bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of phytohemagglutinin to porcine lymphocyte receptors. Time-course studies and comparative binding isotherms using whole cells or cellular receptors incorporated into phospholipid vesicles
Authors: Dupuis Gilles , Bastin Bânû
Published: December 2009

We have studied the binding of 125I-labeled phytohemagglutinin (PHA) to porcine splenic lymphocytes (107 cells∙mL−1) at 37 °C. Our data show that PHA binding is dependent on the incubation period and that a maximum quantity of 4.53 ± 0.13 μg is bound when 200 μg of PHA is added. The binding process is rapid and saturation can readily be achieved in less than 2 min. These observations suggest, in accordance with the mass-action principle, that the binding equilibrium can be rapidly displaced towards PHA–receptor complex formation when sufficient amounts of PHA are added. Our data further suggest that receptor sites are exposed at the cell surface and that putative cryptic receptor sites are unlikely to play a major role in the initial part of the binding process. We have studied this aspect by comparing Scatchard plots for PHA binding to receptors in intact lymphocytes and to lymphocyte-derived receptors incorporated into liposomes. In one case, partially purified plasma membranes were solubilized and incorporated into phosphatidylcholine–phosphatidylserine (PC–PS) vesicles prepared by detergent dialysis. In another case, PHA-receptor glycoproteins were purified by affinity chromatography and reassembled into PC–PS vesicles, using the same technique. Scatchard plot analyses showed nonlinear profiles with a concavity turned upward for PHA binding to receptors of intact cells or of PC–PS vesicles with plasma membranes, and a concavity turned downward for vesicles with purified glycoproteins. These observations suggest that PHA-receptor environment plays a determining role in the binding process of the lectin. Numerical data from binding experiments were obtained by computer-assisted nonlinear regression analysis, using a one ligand–two sites model. The (apparent) high-affinity constant (K1) for intact lymphocytes or partially purified plasma membrane components reassembled into liposomes was 4.6 × 107 M−1 and the (apparent) low-affinity constants (K2) were 4.4 × 106 ± 1.5 × 106 M−1 (intact lymphocytes) and 1.7 × 106 ± 0.6 × 106 M−1 (plasma membranes constituents reassembled into liposomes). The value obtained for reconstituted PHA-glycoprotein receptors (K) was 0.70 × 107 ± 0.10 × 107 M−1. The apparent number of sites was n1 = 0.19 × 106 ± 0.05 × 106 (high affinity) and n2 = 2.50 × 106 ± 0.50 × 106 (low affinity) sites per intact lymphocyte cell. In the case of the reconstituted systems, the number of sites per microgram protein of the PC–PS vesicles were n1 = 0.79 × 1010 ± 0.18 × 1010 and n2 = 31.8 × 1010 ± 8.5 × 1010 for the partially purified plasma membrane and n = 26.6 × 1010 ± 2.9 × 1010 for the purified PHA receptor glycoproteins. Data presented here are discussed in terms of the lymphocyte activation model of Prujansky and colleagues, which suggests that positively cooperative lectin binding is a sine qua non condition for lymphocyte activation.
Spectral and kinetic properties of a cationic peroxidase secreted by cultured peanut cells
Authors: Lambeir Anne-Marie , Dunford H. Brian , Huystee Robert B. van , Lobarzewski Jerzy
Published: December 2009

It is demonstrated that the cationic peroxidase isolated from the growth medium of cultured peanut cells reacts via the same mechanism as other peroxidases, namely conversion of the native enzyme into compound I by reaction with hydrogen peroxide, followed by two reductions by one-electron donors to compound II and then back to the native enzyme. From the pyridine hemochromogen spectrum it is concluded that the prosthetic group of the native enzyme is ferriprotoporphyrin IX. Optical spectra are recorded for (i) the native (ferric) enzyme and its cyanide, azide, fluoride and alkaline forms, (ii) ferrous peroxidase and its cyanide and carbon monoxide complexes, and (iii) compounds I, II, and III. Equilibrium studies show that the ferric cyanide complex has a dissociation constant of 3.0 ± 0.5 μM over the pH range 3–8. The fluoride complex has a dissociation constant which varies from 1.6 μM at pH 4.0 to 28 μM at pH 4.8. Azide has a much lower affinity than fluoride. The alkaline transition occurs with an apparent pKa value of 9.2. Rate constants were recorded for cyanide binding, the alkaline transition, compound I formation, and for the reactions of compound II with a series of substrates. Similarities and differences to horseradish peroxidase are discussed.
Four serine proteases from the latex of Euphorbia tirucalli
Authors: Lynn K. R. , Clevette-Radford N. A.
Published: December 2009

Four proteolytic enzymes, euphorbains t1–t4, were isolated from the latex of the succulent Euphorbia tirucalli ("milk bush") and purified to homogeneity. The enzymes all have molecular weights of 74 000. Each enzyme has several different charged forms; t1, for example, has four with isoelectric points in the range of 5.0–5.5. The four proteases examined are of similar amino acid compositions but yield differing two-dimensional maps of tryptic digests. The euphorbain t's are not closely related in composition to their counterparts isolated from Euphorbia lathyris, Euphorbia pulcherrima, and Euphorbia cyparissias. The euphorbains from E. tirucalli were inhibited by phenylmethylsulphonyl fluoride and by histidine-specific reagents, and so are trypsin-like proteases.
Effect of phospholipids and detergents on transitions and equilibrium between the bleaching intermediates of rhodopsin
Authors: Motoyama Hiroaki , Hamanaka Toshiaki , Kawase Nobuyoka , Boucher François , Kitô Yuji
Published: December 2009

The thermal stability of bleaching intermediates and the metarhodopsins [Formula: see text] equilibrium have been examined for purified rhodopsin in cholic acid and a series of alkyl sucrose esters. The results are compared with rhodopsin contained in its natural phospholipid environment. It is found that detergents modify the transition temperatures of the photointermediates in a complex manner. The equilibrium between metarhodopsins I and II is also strongly dependent on the detergent species. It is shifted according to the micellar rigidity. This effect is reversible and allows direct observation of a back reaction from metarhodopsin II to I.
Citrate chelation as a potential mechanism against aluminum toxicity in cells: the role of calmodulin
Authors: Suhayda Charles G. , Haug Alfred
Published: December 2009

At a molar excess of [citrate]/[aluminum], this organic acid can protect calmodulin from aluminum binding if the metal is presented to the protein in stoichiometric micromolar quantities, as judged by fluorescence and circular dichroism spectroscopy. Similar citrate concentrations are also capable of fully restoring calmodulin's hydrophobic surface exposure to that of the native protein when calmodulin was initially damaged by aluminum binding. Fluoride anions are equally effective in restoring calmodulin's native structure as determined by fluorescence spectroscopy. Measurements of the kinetics of citrate-mediated aluminum removal also indicated that the metal ions are completely removed from calmodulin, consistent with results derived from atomic absorption experiments. On the other hand, results from circular dichroism studies indicated that citrate-mediated aluminum removal from calmodulin can only partially restore the α-helix content to that originally present in apocalmodulin or in calcium–calmodulin, dependent upon the absence or presence of calcium ions. The results that chelators like citrate can protect calmodulin from aluminum injury may provide a conceptual understanding of physiological observations regarding aluminum-tolerant plant species which are generally rich in certain organic acids.
Selective inhibitory effects of thyroid hormones on estrogen-induced protein synthesis in chick embryo liver
Authors: Elbrecht Alex , Lazier Catherine B.
Published: December 2009

We have investigated the effect of thyroid hormones on estrogen-induced responses in embryonic chick liver. Administration of thyroid hormones inhibits estrogen induction of vitellogenin, as well as of apoprotein-II of very low density lipoprotein (VLDL apo-II). A proportionate decrease in the concentration of hepatic salt-soluble nuclear estrogen receptor is also observed. In contrast, estrogen stimulation of apoprotein-B (VLDL apo-B) synthesis is relatively resistant to inhibition. The inhibitory effects of the thyroid hormones could be due to increased metabolism and clearance of estradiol-17β in their presence. The relative resistance of estrogen-induced VLDL apo-B synthesis to thyroid hormone inhibition can be explained by its greater sensitivity to low doses of estradiol. In addition, experiments with the antithyroid agent thiourea suggest that, in vivo, estrogen-induced responses could be balanced by the selective inhibitory effects of thyroid hormones.
Structural studies of the class II histocompatibility antigens of the ACI rat
Authors: Goldner-Sauvé Audrey J. , Fuks Abraham , Guttmann Ronald D.
Published: December 2009

The class II antigens of the ACI rat were studied using both conventional alloantisera and monoclonal antibodies. By sequential immunoprecipitation experiments and cell binding studies, alloantisera were shown to contain antibodies to both the RT1.B and the RT1.D gene products. Using one- and two-dimensional gel electrophoresis, the structures of these gene products were shown to be distinguishable. The importance of these differences for the immune response and antigen presentation is discussed.
Endo-type mode of action of (1 → 4)-β-D-glucan cellobiohydrolase from Sclerotium rolfsii
Authors: Sadana Jai C. , Patil Rajkumar V.
Published: December 2009

Sclerotium (1 → 4)-β-D-glucan cellobiohydrolase has two types of activities: an endo-type mode of action forming insoluble short fibres from native cotton and its endwise action of removing cellobiosyl units from the nonreducing chain ends of β-1,4-glucans.
Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP
Authors: LeBel Denis , Beattie Marlyne
Published: December 2009

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.
Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone
Authors: Dijk Willem van , Boers Willem , Sala Mieke , Lasthuis Anne-Marie , Mookerjea Sailen
Published: December 2009

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20–24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-α1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34–48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethanasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of α-amanitin in the culture media at 0 h. The inhibiting effect of α-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.
The distribution of the stalk cell differentiation inducing factor and other lipids during the differentiation of Dictyostelium discoideum
Authors: Neave Nancy , Kwong Linda , MacDonald James I. S. , Weeks Gerald
Published: December 2009

The production of the stalk cell differentiation inducing factor (DIF) is restricted to the differentiation phase of the Dictyostelium discoideum life cycle. By the migrating pseudoplasmodial stage, the majority of the accumulated DIF is localized outside the pseudoplasmodia and the remaining pseudoplasmodial DIF is distributed between the intracellular and intercellular compartments. These results indicate possible reasons for the failure to detect gradients of DIF along the migrating pseudoplasmodia. De novo synthesized sterol is also localized predominantly outside the pseudoplasmodia. In contrast the majority of the de novo synthesized polar lipid and steryl ester are within the pseudoplasmodium. Of the de novo synthesized intracellular components, the polar lipid is almost entirely membrane bound and, although most of the sterol and steryl ester are also membrane bound, significant amounts are cytosolic. Intracellular DIF is also detectable in both membrane and cytosolic fractions.
Stability of histone mRNAs is related to their location in polysomes
Authors: Bird R. Curtis , Jacobs Frederik A. , Sells Bruce H.
Published: December 2009

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.
An immunochemical study of Neurospora nucleases
Authors: Fraser Murray J. , Chow Terry Y.-K. , Cohen Helga , Koa Helena
Published: December 2009

Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate – DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease.
Hybrid gene synthesis: its application to the assembly of DNA sequences encoding the human parathyroid hormones and analogues
Authors: Sung Wing L. , Zahab Diana M. , Yao Fei-Long , Tam Cherk S.
Published: December 2009

Bypassing any intermediate steps of purification and gene assembly, several synthetic oligonucleotides constituting a DNA duplex with a small base-mismatching region were phosphorylated, annealed, and ligated directly into a linearized plasmid vector. After transformation in bacteria, the two plasmid strands individualy yielded two different plasmids bearing altered versions of the same gene. Via this approach, DNA coding sequences of the human parathyroid hormone and analogues were synthesized and cloned in Escherichia coli.
Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins
Authors: Stan-Lotter Helga , Bragg Philip D.
Published: December 2009

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.
Purification and properties of human placental NADPH–cytochrome P-450 reductase
Authors: Muto Norio , Tan Liat
Published: December 2009

Human placental NADPH–cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 μM 4-androstene-3, 17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35–60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2′, 5′-ADP–Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolyis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 ± 0.7 μmol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.
Taxol-induced assembly and characterization of microtubule proteins from developing brine shrimp (Artemia)
Authors: Rafiee Parvaneh , MacKinlay Sara Ann , MacRae Thomas H.
Published: December 2009

Incubation of Artemia cell-free extracts with taxol, followed by centrifugation through sucrose cushions, yielded pellets composed of short, morphologically normal microtubules which exhibited a tendency to fray at their ends. Immunological staining of protein blots with polyclonal or monoclonal antibodies revealed that the major pellet protein is tubulin and that bovine neural tubulin and Artemia tubulin are antigenically distinct. By several criteria, but prinicipally by their taxol-induced coassembly with tubulin, many of the nontubulin pellet proteins are microtubule-associated proteins (MAP). In spite of extensive morphogenesis, hatching, and the eventual resumption of mitosis during development, no new MAP appear, with reduction in the number of MAP after hatching the only observable change in these proteins. We have yet to demonstrate a function for Artemia MAP but have shown that the rate and extent of assembly of Artemia tubulin, which polymerizes readily in vitro in the absence of MAP, are stimulated by bovine MAP. Electrophoretic analysis revealed that the taxol-assembled microtubules were composed of several isotubulins, these being identical to the isoforms in biochemically purified Artemia tubulin. In addition, a new Artemia α-tubulin was observed, and it was shown that the isotubulin population does not change during the period of development examined. Maintenance of identical isotubulin populations in developing organisms for extended periods, which suggests that all tubulins are functional, in concert with the lack of change in tubulin during cell differentiation, runs counter to the proposal that chemically distinct isotubulins are required for assembly of functionally specific microtubules.
Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment
Authors: Gupta B. D. , Borys T. J. , Deshpande S. , Jones R. E. , Abrahamson E. W.
Published: December 2009

In the presence of exogeneous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition. GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5′-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.
Acid and alkaline ceramidases of rat tissues
Authors: Spence Matthew W. , Beed Stephen , Cook Harold W.
Published: December 2009

Ceramidase (N-acylsphingosine deacylase) was measured in rat tissue homogenates with N-[1-14C]oleoyl- and N-[9,10-3H]palmitoyl-sphingosine. Two activities were observed with pH optima of approximately 5 and 8. The activity at pH 8 exceeded that at pH 5 in all tissues and tissue areas examined, and both activities decreased in the following order: kidney > brain stem > cerebrum > cerebellum > liver > spleen > cardiac muscle > lung > psoas muscle. The distribution of activity is not as tissue specific as that of neutral Mg2+-stimulated sphingomyelinase which is enriched in cells of neural origin and thus suggests a less specialized role for the alkaline ceramidase.
Reaction of the anionic acetylation agent methyl acetyl phosphate with D-3-hydroxybutyrate dehydrogenase
Authors: Kluger Ronald , Tsui Wing-Cheong
Published: December 2009

Methyl acetyl phosphate is a competitive inhibitor of the reduction of acetoacetate by D-3-hydroxybutyrate dehydrogenase. The material also irreversibly inactivates the enzyme. The kinetics of the inactivation are consistent with methyl acetyl phosphate acetylating the conjugate base of a hydrogen bond donor. Protection offered by a substrate analogue (methyl acetonylphosphonate) in the presence of coenzyme implicates reaction at the cationic active site. Reversible protection by the amino group reagent 2,3-dimethylmaleic anhydride suggests that methyl acetyl phosphate reacts with an amino group. Sulfhydryl reagents and acetyl phosphate, a poorer acetylating agent, do not inactivate the enzyme. The pH dependence of the inactivation suggests that the acetylation occurs at a site that has a pKa of 8.2. The utility of methyl acetyl phosphate and other acyl phosphate monoesters in reacting with lysines adjacent to cationic sites of enzymes, hemoglobin, and histones is noted.
Nucleosomes are assembled into discrete size structures by histone H1 in vitro
Authors: Boulikas Teni
Published: December 2009

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller super-nucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supemucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.
Protein–protein and protein–DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation
Authors: Boulikas Teni
Published: December 2009

Nuclear matrices from calf thymus contained 30–50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein–protein and protein–DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide – sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homooligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homooligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.
Aggregation of smooth muscle membranes and its use in the preparation of plasma membrane enriched fraction from gastric fundus smooth muscle
Authors: Kwan Chiu-Yin
Published: December 2009

Microsomal membranes isolated from rat gastric fundus smooth muscle by differential centrifugation aggregate substantially in the presence of the divalent metal ion Mg2+ or Ca2+. The magnitude of cation-induced membrane aggregation is higher for Ca2+ than for Mg2+, but the ion concentration required for half-maximum membrane aggregation (K0.5 value) is similar for Mg2+ and Ca2+. Cation-induced membrane aggregation is suppressed by high ionic strength and low pH of the medium. Cation-induced membrane aggregation of mitochondrial membrane and plasma membrane enriched fractions differ in the rate of aggregate formation, metal ion concentration dependence, and pH dependence. Such different properties of membrane aggregation were used to prepare a plasma membrane enriched fraction by conventional differential centrifugation. Subfractionation of the heterogenous microsomal membranes by free-flow electrophoresis indicated that smooth muscle plasma membranes showed a higher electrophoretic mobility than the intracellular membranes. These results suggest that ionic interactions on the cell membrane surfaces differ from those on the intracellular membrane surfaces and that induction of membrane aggregation by Ca2+ or Mg2+ is a useful procedure for an effective and rapid preparation of plasma membrane enriched fraction from smooth muscle.
Regulation of prostatic genes: role of androgens and zinc in gene expression
Authors: Matusik R. J. , Kreis C. , McNicol P. , Sweetland R. , Mullin C. , Fleming W. H. , Dodd J. G.
Published: December 2009

Gene expression in the rat dorsolateral prostate gland has been studied using cloned cDNA probes to the most abundant expressed mRNAs. One cDNA clone (pM-40) corresponds to two closely homologous mRNAs of about 880 nucleotides which code for two proteins of 23 and 21 kilodaltons (kDa). At least the 23-kDa protein contains a signal peptide. Another clone (pRWB) corresponds to a 1550-nucleotide mRNA which codes for a 52-kDa protein which also contains a signal peptide. The steady-state levels of these specific mRNAs increase in the dorsolateral prostate with sexual maturation. In castrated mature male rats, the M-40 mRNAs are inducible either by androgens or zinc, while the RWB mRNA is only responsive to androgens. In situ cDNA–mRNA hybridization histochemistry has been used to study the localization of the M-40 and RWB gene transcripts. Both M-40 and RWB mRNAs are most abundant in the epithelium of the lateral tip of the dorsolateral prostate. Following castration, the RWB mRNA decreases, while the M-40 mRNAs continue to be expressed in isolated areas of the epithelium. These castration-resistant cells maintain normal morphology in the absence of androgens.
Cultured Nb rat lymphoma cells in endocrine and cancer research
Authors: Gout Peter W. , Noble Robert L. , Beer Charles T.
Published: December 2009

This review outlines the establishment, properties, and use of two lines of cultured Nb rat lymphoma cells. The cultured cells have retained important properties of the cancers of origin, such as dependency on prolactin for growth and a high sensitivity to antineoplastic Vinca alkaloids. The cultures have been useful for defining the hormonal dependency of the lymphomas in the animal and for studying the progression of the lymphomas from hormonal dependency to autonomy. A new, specific and highly sensitive in vitro bioassay for lactogenic hormones has been developed from one of the cultures. The use of the lymphoma cell cultures has revealed unsuspected pharmacological differences between the closely related, clinically useful antineoplastic Vinca alkaloids, vinblastine and vincristine. The lymphoma cell cultures are also useful tools for studying biochemical, cell cycle related events which follow the mitogenic stimulation of cells by a polypeptide growth factor.
Biosynthesis of polyglutamates of folates
Authors: Scrimgeour K. G.
Published: December 2009

Folylpolyglutamate synthetase (FPGS) catalyzes the synthesis of the poly-γ-glutamate forms of tetrahydrofolate and its coenzyme adducts, as well as of the folate-analogue drugs. This paper reviews current knowledge of the preparations of FPGS from mammalian sources (rat, hog, mouse, and beef liver). Kinetic constants for the substrates and activators of FPGS are compared. Tetrahydrofolate and 5-formyltetrahydrofolate are excellent substrates for the enzyme. The Km values for the antifolates and their 7-hydroxy metabolites are much higher than those for the tetrahydrofolates. Aminopterin has higher activity with FPGS than does methotrexate, which partially explains its greater toxicity. 5-Formyltetrahydrofolate, which is used as a rescue agent in high-dose methotrexate-rescue chemotherapy, is a better alternate substrate of FPGS than is methotrexate and therefore is a potent competitive inhibitor of the glutamylation of methotrexate. Thus, low concentrations of the rescue agent prevent formation of cytotoxic polyglutamates of methotrexate. The pathway of the reaction is the addition of a glutamate residue to the terminal γ-carboxyl of the pteridine substrate. That longer folylpolyglutamates are poorer substrates possibly is a result of this addition pathway. Pteroic acid activates FPGS by lowering the Km value of the pteridine substrate. It also greatly increases the activity of the synthetase at physiological pH values.
Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926
Authors: Oost Bernard A. van , Edgell Cora-Jean S. , Hay Colin W. , MacGillivray Ross T. A.
Published: December 2009

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250 000 relative mass (Mr) which was secreted into the medium as a 220 000 Mr protein. A cDNA library was constructed in λgt 11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.
The RP031 gene of Saccharomyces cerevisiae encodes the largest subunit of RNA polymerase III
Authors: Moyle Matthew , Hofmann Theo , Ingles C. James
Published: December 2009

The 5′ end of the RNA transcript of the Saccharomyces cerevisiae RNA polymerase related gene, RP031, was localized on the RP031 locus using Northern analyses and primer extension experiments. The amino terminal protein sequence was determined for the largest (relative mass ~ 160 000) subunit polypeptide of S. cerevisiae RNA polymerase III. The N-terminal amino acid sequence of this polypeptide was shown to be identical to that predicted for a putative RP031 polypeptide initiated at the first ATG codon located 208–216 base pairs 3′ to the major RP031 mRNA start sites. The RP031 locus thus encodes the largest subunit of S. cerevisiae RNA polymerase III.
Chemotaxis-associated properties of separated prestalk and prespore cells of Dictyostelium discoideum
Authors: Mee J. D. , Tortolo D. M. , Coukell M. B.
Published: December 2009

During development, prestalk and prespore cells of Dictyostelium discoideum become organized in multicellular structures. This physical association makes it difficult to characterize the two cell types biochemically and physiologically. In the present study, we have separated prestalk and prespore cells from 16-h slugs by the method of Tsang and Bradbury and have examined a number of chemotaxis-associated properties of these cells. When assayed on phosphate-buffered agar under both gradient and nongradient conditions, isolated prestalk cells responded chemotactically to cAMP and, unexpectedly, to folate and certain folate derivatives. In contrast, separated prespore cells failed to respond appreciably to any of these compounds. Neither prestalk nor prespore cells of strain HC91 exhibited a cAMP-induced increase in intracellular cGMP. However, a cGMP response was observed in both prestalk and prespore cells of strain NP368, a cGMP phosphodiesterase deficient mutant. Both cell types exhibited comparable cAMP-mediated light-scattering changes and possessed similar levels of surface cAMP- and folate-binding sites. On the other hand, prestalk cells had at least fourfold higher cAMP phosphodiesterase and folate deaminase activities than prespore cells, and a large fraction of both activities was on the cell surface. Therefore, the greater chemotactic response of prestalk cells to cAMP and folate on agar might be due, in part, to their increased capacity to generate a chemoattractant gradient. Results obtained in this study demonstrate that prestalk and prespore cells separated by this procedure can be used in certain physiological as well as biochemical experiments.
Further characterization of the posttranslational modifications of core histones in response to heat and arsenite stress in Drosophila
Authors: Desrosiers Richard , Tanguay Robert M.
Published: December 2009

The effects of a heat shock or arsenite treatment on the methylation and acetylation of core histones have been investigated in Drosophila cultured cells. The decrease in H3 methylation, which is observed during a heat shock, is not a demethylation process, but results from methylation arrest. Two-dimensional gel electrophoresis leaves no ambiguity concerning the identity of H2B as a methylated protein, since H2B and D2, a nuclear nonhistone protein, which comigrate on one-dimensional gels, are well separated on these gels. Two-dimensional gel electrophoresis in the presence of Triton X-100 resolves each of the core histones into multiple forms resulting from posttranslational modifications. There are apparently, however, no histone variants in cultured Drosophila cells. At 23 °C, the various forms of the core histones resolved on two-dimensional gels are methylated. Under heat-shock or arsenite treatment, the methylation of all forms of H3 is decreased, while that of the various forms of H2B increases. These stress conditions also induce a generalized diminution in the acetylation of all forms of core histones. In the course of a heat shock, the synthesis of H2B is increased and this newly synthesized histone remains unacetylated during the shock. These changes in the patterns of core histone methylation and acetylation may be correlated with the reorganization of gene activity brought about by the heat shock.
Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium
Authors: O'Connor-McCourt M. , Soley M. , Hayden L. J. , Hollenberg M. D.
Published: December 2009

We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 × 105 ± 0.3 × 105) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks. We conclude that the long-term culture of hepatocytes in serum-free medium yields an altered low molecular form of the EGF-URO receptor that is, nonetheless, functional. The study points to differential changes in receptors for peptide hormones that may occur in long-term hepatocyte cultures and illustrates the feasibility of using such cultures for metabolic studies of the actions of EGF-URO.
Cellular localization of Drosophila 83-kilodalton heat shock protein in normal, heat-shocked, and recovering cultured cells with a specific antibody
Authors: Carbajal M. E. , Duband J.-L. , Lettre F. , Valet J.-P. , Tanguay R. M.
Published: December 2009

To gain insight on the possible functions of heat shock proteins (hsp's) in Drosophila, we have purified the 83-kilodalton hsp (hsp 83) from cultured cells and studied its intracellular localization by immunofluorescence in normal, heat-shocked, and recovering cells. The specificity of the antibody was assessed by one- and two-dimensional gel immunoblotting and by partial proteolytic digestion. The anti-hsp 83 antibody does not show any significant cross-reactivity with hsp's of different avian or mammalian cell lines, but cross-reacts with hsp's of similar molecular masses in other dipteran insects. The partial proteolytic peptide maps of Drosophila hsp 83 differ from those of mouse hsp 89 and chicken hsp 84. Immunoblotting of Drosophila Kc cells heat shocked at different temperatures indicates a maximal expression of hsp 83 at 33 °C. By immunofluorescence, hsp 83 is shown to have a strictly cytoplasmic localization. In unstressed cells, it is distributed in the entire cytoplasm with a slight enrichment in the perinuclear region. After heat shock, it seems to concentrate at the cell periphery close to the plasma membrane and it gradually redistributes to the whole cytoplasm during cellular recovery at normal temperatures.
Investigation of the role of micellar phospholipid in the preferential uptake of cholesterol over sitosterol by dispersed rat jejunal villus cells
Authors: Child P. , Kuksis A.
Published: December 2009

The uptake of radioactive cholesterol and sitosterol by rat jejunal villus cells was examined using mixed micellar solutions containing sodium taurocholate, equimolar mixtures of the two sterols, and a variety of phospholipid types. The addition of phospholipid to the incubation solutions reduced the cellular absorption of both sterols and gave rise to uptake kinetics that were linear with time. In the presence of egg yolk phospholipid, uptake of the sterols by villus cells occurred with a modest preference for cholesterol over sitosterol. The ratio of accumulated cholesterol/sitosterol increased from 1.0 initially to 1.23 ± 0.04 (n = 18) after a 30-min incubation at 37 °C. The selectivity displayed in the villus cells increased significantly as egg phosphatidyléthanolamine was added to the egg phosphatidylcholine (PC) preparation in micellar solution. It was markedly decreased when dipalmitoyl PC or the primarily saturated egg yolk sphingomyelin were incorporated into the micelles. In every case examined, phospholipid was taken up by the cells concurrently with the sterols. The selectivity between cholesterol and sitosterol was maintained when the donor species were multilamellar vesicles composed of egg PC and the sterols, but not when the donor particles were albumin-stabilized sterol dispersions or taurocholate solutions in the absence of PC. The results show that the selective absorption of cholesterol over the plant sterol occurs only in the presence of unsaturated phospholipid. The phospholipid may act by influencing the permeability of the cellular membranes to the two sterols or the rate of sterol desorption from the phospholipid-containing micellar or liposomal carriers. The selective recognition of cholesterol may, therefore, be a general property of phospholipid-containing structures that is expressed at several stages of the absorptive process.
Characterization of lithium-induced enzyme release from human polymorphonuclear leukocytes
Authors: Hart David A. , Groenewoud Yolanda , Chamberland Suzanne
Published: December 2009

Lysozyme release from purified human polymorphonuclear leukocytes was found to be uniquely enhanced by 2.5–20 mM LiCl. This effect was dose dependent and was not detected when the media was supplemented with NaCl, KCl, MgCl2, or CaCl2. The purified isotopes of Li+, 6Li, and 7Li were equally effective in enhancing lysozyme release from the cells at 10 and 20 mM, but 6Li was more effective than 7Li at 5 mM. The enhancement of enzyme release in the presence of Li+ was comparable to the enhancement observed in the presence of N-formylmethionylleucylphenylalanine (fMLP). Addition of LiCl plus fMLP did not result in lysozyme release in excess of each stimulant alone, except when the cells were incubated with 20 mM6Li + 10−5 M fMLP. In addition, enzyme release induced by these two agents could be further enhanced to the same degree by addition of cytochasin D to the incubation mixtures. While similarities between enzyme release induced by LiCl and fMLP were detected, optimal stimulation of enzyme release by Li+ was much more sensitive to inhibition by pertussis toxin than was maximal fMLP stimulation. Therefore, the intracellular events altered by Li+ and the peptide may share some metabolic steps, but they differ in their sensitivity to alterations in cAMP metabolism.