bluuebunny/crossref_metadata_2025 huggingface dataset:
Acyclovir inhibition of Epstein-Barr virus replication.
Authors: Datta A K , Colby B M , Shaw J E , Pagano J S
Published: May 2006

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] triphosphate inhibits Epstein-Barr virus (EBV)-associated DNA polymerase (DNA nucleotidyltransferase; EC 2.7.7.7) to a greater extent than it inhibits host alpha and beta DNA polymerases. The affinity of the compound for viral polymerase is 100-fold higher than for alpha-polymerase. The extent of inhibition is dependent upon the base composition of the template-primer. The inhibition is prevented by increasing concentrations of deoxyguanosine triphosphate. The EBV-associated DNA polymerase reaction in the presence of the inhibitor, although depressed, proceeds at a linear rate over a long period of time. In contrast, the reaction of Escherichia coli DNA polymerase I in the presence of 2',3'-dideoxythymidine 5'-triphosphate, a DNA chain terminator, levels off after initial linearity. Preincubation of acyclovir triphosphate with DNA and enzyme does not increase its inhibitory activity. The virus-producing cell line P3HRF-1 consistently shows reduced viral genome numbers and viral capsid antigen on prolonged exposure to acyclovir. The number of EBV genomes returns to the control level when the cells are grown in drug-free medium. The results suggest that a competitive mechanism is the major mode of acyclovir inhibition of EBV replication.
Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity.
Authors: Benson A M , Hunkeler M J , Talalay P
Published: May 2006

2(3)-tert-Butyl-4-hydroxyanisole (BHA) is one of several widely used antioxidant food additives that protect against chemical carcinogenesis and toxicity. The present report concerns the enhancement of dicoumarol-inhibited NAD(P)H:quinone reductase [NAD(P)H dehydrogenase (quinone); NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2] activity in mouse tissues in response to dietary administration of BHA. Cytosolic quinone reductase specific activity was increased significantly in 10 of 15 tissues examined from BHA-fed mice. The greatest proportionate increase, to 10 times control levels, was observed in liver. BHA also increased the quinone reductase activities of kidney, lung, and the mucosa of the upper small intestine severalfold. The increases of quinone reductase activities in liver and digestive tissues in response to BHA were comparable to the increases previously observed in glutathione S-transferase (EC 2.5.1.18) and epoxide hydratase (EC 3.3.2.3) activities. Quinones are among the toxic products of oxidative metabolism of aromatic hydrocarbons. NAD(P)H:quinone reductase exhibits broad specificity for structurally diverse hydrophobic quinones and may facilitate the microsomal metabolism of quinones to readily excreted conjugates. The protective effects of BHA appear to be due, at least in part, to the ability of this antioxidant to increase the activities in rodent tissues of several enzymes involved in the nonoxidative metabolism of a wide variety of xenobiotics.
Prolonged survival of bovine adrenal chromaffin cells in rat cerebral ventricles.
Authors: Perlow M J , Kumakura K , Guidotti A
Published: May 2006

Dispersed, cultured bovine adrenal chromaffin cells transplanted into the cerebral ventricles of neonatal and adult rats survived at least 2 mo without evidence of immunological rejection. The cells can be identified by their strong yellow fluorescent reaction with glyoxylic acid, which suggests that they maintain intact the capability of synthesizing and storing catecholamines. The cells did not show sprouting or process formation and appeared to be free in the ventricle or aggregated in clusters. This shows that cells from different animal species and from different tissue origins can be transplanted and can survive in the cerebral ventricles.
Localization of androgen-binding protein in proliferating Sertoli cells in culture.
Authors: Kierszenbaum A L , Feldman M , Lea O , Spruill W A , Tres L L , Petrusz P , French F S
Published: May 2006

The peroxidase and immunofluorescent localization patterns of androgen-binding protein (ABP), a biological marker of Sertoli cell function, have been examined in cultured Sertoli cells isolated from 20- to 22-day-old rats. ABP immunoreactivity in the form of cytoplasmic granules of variable diameter was observed in Sertoli cells with characteristic lipid droplets and a colony-forming, epithelial-like growth pattern. Incubation of cultures with [3H]thymidine demonstrated that Sertoli cells continue to produce ABP while retaining their capability for synthesizing DNA and undergoing mitosis. A variable number of cultured Sertoli cells became morphologically transformed after exposure to follitropin (follicle-stimulating hormone) and pharmacological agents acting on cyclic nucleotide metabolism. The induced change in Sertoli cell shape coincided with a disappearance of ABP-containing granules from the cytoplasm. These observations demonstrate that localization of ABP by immunological techniques is a valuable tool for the characterization of structural and functional properties of Sertoli cell in culture.
Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts.
Authors: Anderson W F , Killos L , Sanders-Haigh L , Kretschmer P J , Diacumakos E G
Published: May 2006

A mixture of two recombinant plasmids was microinjected into mouse thymidine kinase-negative fibroblasts (L cells). One plasmid contained the thymidine kinase gene of herpes simplex virus type I and the other contained the human beta globin gene. Seven fibroblast colonies arising from injected cells incubated in hypoxanthine/aminopterin/thymidine medium were analyzed. These microinjected cells were shown to: (i) produce functionally active herpes simplex type I thymidine kinase enzyme, (ii) replicate the human beta globin gene, and (iii) produce human beta globin mRNA sequences at low levels. Thus, the genetic defect (lack of thymidine kinase activity) was corrected by the microinjected thymidine kinase gene, and a coinjected human beta globin gene was replicated and weakly expressed.
Development and characterization of allospecific long-term human cytolytic T-cell lines.
Authors: Reiss C S , Hemler M E , Englehard V H , Mier J W , Strominger J L , Burakoff S J
Published: May 2006

Two long-term human cytolytic T lymphocyte (CTL) lines (VE and JR), whose cytolytic activity was dependent upon both irradiated JY cells (the stimulating alloantigen) and T-cell growth factor, were established. These lines were monitored in culture for 6-8 months. Both lines were specific for HLA-A or B antigens or both and the JR line was allospecific for HLA-B7. These CTL lines killed specific target cells at an effector-to-target ratio of 0.4 (VE) or 0.08 (JR). All of the cells, which grow in suspension, rosetted with sheep erythrocytes and reacted with an antiserum specific for human T cells. The CTL line VE was used to raise rabbit antisera that immunoprecipitated two specific polypeptides (78,000 and 33,000 daltons) from labeled membranes of these CTL lines.
Expression of cloned hepatitis B virus DNA in human cell cultures.
Authors: Hirschman S Z , Price P , Garfinkel E , Christman J , Acs G
Published: May 2006

DNA was isolated from the ayw subtype of hepatitis B virus (HBV) that had been incubated in vitro with all four deoxynucleoside triphosphates in order to complete the circular viral genome by means of the endogenous DNA polymerase. The purified viral DNA was cleaved with EcoRI restriction endonuclease, inserted into the EcoRI site of plasmid pBR322, and cloned in Escherichia coli chi 1776. DNA from a clone, pHBV-1, that contained a 3200-base-pair insert of HBV DNA was cleaved with EcoRI and incubated with phage T4 ligase under conditions favoring intramolecular ligation. HeLa cell cultures exposed to this DNA showed marked cytopathic changes, accompanied by production of hepatitis B core and surface antigens, 11-14 days after subculture. Electron microscopic examination of anti-hepatitis B surface antigen immunoprecipitates from culture media of these cells revealed both 42-nm particles with central cores and 20-nm round particles. Although neither intact circular nor EcoRI-cleaved linear pHBV-1 DNAs evoked these effects in HeLa cells, both cytopathic changes and intranuclear hepatitis B core antigen were detected in HeLa cells infected with Dane particles.
Random walk theory of a trap-controlled hopping transport process
Authors: Scher H. , Wu C. H.
Published: May 2006

A random walk theory of hopping motion in the presence of a periodic distribution of traps is presented. The solution of the continuous-time random walk equations is exact and valid for arbitrary intersite interactions and trap concentration. The treatment is shown to be equivalent to an exact solution of the master equation for this trapping problem. These interactions can be a general function of electric field and are not restricted to nearest neighbors. In particular, with the inclusion of trap-to-trap interactions, as well as trap-to-host interactions, an
exact
treatment of the change from one hopping channel to another has been obtained. The trap-modulated propagator has been derived in terms of a type of Green's function that is introduced. The results are specialized to spatial moments of the propagator, from which expressions for the drift velocity and diffusion coefficient are obtained. Numerical results for the drift velocity are presented and shown to account for the change in hopping channels in recent transport measurements in mixed molecularly doped polymers.
Heterogeneous distribution of filipin--cholesterol complexes across the cisternae of the Golgi apparatus.
Authors: Orci L , Montesano R , Meda P , Malaisse-Lagae F , Brown D , Perrelet A , Vassalli P
Published: May 2006

The Golgi apparatus is a key element in the ordered movement of secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane during secretion. It has been shown that cisternae that receive membranes from the reticulum are morphologically similar to the latter and that cisternae liberating secretory granules resemble that plasma membrane. By using an ultrastructural probe for membrane cholesterol, filipin, on freeze-fractured and thin-sectioned exocrine and endocrine pancreatic cells, we have shown that an enrichment in filipin-cholesterol complexes takes places across the stacked cisternae of the Golgi apparatus; the reticulum-related (forming) cisternae are poor in such complexes, but the secretory granule-related (maturing) cisternae contain numerous complexes. Secretory granule membrane is also richly labeled with filipin-cholesterol complexes. The heterogeneous cholesterol distribution in the membranes of the Golgi apparatus, as shown by filipin, emphasizes the polarity of this organelle, in agreement with its role in organizing the traffic of the secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane.
Cloning of the uvrC gene of Escherichia coli: expression of a DNA repair gene.
Authors: Sharma S , Ohta A , Dowhan W , Moses R E
Published: May 2006

We have cloned the uvrC gene of Escherichia coli, using an F' plasmid carrying the uvrC region as a source of DNA. Two plasmids, pSC101 and pBR322, were used as cloning vectors. The recombinant plasmids were selected for their ability to complement the uvrC defect of E. coli strains AB1884 and N177. We conclude that the uvrC structural gene is contained in a 1.9-kilobase DNA fragment. The protein encoded by the uvrC gene appears to have a monomer molecular weight of 64,500 as analyzed by denaturing polyacrylamide gel electrophoresis. Strains containing multicopy uvrC+ plasmids overproduce a factor that is missing in lysates of uvrC- mutants and required for an in vitro model repair reaction. The expression of uvrC+ hybrid plasmids suggests that the structural gene is separated by at least 0.8 kilobase from the regulatory region.
Membrane glycoproteins involved in cell--substratum adhesion.
Authors: Knudsen K A , Rao P E , Damsky C H , Buck C A
Published: May 2006

A combination of immunological and biochemical methods were used to identify surface membrane components involved in cell-substratum adhesion. Broad-spectrum antiserum, prepared against surface membranes from hamster cells, induced reversible rounding and detachment of hamster fibroblasts from a substratum in vitro. This phenomenon was inhibited by Nonidet P-40 extracts of hamster cells. Therefore, an antibody neutralization assay was developed to detect the presence of antigen during the fractionation of Nonidet P-40 extracts of cells. After two differential precipitation steps, anion exchange chromatography, and sequential lectin affinity chromatography, a fraction greatly enriched in ability to block antiserum-induced changes in cell adhesion and appearance was isolated. Analysis of this fraction by NaDodSO4/polyacrylamide gel electrophoresis revealed a highly restricted group of glycoproteins with Mr approximately 140,000. A lectin-purified glycoprotein fraction was used to raise a higher titer antiserum that was able to induce reversible rounding and detachment of cells from a substratum and, when immobilized on an antibody affinity column, was able to bind and release material capable of blocking antiserum-induced cell rounding. These methods have allowed us to focus attention on a restricted group of glycoproteins that are integral constituents of the surface membrane and which play some as yet undetermined role in the process of cell--substratum adhesion.
Human somatotropin: covalent reconstitution of two polypeptide contiguous fragments with thrombin.
Authors: Gráf L , Li C H
Published: May 2006

Thrombin has been shown to resynthesize the Arg134-Thr135 peptide bond between the 134- and 57-residue thrombin fragments of reduced and carbamidomethylated human somatotropin at pH 6.0 in 90% (vol/vol) glycerol. The maximal amount of synthesis was about 20% as estimated by sodium dodecyl sulfate gel electrophoresis and radioreceptor assay. The resynthesized polypeptide was isolated and shown to be indistinguishable from the reduced and carbamidomethylated hormone when tested by two receptor-binding assays, radioimmunoassay, and rat tibia test.
Desensitization to gonadotropins in cultured Leydig tumor cells involves loss of gonadotropin receptors and decreased capacity for steroidogenesis.
Authors: Freeman D A , Ascoli M
Published: May 2006

The ability of human choriogonadotropin (hCG) to regulate its receptors and target cell responses has been studied in a clonal strain of cultured Leydig tumor cells (MA-10). Exposure of the MA-10 cells to hCG results in decrease in hCG binding activity which is dependent on time and the concentration of hCG. This decrease is due to a change in the number of receptors rather than in the affinity of the receptors, and it is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. Exposure of the MA-10 cells to hCG also resulted in a reduction of the steroidogenic responses to cholera toxin and 8-Br-adenosine cyclic 3',5'-monophosphate. The hCG-induced loss of steroidogenic responses to these stimuli seems to be due to the stimulation of steroidogenesis rather than to the decrease in hCG receptors because it also can be induced when steroidogenesis is stimulated with cholera toxin or 8-Br-adenosine 3',5'-monophosphate under conditions such that the number of hCG receptors is not reduced.
Estimating genetic divergence and genetic variability with restriction endonucleases.
Authors: Engels W R
Published: May 2006

Restriction endonucleases cut DNA at specific sites determined by the local nucleotide sequence. By comparing related DNA segments with respect to where such cuts are made, one can estimate the extent of sequence homology between the segments. Empirical methods are presented here for using these data to measure the proportion of mismatches between two sequences, the proportion of polymorphic positions in a series of sequences, or the degree of heterozygosity in a population. These methods do not require any assumptions concerning the evolutionary or population genetic processes involved. One can also use the data to calculate the precision of each of these estimates. When the positions of the cuts are not determined, these estimates can be made, using only the lengths of the resulting DNA fragments, by means of a maximum likelihood procedure. Several examples demonstrate the usefulness of these methods to study genetic differences in regions of the genome not amenable to study by other methods.
Longer-than-unit-length viroid minus strands are present in RNA from infected plants
Authors: Branch Andrea D. , Robertson Hugh D. , Dickson Elizabeth
Published: May 2006

Nucleic acids isolated from uninfected and potato spindle tuber viroid-infected Rutgers tomato plants were fractionated on agarose gels under two different sets of denaturing conditions and hybridized to
125
I-labeled viroid in a series of blot hybridization experiments. Complementary strand nucleic acids detected in extracts of infected plants were heterogeneous in size, with four discrete bands containing molecules approximately 700, 1050, 1500, and 1800 nucleotides long. Enzymatic studies indicated that these viroid minus strands are composed exclusively of RNA and, as extracted, are present in complexes containing extensive double-stranded regions. After treatment with several RNases under conditions favoring digestion of single-stranded regions, the high molecular weight minus strands can no longer be detected and roughly unit-length minus strands appear. A model for the structure of the viroid replication intermediate is proposed.
Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism.
Authors: Magistretti P J , Morrison J H , Shoemaker W J , Sapin V , Bloom F E
Published: May 2006

Mouse cerebral cortex slices will synthesize [3H]glycogen in vitro. Vasoactive intestinal polypeptide (VIP) stimulates the enzymatic breakdown of this [3H]glycogen. The concentration giving 50% of maximum effectiveness (EC50) is 26 nM. Under the same experimental conditions norepinephrine also induces a concentration-dependent [3H]glycogen hydrolysis with an EC50 of 500 nM. The effect of VIP is not mediated by the release of norepinephrine because it is not blocked by the noradrenergic antagonist d-1-propranolol and is still present in mice in which an 85% depletion of norepinephrine was induced by intracisternal 6-hydroxydopamine injections. Other cortical putative neurotransmitters such as gamma-aminobutyric acid, aspartic acid, glutamic acid, somatostatin, and acetylcholine (tested with the agonist carbamylcholine) do not induce a breakdown of [3H]glycogen. This glycogenolytic effect of VIP and norepinephrine, presumed to be mediated by cyclic AMP formation, should result, at the cellular level, in an increased glucose availability for the generation of phosphate-bound energy. Given the narrow radial pattern of arborization of the intracortical VIP neuron and the tangential intracortical trajectory of the noradrenergic fibers, these two systems may function in a complementary fashion: VIP regulating energy metabolism locally, within individual columnar modules, and norepinephrine exerting a more global effect that spans adjacent columns.
Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells: evidence for discrete structural domains.
Authors: Mally M I , Grayson D R , Evans D R
Published: May 2006

The multifunctional protein that initiates de novo pyrimidine biosynthesis in mammalian cells carries carbamoylphosphate synthetase, aspartate transcarbamylase (aspartate carbamoyltransferase), and dihydro-orotase activities on a single 215,000-dalton polypeptide chain. Kinetic studies of the controlled proteolysis of the molecule by elastase showed that the protein was not attacked at random by the protease but rather was successively cleaved into at least six well-defined proteolytic fragments. The initial cleavage converted the intact molecule into a 190,000-dalton species which appeared to retain all of the catalytic and regulatory functions of the native protein. This species was subsequently cleaved into two fragments, 150,000 and 40,000 daltons. The 40,000-dalton species, which carried the aspartate transcarbamylase activity, was resistant to further proteolysis; the 150,000-dalton polypeptide, which carried carbamoyl-phosphate synthetase and dihydro-orotase activities, underwent further digestion to 140,000 daltons. Continued proteolysis produced two species, 79,000 and 45,000 daltons; like the 40,000-dalton species, these were stable against further elastase digestion. The aspartate transcarbamylase and dihydro-orotase activities and the regulatory functions were preserved throughout the course of digestion; the carbamoylphosphate synthetase activity was more labile. By using sucrose gradient centrifugation and ion exchange chromatography, the 40,000- and 45,000-dalton species have been isolated. The 40,000-dalton fragment was found to have only aspartate transcarbamylase activity; the 45,000-dalton fragment has only dihydro-orotase activity. These experiments showed that this multifunctional protein is organized as discrete structural domains in which regions of the polypeptide chain are autonomously folded into separate functional units.
Reversible unidirectional inhibition of sucrose synthase activity by disulfides.
Authors: Pontis H G , Babio J R , Salerno G
Published: May 2006

Sucrose synthase (UDPglucose:D-fructose 2-alpha-D-glucosyltransferase, EC2.4.1.13), which catalyzes the synthesis and cleavage of sucrose, exhibits differences in some properties between the two reactions. When enzyme previously incubated with oxidized glutathione or oxidized thioredoxin was used, sucrose cleavage was inhibited whereas sucrose synthesis proceeded at a normal rate. Sucrose cleavage activity could be restored by incubation with dithiothreitol or reduced glutathione. The thioredoxin effect was influenced by the presence of cleavage reaction substrates--i.e., sucrose and UDP. Thioredoxin action was rather slow compared with the catalytic reaction. These findings may have important implications for understanding the metabolic role of sucrose synthase and oxidized thioredoxin. Theoretically, the fact that an enzyme catalyzing a reversible reaction is inhibited in one direction only suggests that a modification in the enzyme affinities for its substrates must have occurred.
General solution to the inverse problem of the differential equation of the ultracentrifuge.
Authors: Todd G P , Haschemeyer R H
Published: May 2006

Whenever experimental data can be simulated according to a model of the physical process, values of physical parameters in the model can be determined from experimental data by use of a nonlinear least-squares algorithm. We have used this principle to obtain a general procedure for evaluating molecular parameters of solutes redistributing in the ultracentrifuge that uses time-dependent concentration, concentration-difference, or concentration-gradient data. The method gives the parameter values that minimize the sum of the squared differences between experimental data and simulated data calculated from numerical solutions to the differential equation of the ultracentrifuge.
Changes in the hydrogen exchange kinetics of Escherichia coli aspartate transcarbamylase produced by effector binding and subunit association.
Authors: Lennick M , Allewell N M
Published: May 2006

Large changes in solvent accessibility to aspartate transcarbamylase (aspartate carbamoyltransferase, carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), as monitored by tritium exchange, result from binding of substrates and substrate analogs to the catalytic subunit (c3), binding of nucleoside triphosphates to the regulatory subunit (r2), and subunit association. Rates of exchange are reduced in each of these cases, although to different degrees. Succinate, in the presence of carbamoyl phosphate, retards exchange from c3 no more than carbamoyl phosphate alone, and less than N-phosphonacetyl-L-aspartate, a bisubstrate analog. Larger changes in rates of exchange from r2 are produced by CTP than by ATP; however, both CTP and ATP accelerate exchange from c3 to the same extent. The changes in the kinetics of exchange that result from binding of both substrate analogs and nucleoside triphosphates to the native enzyme (c6r6) are much smaller. Carbamoyl phosphate, with or without succinate, retards exchange only slightly, while the bisubstrate analog has a somewhat larger effect. Experiments with reconstituted enzyme, in which only c3 is tritium labeled, indicate that changes in solvent accessibility produced by active site ligands are largely confined to c3. Neither CTP nor ATP alters the overall rate of exchange from c6r6 significantly. The possibility of opposing changes in the two types of subunits was ruled out in experiments in which only one subunit was labeled. The nonadditive effects of ligation and subunit association imply a set of responsive protons common to both processes and suggest that they are linked not only thermodynamically and functionally but also dynamically.
Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form.
Authors: Enomoto T , Lichy J H , Ikeda J E , Hurwitz J
Published: May 2006

The 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those of HeLa cell DNA polymerases alpha, beta, and gamma. The Ad protein-associated DNA polymerase activity was detected with activated DNA but not with poly(rA).oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.
Induction by cholera toxin of synchronous divisions in vivo in the epidermis resulting in hyperplasia.
Authors: Kuroki T
Published: May 2006

Intracutaneous injection of cholera toxin, exotoxin of Vibrio cholerae, into the dorsal skin of mice, rats, and hamsters at doses of greater than 0.1 ng evoked an acute reaction at the site of injection, which was characterized histologically by an edematous reaction in the dermis and mitotic stimulation in the epidermis. Mitotic and labeling induces of basal cells of the mouse epidermis showed two peaks at 24 and 48 hr after injection, thereby producing epidermal hyperplasia. The thickness of the intrafollicular epidermis increased progressively from 32 hr after toxin injection, being greatest on day 4 and decreasing to normal on day 7. The epidermis on day 4 after injection of 1.0 ng of toxin was about 4- to 6-fold thicker than normal or phosphate buffer-treated control skin. This sequence of events indicated that cholera toxin induced two successive synchronous divisions of the epidermal cells and produced temporary hyperplasia without interfering with epidermal differentiation. The complete structure and function of the cholera toxin are required for induction of epidermal hyperplasia: no mitotic stimulation was induced by injection of the A and B units of the cholera toxin molecule or by preincubation of the toxin with anti-cholera toxin antibody and with the membrane receptor, GM1 ganglioside. Five other agents known to increase the level of intracellular cyclic AMP by different means (dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, isoproterenol, and prostaglandin E1) did not produce a skin reaction.
Regional assignment of genes for human α-globin and phosphoglycollate phosphatase to the short arm of chromosome 16
Authors: Koeffler H. Phillip , Sparkes Robert S. , Stang Howard , Mohandas T.
Published: May 2006

The human α-globin and phosphoglycollate phosphatase (EC 3.1.3.18) genes have been regionally localized to the short arm of human chromosome 16 (HC16). This was accomplished by fusing mouse fibroblasts (A9) to human fibroblasts that contain a reciprocal translocation between the long arms of chromosomes 16 and 11. The murine A9 cells are deficient in adenine phosphoribosyltransferase (APRT), an enzyme present on the long arm of HC16 (HC16q). Hybrid cells were grown in selection culture medium that required the cells to retain human APRT. Therefore, the hybrids exhibited stable retention of the entire HC16 or the rearranged chromosome containing HC16q. We isolated five independent primary and secondary hybrid cell lines which retained either HC16 or HC16q at a high frequency. The presence of human α-globin genes in the various clones was established directly by DNA extraction and hybridization to a cDNA probe for human α-globin genes. Autoradiographs showed that hybrid cells containing the long arm, but not the short arm, of HC16 showed only the background mouse bands. Hybrid cells that retained the entire HC16 demonstrated the band(s) containing the human α-globin genes. Hybrid cells that contained HC16 with its α-globin genes were then placed in culture medium that contained diaminopurine, which is lethal for cells containing APRT. These counter-selected hybrid cells had lost HC16 and also lost the human α-globin genes as determined by blot hybridization. The presence of α-globin gene sequences in the hybrid clones was concordant with HC16 only and not with any other human chromosome. These results confirm the assignment of α-globin genes to HC16 and localize the genes to the short arm. We also assign the locus for phosphoglycollate to the short arm of HC16.
Mutational alteration of membrane phospholipid composition and voltage-sensitive ion channel function in paramecium.
Authors: Forte M , Satow Y , Nelson D , Kung C
Published: May 2006

A behavioral mutant of Paramecium tetraurelia (baA) has been isolated that has an abnormal response when placed in solutions containing Ba2+. This mutant is shown here to have a dramatic alteration of the sphingolipid and phosphonolipid composition of its ciliary membrane. This biochemical defect is present in independently isolated alleles at baA locus and segregates in crosses with the behavioral phenotype. Electrophysiologically, the mutation reduces significantly conductance of both voltage-sensitive Ca2+ channels and voltage-sensitive K+ channels. When the mutant is grown in sterol-supplemented medium, its behavior, electrophysiological properties, and lipid composition are hardly distinguishable from wild type grown under similar conditions. This mutant then, provides strong evidence that membrane lipids significantly influence the function of the membrane molecules responsible for the generation of action potentials.
Human lymphocyte production of corticotropin and endorphin-like substances: association with leukocyte interferon.
Authors: Smith E M , Blalock J E
Published: May 2006

Human leukocyte interferon (hIFN-alpha) preparations contain immunologically and biologically recognizable endorphin and corticotropin-like (ACTH-like) activities. The ACTH bioactivity was demonstrable only after pepsin or acid treatment. Highly purified hIFN-alpha was composed of two molecular species of interferon (18,500 and 23,000 daltons). Endorphin activity was associated with both of these molecules. Pepsin treatment of the 23,000-dalton but not the 18,500-dalton hIFN-alpha generated ACTH activity. In acid, the 23,000-dalton hIFN-alpha broke down into the 18,500-dalton form and ACTH (4500 daltons). The ACTH derived from hIFN-alpha by pepsin digestion comigrated with a purified ACTH standard in NaDodSO4/polyacrylamide gel electrophoresis. hIFN-alpha-producing lymphocytes showed positive immunofluorescence after staining with highly specific antisera to ACTH alpha-(1-13) and gamma-endorphin. Essentially 100% of the human peripheral lymphocytes were capable of producing both ACTH and gamma-endorphin-related substances, presumably associated with hIFN-alpha. These results strongly suggest a circuit between the immune and neuroendocrine systems which involves neuroendocrine hormone-like substances, some of which are associated with hIFN-alpha
Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval.
Authors: Davis B K
Published: May 2006

A survey of species differences in the duration of capacitation, T, has revealed that they closely correlate with sperm cholesterol/phospholipid mole ratios, R : T = 8R - 1 (r2 = 0.97, in which r is Pearson's correlation coefficient). Because uterine cells displayed low relative cholesterol concentrations, spermatozoa evidently experience a negative external cholesterol gradient (positive phospholipid gradient) during capacitation. A decrease in sperm R-value is suggested, therefore, to accompany capacitation. The idea received strong support from a kinetic analysis of capacitation intervals, based on the rate of cholesterol efflux from sperm cells in utero. Lipid-binding serum proteins in uterine fluid are attributed with removing a sterol barrier to the Ca2+-facilitated membrane fusion that initiates the acrosome reaction. Tight cell junctions prevent permeation of the male generative tract by these proteins (capacitation factors). Furthermore, seminal plasma contains a decapacitation factor, identified as a membrane vesicle (cholesterol donor) component of this fluid, that reverses capacitation. Initiation of the sperm acrosome reaction among mammals could be the first fusion process found to be physiologically modulated through the membrane bilayer cholesterol level.
Immunohistochemical localization of renin in luteinizing hormone-producing cells of rat pituitary.
Authors: Naruse K , Takii Y , Inagami T
Published: May 2006

The location of renin (EC 3.4.99.19) in rat pituitary was determined by the peroxidase-antiperoxidase immunohistochemical technique. By using antisera prepared with purified rat renal renin, an immunoreactive substance was localized within ovoid cells scattered throughout the anterior pituitary. These cells were shown to be luteinizing hormone-producing cells by staining with anti-luteinizing hormone antisera in adjacent sections. By using the double staining method, the renin-containing cells were differentiated from cells containing corticotropin, thyrotropin, growth hormone (somatotropin), and prolactin (mammotropin). These results suggest a possible local role for renin in the anterior pituitary.
Deletions in the alpha-globin gene complex in alpha-thalassemic mice.
Authors: Whitney J B , Martinell J , Popp R A , Russell L B , Anderson W F
Published: May 2006

Three induced, heritable mutations in the mouse cause alpha-thalassemias. The adult alpha-globin genes on each mutant chromosome are no longer expressed. Embryos heterozygous for one normal and any of the three mutant chromosomes also seem to be deficient in embryonic alpha-globin-like x-globin, suggesting that the x-globin gene is nearby and also inactivated. A normal genetic polymorphism for a specific EcoRI site in or around the mouse alpha-globin gene complex has been used here to show that each of the three mutated chromosomes has a deletion that includes the segment of a 12-kilobase EcoRI band which normally carries one of the two adult alpha-globin genes. The deletion of the comparable part of the second alpha-globin gene site is also inferred. Nonetheless, a 4.7-kilobase EcoRI segment which carries a characterized alpha-globin-like pseudogene is still present in each mutant. These mutations were recovered after triethylenemelamine or x-ray treatments.
Skin tumor promotion by phorbol esters is a two-stage process.
Authors: Fürstenberger G , Berry D L , Sorg B , Marks F
Published: May 2006

In the semisynthetic compound phorbol 12-retinoate 13-acetate (PRA), the antipromoting principle of vitamin A acid is combined with the structure of a phorbol ester tumor promoter. In skin of NMRI mice, a single topical application of PRA induces skin inflammation, epidermal proliferation, and sustained hyperplasia to a similar extent and apparently along the same pathway as an equimolar dose of the strong tumor promoter phorbol 12-myristate 13-acetate (PMA). The mitogenic effects of both PRA and PMA are mediated by prostaglandin E synthesis. However, in mouse skin initiated with 7,12-dimethylbenz[a]anthracene, PRA does not promote tumor development, even at a high dose. Under continuous PRA treatment, however, one to four applications of PMA (insufficient by itself to promote tumor growth) gave a strong tumor response. Thus, it can be demonstrated that the effects necessary for tumor promotion can be brought about by a single application of PMA and that the subsequent chronic hyperproliferation of epidermis is probably necessary only to make the tumors visible. By using the nonpromoting irritant mitogen PRA, the concept of two-stage tumor promotion can thus be strongly supported. Furthermore, in the NMRI mouse, PRA is a much more potent second-stage promoter than mezerein, recently reported to be an incomplete promoter in the Sencar mouse.
erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities.
Authors: Bouchard P , Penningroth S M , Cheung A , Gagnon C , Bardin C W
Published: May 2006

Protein carboxylmethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24.) is believed to be involved in the regulation of sperm motility. To test this hypothesis, we investigated the effects of erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) which, in combination with adenosine and homocysteine thiolactone, inhibits protein carboxylmethylase activity in monocytes. This group of compounds inhibited sea urchin sperm motility. Unexpectedly, EHNA alone inhibited the motility., This observation was confirmed in intact spermatozoa from rats, rabbits, and humans. EHNA also inhibited the motility of demembranated, reactivated sea urchin and rat spermatozoa from which protein carboxylmethylase had been extracted. In these preparations, motility was restored by ATP. These observations suggested that EHNA arrests sperm motility by inhibiting the axonemal dynein ATPase on which motility depends. Kinetic analysis demonstrated that EHNA produced mixed inhibition of both the axonemal ATPase and the partially purified dynein 1 from sea urchin sperm tails, as well as the axonemal ATPase of rat sperm tails. These observations also provide evidence for the similarity of the active site of the dynein ATPase in sea urchin and rat spermatozoa.
Selective killing of human malignant cell lines deficient in methylthioadenosine phosphorylase, a purine metabolic enzyme.
Authors: Kamatani N , Nelson-Rees W A , Carson D A
Published: May 2006

Seven out of 31 (23%) human malignant tumor cell lines had no detectable methylthioadenosine phosphorylase activity (less than 0.001 nmol/min per mg of protein), assayed with 5'-chloroadenosine as substrate. The enzyme-deficient cell lines were derived from five leukemias, one melanoma, and one breast cancer. None of 16 cell lines of nonmalignant origin, derived from lymphocytes, fibroblasts, and epithelial cells, lacked the enzyme (range, 0.156-1.447 nmol/min per mg of protein). As detected by autoradiography, intact enzyme-positive cell lines normal immature bone marrow cells, and four specimens of malignant tumor cells incorporated the adenine moiety of 5'-chloroadenosine into nucleic acids; however, no enzyme-deficient cell lines used 5'-chloroadenosine. When both types of cell lines were cultured in a medium containing 0.4 microM methotrexate, 16 microM uridine, and 16 microM thymidine (or 10 microM azaserine alone), no cells grew. If methylthioadenosine was added to the same medium, only enzyme-positive cells increased in number; most enzyme-deficient cells were dead after 3 days. Thus, human malignant tumor cell lines naturally deficient in methylthioadenosine phosphorylase could be selectively killed when de novo purine synthesis was inhibited and methylthioadenosine was the only exogenous source of purines.
Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.
Authors: Rast D M , Bartnicki-Garcia S
Published: May 2006

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.
Target neuron-specific process formation by embryonic mesencephalic dopamine neurons in vitro.
Authors: Hemmendinger L M , Garber B B , Hoffmann P C , Heller A
Published: May 2006

Mesencephalic dopamine neurons from the embryonic mouse brain were dissociated, aggregated in vitro in the presence of dissociated cells from appropriate or inappropriate target neuron areas, and visualized by the Falck-Hillarp histofluorescence technique after exposure to 1 microM exogenous dopamine. When aggregated with the surrounding rostral mesencephalic tegmentum cells only or with the addition of rostral tectum cells, the dopamine neurons formed a dense dendritic arborization, but no axons were observed. In the presence of dopamine-neuron target cells from the corpus striatum, a dense axonal plexus characteristic of that formed in this area in vivo was observed. In contrast, in aggregates formed with target cells from the frontal cortex, branching fluorescent axons bearing irregularly spaced and shaped varicosities were found coursing through the neuropil, as is characteristic of the dopaminergic innervation to the frontal cortex in vivo. Only proximal dendrites were observed in the presence of these axonal target cells. Dopamine neurons cultured with inappropriate target cells from the occipital cortex did not form either extensive axonal or dendritic processes. Thus, the presence, type, and distribution of dopamine neuronal processes are dependent on the presence of appropriate target cells. The formation of unique patterns of neuronal processes by dissociated neurons in vitro suggests that the information necessary for this differentiation is intrinsic to the dopamine neurons and their target cells. This system provides a useful model with which to study basic mechanisms underlying neuronal recognition.
Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.
Authors: Kawamura M , Jensen D F , Wancewicz E V , Joy L L , Khoo J C , Steinberg D
Published: May 2006

Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation.
Early events in herpes simplex virus type 1 infection: photosensitivity of fluorescein isothiocyanate-treated virions.
Authors: DeLuca N , Bzik D , Person S , Snipes W
Published: May 2006

Herpes simplex virus type 1 is photosensitized by treatment with fluorescein isothiocyante (FITC). The inactivation of FITC-treated virions upon subsequent exposure to light is inhibited by the presence of sodium azide, suggesting the involvement of singlet oxygen in the process. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that treatment with FITC plus light induces crosslinks in viral envelope glycoproteins. Treatment of virions with high concentrations of FITC (50 micrograms/ml) plus light causes a reduction in the adsorption of the virus to monolayers of human embryonic lung cells. For lower concentrations of FITC (10 micrograms/ml) plus light, treated virions adsorb to the host cells, but remain sensitive to light until entry occurs. The loss of light sensitivity coincides with the development of resistance to antibodies. These results are most consistent with a mechanism of entry for herpes simplex virus involving fusion of the viral membrane with the plasma membrane of the host cell.
Circulant orbitals for atoms and molecules
Authors: Parr Robert G. , Chen Min-Bo
Published: May 2006

Circulant orbitals ϕ

n

for a closed-shell system are the orbitals obtained when the
N
canonical orthonormal Hartree-Fock orbitals λ
[unk]
are subjected to a unitary transformation which is the discrete Fourier transformation: ϕ

n

= 1/√
N
Σ
[unk]
λ
[unk]
ω
(n-1)([unk]-1)
, where ω = exp(2πi/
N
). Electron densities associated with the orbitals ϕ

n

are each close to the average total electron density. The Fock matrix, diagonal for canonical orbitals, for circulant orbitals is a Hermitian circulant matrix, ε

m, m+q

= 1/
N
Σ
[unk]
ε
[unk]
ω

q
([unk]-1)

, where the ε
[unk]
are the canonical orbital energies. The states
^F
ϕ

n

are uniformly distributed on the surface of a sphere in Hilbert space.
N-Methylprotoporphyrin IX: chemical synthesis and identification as the green pigment produced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment.
Authors: Ortiz de Montellano P R , Beilan H S , Kunze K L
Published: May 2006

The hepatic pigment accumulated in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated rats, which has been reported to inhibit ferrochelatase, has been isolated and purified. The pigment has been resolved into one major, one minor, and two trace components, all of which appear to be isomeric porphyrins. The major fraction has been unambiguously identified by spectroscopic methods as the isomer of N-methylprotoporphyrin IX (isolated as the dimethyl ester) in which vinyl-substituted pyrrole ring A is methylated. The minor product appears to be an isomer of the same porphyrin with the N-methyl group on propionic acid-substituted ring C, and the trace components have the same high-pressure liquid chromatography retention times as the other two possible isomers of the porphyrin. The four isomers of N-methylprotoporphyrin IX have been chemically synthesized, independently characterized, and used to confirm the structures of the biologically products.