similar to the corresponding media. The increase in frequency of hyperploid smooth muscle cell nuclei correlates with the normal growth, development, and aging of human artery wall. We suggest that the regular existence of a subset of polyploid smooth muscle cells may indicate an important functional role for this phenotype.
Relaxation processes for internal waves in mesoscale flow
Authors: Watson Kenneth M.
Published: May 2006

A dynamical “test-wave” model has been developed to study transport phenomena within oceanic internal wave fields. This model is extended here to describe effects of a mesoscale flow field on internal wave transport. Previous work with weak-interaction perturbation theory has suggested a substantial enhancement due to mesoscale currents. Extension to the strong-interaction regime in the present paper suggests a relatively small effect due to mesoscale interactions.
Homological versus algebraic equivalence in a jacobian
Authors: Harris Bruno
Published: May 2006

Let Z be an algebraic
p
cycle homologous to zero in an algebraic complex manifold
V
. Associated to Z is a linear function ν on holomorphic (2
p
+ 1)-forms on
V
, modulo periods, that vanishes if Z is algebraically equivalent to zero in
V
. I give a formula for ν for the case of
V
the jacobian of an algebraic curve
C
and Z=
C - C
′ (
C
′ = “inverse” of
C
′) in terms of iterated integrals of holomorphic 1-forms on
C
. If
C
is the degree 4 Fermat curve, I use this formula to show that
C - C
′ is not algebraically equivalent to zero.
Receptor-mediated phosphorylation of the hepatic insulin receptor: Evidence that the
M
r
95,000 receptor subunit is its own kinase
Authors: Van Obberghen Emmanuel , Rossi Bernard , Kowalski Aline , Gazzano Helene , Ponzio Gilles
Published: May 2006

Insulin stimulates the phosphorylation of its own receptor. In the work reported here, the kinase activity responsible for the insulin-stimulated phosphorylation of the insulin receptor was localized. In a first approach, partially purified insulin receptors derived from normal rat hepatocytes were immunoprecipitated with antibodies specific for the insulin receptor; thereafter, the immunoprecipitates were incubated with [γ-
32
P]-ATP in the absence or presence of insulin (1 μM). NaDodSO
4
/polyacrylamide gel electrophoretic analysis of the immunoprecipitates under reducing conditions revealed autophosphorylation of the β subunit (
M
r
95,000) of the insulin receptor; the α subunit (
M
r
130,000) was not phosphorylated. Further, insulin specifically increased 3- to 4-fold the labeling of its own receptor β subunit, indicating that anti-receptor antibodies precipitate a functional and insulin-stimulable protein kinase that appears to be independent of cyclic AMP and calcium. To localize more precisely the insulin receptor-related kinase activity, we searched for an ATP-binding site on solubilized insulin receptors. By using covalent labeling with oxidized [α-
32
P]ATP, a labeled polypeptide with precisely the same electrophoretic mobility as that of the β subunit of the insulin receptor (
M
r
95,000) was specifically immunoprecipitated with anti-receptor antibodies. Further, its appearance was prevented when the immunoprecipitation was preceded by incubation with unlabeled insulin. In conclusion, we have shown that an insulin-stimulated phosphorylation site and an ATP-binding site coexist on the β subunit of the insulin receptor. The simultaneous presence of these two sites on the same receptor subunit indicates that the insulin receptor acts as its own protein kinase.
Energetics of photosynthetic glow peaks
Authors: DeVault Don , Govindjee  , Arnold William
Published: May 2006

By postulating temperature-dependent equilibria between two or more electron carriers acting as traps for electrons or holes, it is possible to modify the Randall-Wilkins theory of thermoluminescence so as to explain the abnormally large apparent activation energies and apparent frequency factors observed in photosynthetic glow curves when fitted by unmodified Randall-Wilkins theory. The equilibria serve to inhibit the formation of the light-emitting excited state by withholding the needed precursor state. When the inhibition is released at higher temperature by shift of equilibrium with temperature, the rise of the glow peak can be much faster than would result from Arrhenius behavior based on the true activation energy and so appears to correspond to a higher activation energy accompanied by a larger frequency factor. From another viewpoint, the enthalpy changes, Δ
H
, of the equilibria tend to add to the activation energy. Similarly the entropy changes, Δ
S
, of the equilibria tend to add to the entropy of activation, giving the large apparent frequency factors. The positive values of Δ
S
needed would correspond to entropy decreases in the forward early electron transport. A comparison of the glow peaks obtained by different workers is also presented.
Antibodies of predetermined specificity for the NH2 terminus of a cellular protein p53 react with the native molecule: evidence for the presence of different p53s.
Authors: Luka J , Sternås L , Jörnvall H , Klein G , Lerner R
Published: May 2006

Two synthetic peptides corresponding to residues 1-20 and 10-20, respectively, of one type of a cellular protein called "p53" have been linked to a carrier protein and injected into rabbits to raise antibodies. The antibodies obtained were capable of reacting with the native protein, as judged by an enzyme-linked immunosorbent assay, protein A-linked staining of immunoblots after NaDodSO4 gel electrophoresis, and immunoprecipitation. The immunoassay titers against the protein were lower for these antibodies than for antisera derived from immunization with purified p53. However, staining with the immunoblot method showed that the antipeptide antibodies against p53 were uniquely specific. The data suggest that at least two different types of p53 molecules occur. The cellular protein previously isolated from human cells transformed by Epstein-Barr virus and from murine tumors induced by methylcholanthrene appears to be larger than the p53 reported in relation to simian virus 40- or adenovirus-transformed cells and to some other tumors. Some interrelationships have not been excluded, but it is clear that the two protein molecules do not behave identically. The reactions of the antipeptide antibodies with the intact protein have implications in regard to protein conformations. The strict specificities of such antibodies allow the generation of distinct sets of reagents useful for quantitation, purification, and cloning.
Antibodies of predetermined specificity detect two retroviral oncogene products and inhibit their kinase activities
Authors: Sen Stacy , Houghten Richard A. , Sherr Charles J. , Sen Arup
Published: May 2006

Oligopeptides predicted from the nucleotide sequence of the oncogene v-
fes
of feline sarcoma virus (FeSV) were synthesized chemically and used to generate specific antibodies. Antisera against a 12-amino-acid-long oligopeptide (12-mer) located 42 residues from the carboxyl terminus of the v-
fes
coding sequence efficiently recognized the transforming proteins encoded by Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of FeSV. This 12-mer also contains 10 amino acid residues homologous in order and position to those predicted from the nucleotide sequence of the oncogene v-
fps
of avian Fujinami sarcoma virus (FSV). The anti-12-mer immunoprecipitated the FSV-specific transforming protein molecules from FSV-transformed cells. Binding of these antipeptide antibody molecules to the v-fes and the v-fps gene products inhibited their associated tyrosine-specific protein kinase (EC 2.7.1.37) activities. The ability to generate such site-specific antisera to the products of related oncogenes will be valuable in the molecular characterization of retroviral transforming proteins and their normal cellular homologs.
Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding.
Authors: Young J D , Unkeless J C , Kaback H R , Cohn Z A
Published: May 2006

We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (delta psi) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in delta psi, but a sustained depolarization lasting approximately equal to 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+,K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.
Characterization of human serum spreading factor with monoclonal antibody.
Authors: Barnes D W , Silnutzer J , See C , Shaffer M
Published: May 2006

Serum spreading factor is a glycoprotein isolated from human serum that promotes spreading of a variety of cell types on culture dishes. We developed mouse hybridoma lines secreting monoclonal antibody to serum spreading factor that markedly inhibited the rate of serum spreading factor-promoted spreading of both fibroblastic and epithelial cells in culture. Fibronectin-promoted cell spreading was unaffected by monoclonal antibody to serum spreading factor, and the factor appeared to be distinct by several criteria from fibronectin or laminin. Human serum-promoted cell spreading was partially inhibited by monoclonal antibody to serum spreading factor. The antibody recognized primarily two forms of serum spreading factor that migrated in NaDodSO4/polyacrylamide gel electrophoresis in a manner consistent with molecular weights of 65,000-70,000 and 75,000-78,000. In addition to being found in plasma, serum spreading factor was also found associated with washed human platelets.
Reward learning in normal and mutant
Drosophila
Authors: Tempel Bruce L. , Bonini Nancy , Dawson Douglas R. , Quinn William G.
Published: May 2006

Hungry fruit flies can be trained by exposing them to two chemical odorants, one paired with the opportunity to feed on 1 M sucrose. On later testing, when given a choice between odorants the flies migrate specifically toward the sucrose-paired odor. This appetitively reinforced learning by the flies is similar in strength and character to previously demonstrated negatively reinforced learning, but it differs in several properties. Both memory consolidation and memory decay proceed relatively slowly after training with sucrose reward. Consolidation of learned information into anesthesia-resistant long-term memory requires about 100 min after training with sucrose compared to about 30 min after training with electric shock. Memory in wild-type flies persists for 24 hr after training with sucrose compared to 4-6 hr after training with electric shock. Memory in
amnesiac
mutants appears to be similarly lengthened, from 1 hr to 6 hr, by substituting sucrose reward for shock punishment. Two other mutants,
dunce
and
rutabaga
, which were isolated because they failed to learn the shock-avoidance task, learn normally in response to sucrose reward but forget rapidly afterward. One mutant,
turnip
, does not learn in either paradigm. Reward and punishment can be combined in olfactory discrimination training by pairing one odor to sucrose and the other to electric shock. In this situation, the expression of learning is approximately the sum of that obtained by using either reinforcement alone. After such training, memory decays at two distinct rates, each characteristic of one type of reinforcement.
Homologous upstream sequences near Epstein-Barr virus promoters.
Authors: Farrell P J , Deininger P L , Bankier A , Barrell B
Published: May 2006

The sequence of the 17,166-base-pair EcoRI C fragment of Epstein-Barr virus DNA, cell line B95-8, was determined. In vitro transcription was used to identify three RNA polymerase II promoters within this fragment of the virus. Cytoplasmic poly(A)+ RNAs starting at these points were demonstrated in B95-8 cells induced into virus production with 12-O-tetradecanoylphorbol 13-acetate. Uninduced B95-8 cells contained much less of these RNAs. The upstream sequences near the three promoters show striking homologies that may be involved in transcriptional control.