bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucin synthesis. The action of pig gastric mucosal UDP-GlcNAc:Galβ1-3(R1)GalNAc-R2 (GlcNAc to Gal) β3-N-acetylglucosaminyltransferase on high molecular weight Substrates
Authors: Brockhausen Inka , Orr Jeanne , Schachter Harry
Published: December 2009

Membrane preparations from pig gastric mucosa were shown to transfer [14C]GlcNAc from UDP-[14C]GlcNAc to blood group A-negative porcine submaxillary mucin previously subjected to mild acid hydrolysis to remove terminal sialyl and fucosyl residues. O-Glycosyl oligosaccharides were removed from enzyme product by alkaline borohydride treatment and, after purification, were subjected to high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Two trisaccharide products were detected: [14C]GlcNAcβ1-3Galβ1-3GalNAcOH and Galβ1-3([14C]GlcNAcβ1-6)GalNAcOH. We have previously reported the in vitro synthesis of the latter compound, a branched trisaccharide, by UDP-GlcNAc:Galβ1-3GalNAc-R (GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase from canine submaxillary glands. However, this is the first report of the in vitro synthesis of the linear trisaccharide GlcNAcβ1-3Galβ1-3GalNAc. Pig gastric mucosal β3-N-acetylglucosaminyltransferase catalyzed the formation of this trisaccharide by incorporation of GlcNAc into the terminal Gal of Galβ1-3GalNAc-α-R when R was a polypeptide from either mucin or antifreeze glycoprotein, but not when R was o-nitrophenyl. We have previously reported the in vitro synthesis by pig gastric mucosa of the tetrasaccharide GlcNAcβ1-3Galβ1-3(GlcNAcβ1-6)GalNAc-α-R when R was o-nitrophenyl or benzyl. We show in this report that pig gastric mucosa can synthesize this tetrasaccharide in vitro when R is a polypeptide from either porcine submaxillary mucin or antifreeze glycoprotein. Pig gastric mucosa therefore contains a β6-N-acetylglucosaminyltransferase capable of converting Galβ1-3GalNAc-α-R to Galβ1-3(GlcNAcβ1-6)GalNAc-α-R and one or more β3-N-acetylglucosaminyltransferases which can add GlcNAc in β1-3 linkage to a terminal Gal residue to form either GlcNAcβ1-3Galβ1-3GalNAc-α-R or GlcNAcβ1-3Galβ1-3(GlcNAcβ1-6)GalNAc-α-R where R is the polypeptide backbone of either mucin or antifreeze glycoprotein.
The development of biochemistry in the 20th century
Authors: Quastel J. H.
Published: December 2009

This lecture consists of a short appraisal of some of the main features that have characterized the growth of biochemistry during the course of the 20th century. It dwells on the early impacts of vitalism, the emergence and elucidation of the vitamins, the discovery of coenzymes, the concept of active centres of enzymes, the development of experimental techniques (including the use of isotopes), the genetic code, and on the development of molecular biology and closely allied fields of investigation. It concludes with a consideration of the influence of the study of membranes and of neurochemistry on current biochemical thought.
The isolation of surface array proteins from bacteria
Authors: Koval S. F. , Murray R. G. E.
Published: December 2009

The methods used for the isolation of regularly structured (RS) surface array proteins of a range of prokaryotes are described. Most RS proteins can be selectively solubilized from envelope preparations with low concentrations of urea or guanidine hydrochloride. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis of the protein extracts shows that most RS arrays are composed of a single polypeptide that may contain carbohydrate. The molecular weight of the proteins varies from 41 000 to 200 000. Possible reasons for the presence of more than one polypeptide in RS protein preparations are discussed, as well as the evidence for proteolytic degradation of some RS proteins during isolation. Structural features of the RS proteins are described and the importance of protein conformation to assembly of the arrays is indicated.
Anti-idiotypic antibodies as probes for hormone–receptor interaction
Authors: Farid Nadir R. , Briones-Urbina Rosario , Islam M. Nazrul
Published: December 2009

On the premise that an antibody combining site is a mirror image of its antigen epitope, it is expected that an anti-idiotypic antibody (i.e., an antibody specific for the combining site of the first antibody) will be homologous to the epitope. Anti-idiotypic antibodies raised against hormones or drugs would, therefore, be anticipated to interact with their respective receptors. According to this schema, anti-idiotypic antibodies could either be antagonists or agonists. Most of the anti-idiotypic antibodies raised against hormones and neurotransmitters to date have proved, however, to be agonists. We have raised antithyrotropin (anti-TSH) anti-idiotypic antibodies and found these to interact with the high affinity binding site for TSH on thyroid plasma membranes and to induce cGMP-dependent adenylate cyclase activation and iodide transport into dispersed thyroid cells, as well as to promote their organization into follicular structures. The anti-TSH anti-idiotypic antibody interacted with a holoreceptor band of relative mass (Mr) ~ 200 000, resolved on sodium dodecyl sulfate – polyacrylamide gel electrophoresis from thyroid membranes and transferred to nitrocellulose paper. In another set of experiments we raised anti-idiotypic antibodies against monoclonal antibodies specific, respectively, for the α and β subunits of TSH. Neither the α nor β monoclonal antibody specific anti-idiotypic antibodies interacted with the TSH holoreceptor. The combinations of the two anti-idiotypic antibodies, however, did so and increased basal cyclase activity significantly compared with normal immunoglobulin G. As a result of the second set of experiments, we propose that the interaction of TSH with its receptor involves two signals delivered by the two subunits rather than a single signal requiring their combination. Anti-idiotypic antibodies raised against highly purified hormones can be obtained in large amounts. They facilitate simple isolation of hormone receptors and are useful as probes for hormone–receptor interactions.
Rapid sensitive fluorescence assays for DNA endonucleases and DNA glycosylases
Authors: Evans D. H. , Bugeja F. X. , Yacyshyn B. J. , Morgan A. R.
Published: December 2009

Using ethidium fluorescence as a probe and the unique topological properties of covalently closed circular (CCC) DNA's, picogram quantities of pancreatic DNase I can be detected. By chemically modifying CCC DNA's without the introduction of nicks, various DNA repair enzymes can be detected even in crude cell extracts. This has previously been done for AP endonucleases from yeast. In this paper we describe the formation of ultraviolet (UV) and bisulfite modified CCC DNA's for the detection of T4 UV endonuclease (a DNA glycosylase + AP endonuclease) and uracil-DNA glycosylases, respectively. These assays should be useful in the isolation of DNA repair mutants.
Studies on leukotriene B4 ω-oxidation in human leukocytes
Authors: Nadeau Marie , Laclos Bernard Fruteau de , Picard Serge , Braquet Pierre , Corey E. J. , Borgeat Pierre
Published: December 2009

The kinetics of the transformation of arachidonic acid into 5-lipoxygenase products in human blood leukocytes stimulated with the ionophore A23187 (2 μM) was studied using high performance liquid chromatography. The levels of 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), Δ6-trans-UTB4, and leukotriene C4 (LTC4) were maximum after 4–5 min of incubation; at longer incubation times, the levels of Δ6-trans-LTB4 and LTC4 remained unchanged, whereas those of 5-HETE and LTB4 decreased rapidly. The disappearance of LTB4 was concomitant with the formation of ω-hydroxy-LTB4 and ω-carboxy-LTB4. When synthetic LTB4 was added to a suspension of unstimulated human leukocytes, a similar pattern of ω-oxidation products was observed. This ω-oxidation process showed some specificity for LTB4, as indicated by the slower reaction of three stereoisomers of LTB4. Studies with subpopulations of human blood cells and human plasma clearly indicated that the polymorphonuclear leukocytes were the main source of enzymic activity for the ω-oxidation of LTB4. The liquid chromatographic behaviors of natural ω-hydroxy-LTB4 and ω-carboxy-LTB4 were studied in two systems.
Effect of 12-O-tetradecanoylphorbol-13-acetate on cell-surface glycosaminoglycans in human diploid fibroblasts
Authors: Wolff Bruce R. , Glick Bernard R. , Bols Niels C.
Published: December 2009

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the loss of glycosaminoglycans (GAGs) from the pericellular compartment of human skin fibroblasts was studied. GAGs from this compartment were analyzed by anion-exchange chromatography after cultures had been labelled with both [3H]glucosamine and [35S]sulfate and then chased in either the presence or absence of TPA. In both control and TPA cultures radioactivity was found in glycopeptides, hyaluronic acid, heparan sulfate (HS), and chondroitin sulfate. The amount of radioactivity that was found in HS was reduced in the TPA cultures, whereas the amounts in the other GAGs were essentially unchanged when control and treated cultures were compared. These results suggest that TPA stimulates the loss of HS from the surface of human fibroblasts.
Phospholipid-dependent changes in membrane permeability induced by staphylococcal δ-lysin and bee venom melittin
Authors: Bhakoo M. , Birkbeck T. H. , Freer J. H.
Published: December 2009

The effect of purified staphylococcal δ-lysin and bee venom melittin on the permeability of large unilamellar vesicles (liposomes) composed of structurally defined lipids was investigated by following release of sequestered ATP using a bioluminescent assay. ATP release was linearly related to the logarithm of the peptide concentration. Sensitivity to the lytic peptides, as measured by the release of ATP and the slope of the dose–response curve, depended on the chain length and the degree of saturation of the hydrocarbon substituents of the phospholipid in the vesicles. Neither the sign of the surface charge nor the presence of sterol had any significant effect on the sensitivity of liposomes to peptide-induced lysis. Release of ATP and haemoglobin from intact sheep and cod erythrocytes was also determined after their exposure to δ-lysin and melittin. Both cell types showed similar degrees of sensitivity to each lytic peptide when ATP release was followed, but the release of haemoglobin from sheep erythrocytes by δ-lysin was much less than from cod erythrocytes. The implications of these results on the mode of action of the two agents are discussed.
The effect of the divalent cations Mg2+ and Mn2+ on adenylate cyclase activity in white and brown adipose tissue of lean and obese (ob/ob) mice
Authors: Bégin-Heick Nicole
Published: December 2009

The divalent cations Mg2+ and Mn2+ in excess of the concentrations required to complex with ATP (excess Mg2+ or Mn2+) modulate the activity of adenylate cyclase. As a substrate, Mn∙ATP was at least as effective as Mg∙ATP in supporting adenylate cyclase activity in white and brown adipose tissue membranes. Both excess Mg2+ and Mn2+ had quantitatively different effects on the enzyme of lean and ob/ob mice and qualitatively different effects in white and brown adipose tissue. In white adipocyte membranes excess Mg2+ increased basal activity, as well as activity owing to guanylylimidodiphosphate (Gpp(NH)p) (with or without isoproterenol) and NaF. Maximal activation by Gpp(NH)p + isoproterenol required a higher concentration of Mg2+ in tissue from ob/ob than lean mice. Excess Mn2+ prevented the activation of the enzyme by Gpp(NH)p or Gpp(NH)p + isoproterenol in a dose-dependent manner. Mn2+ inhibited even in the presence of maximally effective Mg2+ concentrations. The enzyme of the ob/ob mouse membrane required a significantly higher dose of Mn2+ to achieve 50% inhibition. In brown adipose tissue, specific activities of the isoproterenol + Gpp(NH)p and NaF stimulated enzyme were significantly lower in the obese mice under all conditions studied. Except that NaF-stimulated activity was increased significantly more in the membranes of lean mice by the combination of Mg2+ + Mn2+, these cations did not produce significantly different dose-dependent effects in membranes from lean and ob/ob mice. Maximal activation occurred at lower concentrations of MgCl2 (3–5 versus 10–20 mM) and required higher concentrations of MnCl2 (3–5 versus 1 mM) in brown than in white adipose tissue membranes. Furthermore, Mn2+ in excess of the concentration required to activate maximally produced little or no inhibitory effect on the brown adipose tissue enzyme. These studies illustrate the diversity of adenylate cyclase modulation in different tissues. Further studies with bacterial toxins will be necessary to verify whether there are differences in the equilibrium of association of the stimulatory and inhibitory components of guanine nucleotide regulatory proteins in tissues of lean and obese mice.
Metallothionein synthesis and localization in relation to metal storage in rat liver during gestation
Authors: Templeton Douglas M. , Banerjee Diponkar , Cherian M. George
Published: December 2009

The metallothionein (MT) content of fetal rat liver was measured daily during the final week of gestation, by both cadmium saturation and polarographic methods. MT levels rise sharply at day 18 of gestation and continue to increase into the neonatal period. In late gestation, MT serves to bind Cu and Zn from the preexisting hepatic pools of these metals, as well as to accumulate additional amounts of both metals The fetal MT is similar to the adult rat protein both in terms of its protein composition and metal-binding properties. Perinatally the Zn/MT ratio remains constant for several days suggesting a carefully regulated process. At birth, most of the hepatic Zn and a significant amount of hepatic Cu are bound to MT. Immunohistochemical localization of MT shows a progressive increase in cytoplasmic MT with the appearance of nuclear MT by day 20 of gestation in fetal rat liver. The results are discussed in terms of a model for regulation of MT synthesis and for the metal storage role of MT in perinatal development.
Development of a monoclonal antibody to the rabbit 8.5S uterine progestin receptor
Authors: Nakao Kiyohide , Myers John E. , Faber Lee E.
Published: December 2009

Nonactivated (8.5S) rabbit uterine progestin receptor was enriched 10- to 30-fold by chromatography on columns of spheroidal hydroxylapatite and DEAE-cellulose. A total of`~25 μg of receptor (purity ~1%) was injected at multiple sites into a BALB/c mouse. After several injections, splenic lymphocytes were fused with P3x63Ag8.653 mouse myeloma cells. This fusion produced in excess of 240 hybridomas, which were screened by an enzyme-linked immunosorbent assay (ELISA), solid-phase radioimmunoassay, and sucrose gradient centrifugation. One colony (KN 382/EC1) produced a mouse immunoglobulin G1 which bound rabbit 8.5S uterine progestin receptor. The cell line has been repeatedly cloned under conditions of limiting dilution and expanded in mice as ascitic tumors. Antibody was purified by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, and affinity chromatography with protein A – Sepharose CL-4B. Specificity of the antibody was determined by sucrose gradient centrifugation and solid-phase radioimmunoassay. The antibody bound to progestin receptors from rabbit uterus and MCF-7 breast cancer cells. It did not react with progestin receptors from rat uterus, guinea pig uterus, or chick oviduct, nor did it bind to estrogen receptors from any of the tissues we tested.
Isolation, purification, and characterization of a peptide that contains the β-ketoacyl reductase, enoyl reductase, and β-hydroxyacyl dehydrase activities of the pigeon liver fatty acid synthetase
Authors: Puri Rajinder N. , Porter John W.
Published: December 2009

Controlled proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides that are designated II and IV. After 5 h of proteolysis the incubation mixture containing these peptides retains all of the component enzyme activities of the fatty acid synthetase complex. The two peptides are then separated by chromatography on an Affi-Gel Blue column. Gel filtration of the fraction containing peptide II yields a homogeneous peptide as shown by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of this peptide has been estimated to be 130 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, size exclusion chromatography, and amino acid analysis. The sedimentation coefficient for peptide II is approximately 7.4S. Peptide II contains the domains for the β-ketoacyl and enoyl reductases and β-hydroxyacyl dehydrase activities of the fatty acid synthetase complex.
Effects of bovine serum albumin on the interaction of concanavalin A and succinyl-concanavalin A with phospholipid bilayers
Authors: Chicken Christina A. , Sharom Frances J.
Published: December 2009

Under physiological conditions, concanavalin A interacts with the surface of phospholipid liposomes through two distinct classes of binding sites, a relatively small number of high affinity sites and a much larger number of lower affinity sites. Addition of bovine serum albumin induces extensive additional binding of concanavalin A to liposomal membranes and this binding is saturable and "specific" (α-methyl mannoside inhibitable). Fraction V and high purity albumin both induce almost identical levels of concanavalin A binding to liposomes. Scatchard plots of the binding data demonstrate the induction of a large number of new, relatively high affinity lectin-binding sites on addition of albumin. Albumin-induced binding of concanavalin A to the bilayer surface shows a broad pH optimum and is not inhibited by 40% (w/v) ethylene glycol, suggesting that hydrophobic forces are relatively unimportant. In contrast, divalent succinyl-concanavalin A shows very little tendency to bind to liposomes, either in the absence or presence of albumin. Passage of high purity albumin down a concanavalin A affinity column or treatment with periodate completely eliminates the additional lectin binding. It thus seems likely that albumin-induced concanavalin A binding to liposomes is related to the presence of a concanavalin-A-binding component. This phenomenon may have important implications for lectin-binding studies carried out on membranes which have been exposed to serum proteins.
Effect of adroxazine, a heterocyclic compound of the adrenals, on the rate of replication of lymphoblastoid cells
Authors: Rice F. A. H. , McCurdy J. D. , Oresajo C.
Published: December 2009

It has been found that the addition of 10−2–10−8 μL/mL of adroxazine, a heterocyclic compound of the adrenals, to the tissue culture medium increases the rate at which three strains of lymphoblastoid cells replicate. For example, the addition of 10−5 μg/mL of adroxazine to cultured L5178Y cells decreases their doubling time from approximately 11 to 8 h and addition of 10−3 μg/mL of adroxazine to the tissue culture medium of Huly-16 or Huly-29 cells decreases their doubling time from approximately 43 to 34 and 36 h, respectively. Other concentrations of adroxazine, from 10−2 to 10−8 μg/mL, cause a less but measurable decrease in doubling time. The effect of adroxazine on the rate of incorporation of [3H]thymidine into acid-precipitable material by lymphoblastoid cells was found to parallel the effect of adroxazine on doubling time. The effect of adroxazine on the length of DNA synthesis and mitosis was calculated from the percent of cells that are labeled with [3H]thymidine during a 20-min interval and the percent of cells in mitosis, respectively. Results showed that adroxazine does not decrease the length of DNA synthesis or mitosis, but markedly decreases the sum of pre- and post-mitotic resting times [Formula: see text]. Addition of 10−3 μg/mL of adroxazine to the medium decreases the resting times of Huly-16 cells from approximately 20 to 10 h and Huly-29 cells from approximately 12 to 5 h. Addition of 10−5 μg/mL of adroxazine to the medium decreases the resting time of L5178Y cells from 3.3 to 0.1 h. The effect of adroxazine on [Formula: see text] and [Formula: see text] was calculated by using the fraction of labeled cells in mitosis method. It was found that adroxazine markedly decreases the mean duration of both [Formula: see text] and [Formula: see text].
Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli
Authors: Brown Deborah A. , Cook Robert A.
Published: December 2009

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.
The nucleotide sequence of and from Drosophila melanogaster
Authors: Addison W. R. , Gillam I. C. , Hayashi S. , Tener G. M.
Published: December 2009

The nucleotide sequences of two valine tRNA's of Drosophila melanogaster are the following: tRNA[Formula: see text], pGGUUmCCAUAGUGψAGCGGDU*AUCACG(m2G)ψCUGCψUU*ACACGCAGAAGm7GDC(m5C)UCCGGTψCGm1AUCCCGGAUGGAACCACCA; [Formula: see text], pGUUUCCGUAGUGψAGCGGDacp3UAUCACGψGUGCUUCACACGCACAAGm7GDC(m5C)CCCGGTψCGm1AACCCGGGCGGGAACACCA. [Formula: see text] shows greater similarity to prokaryotic and eukaryotic organelle tRNAVal,s than does [Formula: see text].
Properties of purified bovine platelet-derived growth factor stimulating proliferation of human and mouse fibroblasts
Authors: Narczewska Barbara , Czyrski Józef A. , Inglot Anna D.
Published: December 2009

Platelet-derived growth factor (PDGF) was isolated from platelets obtained from fresh bovine blood. The platelet lysates were extracted with acid–ethanol, according to the classic procedure that has been applied to the preparation of various protein hormones and transforming growth factors, but not until now to PDGF isolation. Bovine PDGF was further purified by ion-exchange chromatography on DEAE-Sephadex, CM-Sephadex, and finally by molecular sieving on Sephadex G-100. Bovine PDGF has an isoelectric point of 9.45–10.6 and a molecular weight ranging from 28 000 to 31 000. Reduction of bovine PDGF with 2-mercaptoethanol revealed the presence of two polypeptides with molecular weights of approximately 14 000 and 15 000. A simple biologic microassay of cell multiplication-stimulating activity has been developed in our laboratory. Purified bovine PDGF stimulated the proliferation of BALB/c 3T3 or human fibroblasts at minimal concentrations of 0.36–1.5 ng/mL.
The interaction of rabbit skeletal muscle troponin-T fragments with troponin-I
Authors: Pearlstone Joyce R. , Smillie Lawrence B.
Published: December 2009

The interactions of troponin-I (Tn-I) with a variety of fragments spanning the length of the troponin-T (Tn-T) polypeptide chain have been reinvestigated at physiological ionic strength by affinity chromatographic, gel filtration, and circular dichroism methodologies. Strong binding was observed with fragment T2 (residues 159–259) mimicking that observed with whole Tn-T and Tn-I. Partial binding was seen with the shorter cyanogen bromide (CB) fragments of Tn-T in the order CB4 (residues 176–230) > CB6 (residues 239–259) or CB5 (residues 152–175). No interaction with Tn-I was observed with fragments (CB2, CB3, T1) encompassing residues 1–158 of Tn-T. Based on the present results and the work of others, the binding region for Tn-I includes residues 159–259 and perhaps extends into the highly helical CB2 region (residues 71–151) of Tn-T. No evidence has been obtained by ourselves or others for the interaction of the CB3 region (1–70) with Tn-I. A significant increase (11.6%) in α-helical content was observed when an equimolar amount of fragment T2 (residues 159–259) was mixed with Tn-I, a result similar to that seen with whole Tn-T and Tn-I.
Discrimination of the isozymes of human placental hexosaminidase by kinetic parameter estimation
Authors: Tommasini R. , Mahuran D. J. , Lowden J. A.
Published: December 2009

Human placental β-N-acetylhexosaminidase (EC 3.2.1.52) (HEX) is a lysosomal glycosyl hydrolase with an acidic pH optimum. Four isozymes (HEX B, HEX I1, HEX I2, and HEX A) have been isolated from human placenta. HEX BA derived from the subunit rearrangement of HEX A was also prepared. To determine if the isozymes of hexosaminidase differ in their kinetic parameters, the conditions for 4-methylumbelliferyl-β-D-N-acetylglucosaminide hydrolysis were optimized for each isozyme. The Km values were essentially similar and varied from 0.64 ± 0.06 for HEX BA to 0.85 ± 0.13 for HEX I1. The Vmax values were similar only for HEX I1 (3.90 ± 0.28 kat kg−1) and HEX I2 (4.40 ± 0.17). Vmax values varied significantly between HEX A (9.68 ± 0.52), HEX B (8.00 ± 0.75), HEX BA (4.81 ± 0.17), and the HEX I values.
Recovery of normal protein synthesis in heat-shocked chicken myotubes by liposome-mediated transfer of mRNAs
Authors: Bag Jnanankur
Published: December 2009

Exposure of chicken myotube culture to 45 °C induced the synthesis of three heat-shock polypeptides of 25 000, 65 000, and 81 000 daltons. Recovery to the normal pattern of protein synthesis was judged by the decrease in the synthesis of heat-shock polypeptides. This recovery to normal protein synthesis required de novo synthesis of mRNAs for normal cellular proteins. Inhibition of RNA synthesis by actinomycin D during recovery at 37 °C blocked the recovery process and resulted in the continued synthesis of heat-shock polypeptides. Large unilamellar vesicles were used to examine the effect of delivery of mRNAs isolated from both normal and heat-shocked myotubes on the recovery of these cells from heat-shock treatment. The results presented here show that liposome-mediated delivery of normal mRNAs to heat-shocked cells relieved the block of recovery by actinomycin. On the other hand, when mRNAs from heat-shocked cells were used during recovery, the synthesis of heat-shock polypeptides was stimulated. These observations suggest that the relative abundance of mRNAs in the cytoplasm plays a critical role in regulating protein synthesis in chicken myotube cultures.
In vitro recombination of bacteriophage T7 DNA: Further characterization of the reaction using plasmid DNA
Authors: Lee Donald , Vetter Dan , Beatty Linda , Sadowski Paul
Published: December 2009

We have used a plasmid which contains a cloned fragment of T7 DNA to study the properties of general recombination of phage T7 in vitro. It was shown that T7-infected cell extracts promote recombination by the exchange of double strands of DNA. While both products of these double-strand exchanges were detected, we were unable to show that they were formed during a single recombination event.
Assignment of lectins specific for D-galactose or N-acetyl-D-galactosamine to two groups, based on their circular dichroism
Authors: Young N. Martin , Williams Ross E.
Published: December 2009

The circular dichroism (CD) spectra of thirteen lectins, most being specific for D-galactose or N-acetyl-D-galactosamine, were compared. Two groupings are proposed on the basis of the CD in the near-ultraviolet region. Group one comprises the lectins from Arachis hypogaea, Glycine max, Phaseolus coccineus, P. lunatus, P. vulgaris, and Vicia villosa, and group two comprises lectins from Caragana arborescens, Cytisus sessilifolius, Dolichos biflorus, Griffonia simplicifolia, and Wisteria floribunda. The CD spectra of lectins from Bauhinia purpurea and Sophora japonica were different from any of the other lectins. CD difference spectra produced by the two sugars were distinctive for each protein, suggesting that the combining sites of these lectins are not homologous despite similarities in specificity and that the CD spectral similarities arise from residues in other, more homologous regions of the proteins.
Studies on the primary structure and antigenic determinants of pilin isolated from Pseudomonas aeruginosa K
Authors: Sastry Parimi A. , Pearlstone Joyce R. , Smillie Lawrence B. , Paranchych William
Published: December 2009

The complete amino acid sequence of Pseudomonas aeruginosa K (PAK) pilin was determined using a combination of automated and manual Edman degradation techniques. Suitable peptides were derived from cyanogen bromide, tryptic, chymotryptic, peptic, thermolytic, and citraconylated tryptic cleavages of unmodified or carboxymethylated pilin. The protein, a single polypeptide chain, has N-methylphenylalanine at the NH2-terminus, a total of 144 residues, a molecular weight of 15 013, and an equal number of acid and basic amino acids. The NH2-terminal region (residues 1–43) is very hydrophobic with only three charged residues, suggesting a possible role in subunit–subunit interaction. The two half-cystines, residues 129 and 142, are shown to be linked through a disulfide bridge in the native protein. To delineate the antigenic regions of pilin, the protein was cleaved at Arg-30, Arg-53, and Arg-120 to produce peptide fragments cTI (residues 1–30), cTII (residues 31–53), cTIII (residues 54–120), and cTIV (residues 121–144). cTIII and cTIV were further degraded into several subfragments. The purified peptides were subjected to immunological analysis using direct and competitive enzyme-linked immunosorbent assay procedures. A major antigenic determinant was delineated in a region of the protein encompassing residues 82–101. Three other epitopes were also identified, but reacted with only minor amounts of antibody in the rabbit polyclonal antiserum.
Biosynthesis of 6,7-dihydroxycoumarin in Cichorium intybus
Authors: Brown Stewart A.
Published: December 2009

Labelled 7-hydroxycoumarin (umbelliferone) was converted efficiently to 6,7-dihydroxycoumarin (aesculetin), the aglycone of the glucoside cichoriin, in Cichorium intybus L., and earlier studies contraindicating caffeic acid as a precursor were confirmed. Umbelliferone, which occurs in this species, is thus indicated to be a natural intermediate in aesculetin biosynthesis. Administered umbelliferone was readily converted to its 7-O-glucoside, skimmin, an observation consistent with participation of skimmin in the biosynthetic pathway to cichoriin. This evidence for derivation of aesculetin from umbelliferone provides further support for the theory that polyoxygenated coumarins are, in general, elaborated by additional oxygenation of umbelliferone. Implications of this concept for the evolution of the biosynthesis of coumarins are discussed.
Association of fodrin with brain microtubules
Authors: Fach Barbara L. , Graham Susan F. , Keates Robert A. B.
Published: December 2009

We have compared the polypeptide composition of microtubules isolated from bovine brain by the conventional in vitro reassembly method with those obtained by direct isolation of brain microtubules into a stabilizing buffer. The stabilizing buffer included 6.7 M glycerol to limit the rate of subunit exchange between assembled and unassembled states. The microtubule-associated proteins normally found by in vitro reassembly are also found in the stabilized preparation, but in smaller proportions. Fodrin, a brain membrane-associated protein believed to be homologous to spectrin, was found to be the most abundant component after tubulin in the stabilized microtubules. The ratio of tubulin to fodrin, 16:1 by mass, was almost constant at each stage of the preparation. Some actin was initially present in the stabilized microtubules, but was gradually lost during purification. When stabilized microtubules were diluted into cold aqueous buffer, they depolymerized and the recovered microtubule protein could then be purified by in vitro reassembly. The composition after this treatment resembled that of microtubules prepared initially by reassembly in vitro. The missing fodrin was found to be removed in the preliminary centrifugation and was unavailable for incorporation into growing microtubules during the in vitro assembly step. This suggests that the standard in vitro reassembly procedure for purification of microtubules may distort the composition of microtubule-associated proteins.
Studies on lectins. LIX. Isolation and properties of lectins from fruiting bodies of Xerocomus chrysenteron and Lactarius lignyotus
Authors: Sychrová Hana , Tichà Marie , Kocourek Jan
Published: December 2009

The lectins of fruiting bodies of Xerocomus chrysenteron and Lactarius lignyotus were purified on Sepharose 4B containing immobilized fetuin. Both lectins agglutinate human erythrocytes nonspecifically at limit concentrations of 15 μg/mL. Their erythroagglutinating activities are not inhibited by simple sugars; desialyzed fetuin, desialyzed glycoprotein from edible bird's nest, and desialyzed mucin from porcine submaxillary glands are the most effective inhibitors.
In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product
Authors: Chapdelaine Pierre , Dubé Jean Y. , Frenette Gilles , Tremblay Roland R.
Published: December 2009

Poly(A)+ rich RNA was isolated from prostate of adult dogs and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. Two-dimensional gel electrophoresis of the translation products showed that a protein with a molecular weight of 31 000 was predominantly synthesized. This protein was immunoprecipitated with antibodies directed against purified arginine esterase from dog seminal plasma. mRNA isolated from the prostate of animals castrated for 1 or 2 weeks was unable to direct the synthesis of arginine esterase. However, the synthesis of the enzyme could be stimulated by androgens in castrated animals, presumably by increasing prostatic concentrations of arginine esterase mRNA. The single chain translation product could be further processed in vitro by the addition of dog pancreas microsomes and purified arginine esterase. This procedure yielded split chains of arginine esterase which had identical electrophoretic mobilities as seminal plasma enzyme by two-dimensional gel electrophoresis. When prostatic tissue slices were incubated with tunicamycin, the unglycosylated arginine esterase obtained had a lower molecular weight than the in vitro translation product, suggesting that a signal peptide had been removed in the living cells. These results indicate that arginine esterase processing may include the following steps: removal of a signal peptide, glycosylation, and splitting of the polypeptide chain by active arginine esterase in the secretory granules or outside the cell.
Effects of cytochalasin B on steroid-induced oocyte meiosis and centrifugally induced nuclear movement in the goldfish Carassius auratus
Authors: Habibi Hamid R. , Lessman Charles A.
Published: December 2009

In goldfish, 17α, 20β-dihydroxyprogesterone (DHP) induced oocyte nucleus or germinal vesicle migration (GVM) and dissolution (GVD) in a dose-related fashion. Administration of cytochalasin B (CB) in the presence of DHP inhibited the steroid-induced GVM and GVD after 24 and 48 h of incubation. The presence of CB alone, at concentrations below 25 μg/mL, had no effect on GVM or GVD. Furthermore, CB, either alone or in combination with DHP, elicited significant increases in follicular diameter after 24 and 48 h of incubation. To test the effect of CB on ooplasmic viscoelasticity, fully grown follicles were centrifuged and the centrifugally induced germinal vesicle (GV) displacement was determined. Pretreatment (24 h) of follicles with high doses of CB (25 and 50 μg/mL) increased the movement of GV in a centripetal direction. However, at lower concentrations (0.005–5 μg/mL), CB treatment was without an effect on the centrifugally induced GV movement in the oocyte. The present study suggests involvement of microfilaments or other cytoskeletal components, sensitive to CB, in the mechanisms of GVM and GVD in goldfish oocytes. In addition, a simple technique has been described for testing ooplasmic viscoelasticity determined by movement of the GV under a centrifugal force.
Tc1(Hin): a form of the transposable element Tc1 in Caenorhabditis elegans
Authors: Rose A. M. , Harris L. J. , Mawji N. R. , Morris W. J.
Published: December 2009

In this paper we describe the coexistence of two forms of the transposable element Tc1 in the genome of the nematode Caenorhabditis elegans. A copy of the variant form has been isolated from the Bergerac genome and characterized. Restriction mapping and DNA sequencing have shown that a G to T transversion generated a HindIII restriction site to form the variant Tc1(Hin). The presence of this new restriction site makes this variant easily detectable on genomic blot hybridizations. There are approximately 20 copies of Tc1(Hin) amongst the Tc1's present in the Bergerac genome. Bergerac has approximately 250 copies of Tc1 per genome, whereas Bristol has about 30. In the Bristol strain we detected at least one copy Tc1(Hin). The ratio of Tc1 (Hin) to total Tc1's is similar in the genomes of Bristol and Bergerac, even though they have markedly different total numbers of Tc1. Our results suggest that a trans-acting change in either the elements or the host genome was responsible for the expansion of Tc1 copy number in the Bergerac genome.
On the interaction between 5-hydroxytryptamine and N-acetylneuraminic acid under aqueous conditions
Authors: Berry Leslie R. , Puzzuoli Frank V. , Hatton Mark W. C.
Published: December 2009

A complex designated 5-HT–NeuAc was formed between 5-hydroxytryptamine (5-HT) and N-acetylneuraminic acid (NeuAc) under aqueous conditions. Complex formation was encouraged by exposure to light (3000–3800 Å; 1 Å = 0.1 nm) and freeze-drying and the freeze-dried complex was isolated by gel filtration chromatography. Although stable to rechromatography on Bio-Gel P-2 if H2O was the eluent, 5-HT–NeuAc dissociated into the free components when placed in 0.1 M NaCl. Chemical analyses of the isolated complex showed that an equimolar amount of 5-HT and NeuAc was present and that all group functions were intact; these data suggested that the association between 5-HT and NeuAc was noncovalent. Spectrophotometric measurements demonstrated a small increase (approximately 12%) in extinction coefficient (275 nm) and a large increase (340- to 440-fold) in fluorescence emission (340 nm) compared with 5-HT alone. Data obtained from 1H nuclear magnetic resonance spectroscopy (250 MHz) of 5-HT and NeuAc standards compared closely to published reports. In comparison, measurements made with 5-HT-NeuAc showed that all 5-HT protons were slightly deshielded; of the NeuAc protons, slight deshielding of H8 and significant shielding of H3eq, H3ax, and H6 was observed. From these observations, a model describing the association between 5-HT and NeuAc is proposed.
Poly(ADP-ribosyl)ation of chromatin: kinetics of relaxation and its effect on chromatin solubility
Authors: Frechette André , Huletsky Ann , Aubin Rémy J. , Murcia Gilbert de , Mandel Paul , Lord André , Grondin Gilles , Poirier Guy G.
Published: December 2009

We have studied the kinetics of relaxation of poly(ADP-ribosyl)ated polynucleosomes produced by endogenous enzyme activity by comparing the generation of hyper(ADP-ribosyl)ated histone H1 and its effect on the chromatin structure as revealed by electron microscopy. A correlation can be established between the appearance of histone H1 modified forms and the localized relaxation of the chromatin. We have also noticed, in parallel, that poly(ADP-ribosyl)ated chromatin showed increased solubility in the presence of Mg2+ and 0.2 M NaCl. Electron microscopic examination of the solubilized chromatin produced by poly(ADP-ribosyl)ation shows polynucleosomes exhibiting more relaxed conformation, whereas an increasing amount of hyper(ADP-ribosyl)ated histone H1 is found in the pellet, as shown by acid–urea–polyacrylamide electrophoretic separation of histone extracts.
Activation of folylpolyglutamate synthetase by pteroic acid
Authors: Vickers P. J. , Cecco R. Di , Pristupa Z. B. , Scrimgeour K. G.
Published: December 2009

The glutamylation of methotrexate catalyzed by beef liver folylpolyglutamate synthetase (FPGS) is activated by addition of pteroic acid. Pteroic acid causes greater stimulation of FPGS, including glutamylation of tetrahydrofolate, at neutral pH values (i.e., below the pH optimum of 8.4). We have attributed this activation to a conformational change of FPGS induced by pteroic acid.
The modulation by serum albumin of rabbit platelet aggregation in response to exogenous sodium arachidonate
Authors: Dratewka-Kos Ella , Kindl Brigitte , Tinker David O. , Hsia J. Carlton
Published: December 2009

This work presents a quantitative study of the modulation of platelet responsiveness to sodium arachidonate by serum albumin. Rabbit platelets suspended in protein-free buffer containing dextran aggregate reversibly in response to micromolar amounts of ADP and sodium arachidonate. The optimal concentration of arachidonate for aggregation response is 5 μM. Inhibition occurs at higher concentrations and is not related to thromboxane A2 formation since arachidonate inhibits ADP-induced aggregation of aspirin-treated platelets. Thin-layer chromatographic studies show that, at the high arachidonate levels sufficient to almost completely abolish platelet aggregation, the synthesis of thromboxane A2 persists. Albumin relieves the inhibition caused by excess arachidonate, whether the stimulus is arachidonate itself or ADP. This effect is due to arachidonate binding and is optimal at fatty acid/protein ratios near 4; no stimulation of platelets was observed at ratios less than 2. The optimal concentration of arachidonate for stimulation of platelet aggregation occurs in the range where albumin buffers the free arachidonate concentration most effectively, hence the extremely narrow range of total arachidonate concentrations effective for platelet response seen in the absence of albumin is enormously broadened in the presence of albumin. Albumin inhibits conversion of arachidonate to thromboxane A2 and hydroxy acids, especially at ratios of arachidonate/albumin below 10. Bilirubin (an albumin ligand) has no effect on albumin modulation of platelet response until the bilirubin/protein ratio exceeds 2. Palmitate progressively displaces arachidonate from albumin and affects the range of effective arachidonate concentrations but not the maximum response.
Partial characterization of chromosomal proteins tightly bound to chicken erythroid DNA
Authors: Liew C. C. , Hentzen Peter C. , Bekhor Isaac
Published: December 2009

Extraction of chicken reticulocyte and erythrocyte chromatins with 2 M NaCl yields a small fraction (about 5%) of the total DNA which is very tightly bound to a class of nonhistone chromatin proteins (DNA–P). This DNA fraction has previously been shown to be significantly enriched in active gene sequences. The proteins associated with reticulocyte and erythrocyte DNA–P were analyzed by two-dimensional gel electrophoresis. Reticulocyte DNA–P yield predominantly three major proteins, designated G1, G2, and G3 with relative masses of 80 000, 50 000, and 58 000, respectively. Erythrocyte DNA–P show only two proteins which appear to be similar to the reticulocyte G1 and G2 proteins, except in much reduced quantities as revealed by two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis of the three reticulocyte proteins revealed that the ratio of acidic to basic amino acid residues increased in the order G1 < G2 < G3, while the respective isoelectric points also increased in that order.
The effects of estrogen on apolipoprotein synthesis
Authors: Deeley Roger G. , Tam Shui-Pang , Archer Trevor K.
Published: December 2009

Sex steroids are important regulators of apolipoprotein synthesis and metabolism in a wide variety of species. In man, epidemiological studies have established strong correlations between sex steroid levels, circulating apolipoprotein profiles, and atherosclerotic risk. In this review, we compare data obtained from studies on two model systems that are being used to examine molecular mechanisms by which estrogens and androgens may influence hepatic lipoprotein synthesis. The first involves estrogen-dependent induction of lipoprotein synthesis in avian liver and the second involves estrogen-dependent modulation of lipoprotein synthesis in the human hepatocarcinoma cell line HepG2.
Protein kinase and ATP-binding activity associated with the 72-kdalton single-stranded DNA-binding protein from early region 2A of human adenovirus type 5
Authors: Branton Philip E. , Evelegh Mike , Rowe David T. , Graham Frank L. , Bacchetti Silvia
Published: December 2009

We have developed monoclonal antibodies which react specifically with the human adenovirus type 5 early region 2A 72-kdalton phosphoprotein (72 kDa) and its 48-kdalton proteolytic cleavage product (48 kDa) and have used these antibodies to study a number of properties of these viral polyeptides. Fluorescent antibody staining indicated that the 72 kDa was found almost entirely in the nucleus, generally in discreet patches. Preparations of the 72 kDa, purified by immunoprecipitation or by single-stranded DNA-cellulose column chromatography and incubated with [γ-32P]ATP, were found to contain protein kinase activity. Using photoaffinity labelling with 8-azido-[α-32P]ATP, the 72 kDa was shown to be an ATP-binding protein. The ATP-binding site was probably in the amino terminal region because the 48 kDa which lacks approximately 120 residues at the amino terminus failed to bind ATP. Experiments carried out with immunoprecipitates from wild-type and temperature-sensitive (ts125) infected cells suggested that the ts mutant-induced kinase activity was not more thermolabile than the wild-type protein. Thus although the present results indicated that protein kinase activity is associated with the 72 kDa, it is still unclear whether such activity is intrinsic to the 72 kDa or present in an enzyme associated with it. While it is probably not intrinsic to the carboxy terminal region affected by the ts125 mutation, it is still possible that it resides in an amino terminal domain.
Chinese hamster ovary cell mutants specifically affected in the phosphorylation of C-purine nucleosides
Authors: Mehta Kamal D. , Gupta Radhey S.
Published: December 2009

In Chinese hamster ovary cells, two different types of mutants resistant to purine nucleoside analogs have been isolated. One type of mutants selected in presence of C-nucleosides formycin A and formycin B (FomR mutants) exhibited a high degree of cross resistance to different C-nucleosides but showed very slight to no cross resistance to various N-nucleosides. In contrast, mutants selected in presence of toyocamycin (Toyr mutants) exhibited a high degree of cross resistance to all C- and N-adenosine analogs. Studies on the cellular uptake and phosphorylation of [3H]adenosine, [3H]tubercidin, and [3H]formycin A show that, unlike the Toyr mutants which show reduced phosphorylation of all three compounds, the FomR mutants show reduced cellular phosphorylation of only [3H]formycin A. It is suggested that the genetic lesion in the FomR mutants affects adenosine kinase in a novel manner that specifically affects the phosphorylation of C-purine nucleosides.