bluuebunny/crossref_metadata_2025 huggingface dataset:
The acquisition of thermal tolerance in larvae of Calpodes ethlius (Lepidoptera) and the in situ and in vitro synthesis of heat-shock proteins
Authors: Dean Rob. L. , Atkinson Burr G.
Published: December 2009

Larvae of Calpodes ethlius (Lepidoptera) reared at 22 °C die after 1 h at 45 °C, but they acquire the ability to tolerate 1 h at 45 °C if they are first exposed to 37 °C for 1 or 2 h. Incubation of intact larvae at 37 °C for 1 h induces the new and (or) enhanced synthesis of a similar family of polypeptides (relative masses (Mrs) ~ 22 000) in all tissues examined (silk gland, fat body, wing discs, central nervous system, and muscle). When these same tissues are cultured at 37 °C for 1 h, the new and (or) enhanced synthesis of at least 5 polypeptides (Mrs ~ 95 000, 81 000, 74 000, 26 000, and 22 000) is evident. The more dramatic response elicited from cultured tissues by heat shock results, presumably, from the added stress of dissection. The new and (or) enhanced synthesis of polypeptides with Mrs of ~22 000 by tissues of intact larvae, incubated at 37 °C for 1 h, may be related to the acquisition of thermal tolerance by similarly treated larvae. These results suggest that the induced synthesis of these polypeptides is an integral part of a ubiquitous molecular mechanism involved in the development of thermal tolerance.
The possible role of juvenile hormone esterase in the regulation of juvenile hormone titre in the female cockroach Diploptera punctata
Authors: Rotin Daniela , Tobe Stephen S.
Published: December 2009

Juvenile hormone (JH) degradation in vitro and in vivo was studied in the viviparous cockroach Diploptera punctata. In vitro studies with juvenile hormone esterase (JHE) and "general" or 1-naphthyl acetate esterase (NAcE) revealed that diethyl-p-nitrophenylphosphate (paraoxon) inhibited both JHE and NAcE activity, but the latter was more sensitive and was completely inhibited at 0.1 mM. NAcE activity was resistant to inhibition with Triton X-100, whereas JHE activity in haemolymph of adult females was inhibited 100% at Triton X-100 concentration of 0.25%. Eighty percent inhibition of JHE activity in vivo was observed following injection of 0.2 μL Triton X-100. In contrast to the previously observed dose-dependant increase in JHE activity, NAcE activity did not increase following treatment of allatectomized females with the JH analogue ethyl-2E,4E-3,7,11-trimethyl-2,4-dodecadienoate (hydroprene). Hydroprene was not catabolized in haemolymph of D. punctata in vitro. The half-life of C16 JH (JH III) in the haemolymph, in vivo, was 1.65 h for day 5 females and 2 h for day 6 females.
A novel form of DOPA decarboxylase produced in Drosophila cells in response to 20-hydroxyecdysone
Authors: Spencer Charlotte A. , Stevens Bryn , O'Connor John D. , Hodgetts Ross B.
Published: December 2009

Two cloned derivatives of the Kc cell line of Drosophila were shown to produce DOPA decarboxylase following administration of the steroid moulting hormone 20-hydroxyecdysone. In the continuous presence of the hormone at a concentration of 2 × 10−7 M, DOPA decarboxylase activity first appeared between 48 and 72 h. Because of this lag, the tissue culture system promises to serve as a useful model for those in vivo situations where increases in the hormone titre precede increases in DOPA decarboxylase activity. In clone 7C4, after maximal enzyme activity was achieved at 144 h, the enzyme activity per cell decreased as the cells resumed division following the hormone-induced division arrest. In clone 7E10, cell division never resumed in the presence of 20-hydroxyecdysone and DOPA decarboxylase activity per cell increased continuously from the time it first appeared. When line 7E10 was exposed to a 6-h pulse of the steroid, enzyme activity appeared about 18 h earlier than in the presence of continuous hormone and, further, the cells were released from division arrest. Enzyme activity per cell then declined from an early 96-h maximum. The enzyme produced by the cell lines was immunologically distinct from the enzyme produced in vivo and ion-exchange column chromatography resolved the enzyme from cells and intact organisms into two species. Although the cells clearly produce a novel form of enzyme, it nevertheless shares many properties with the in vivo enzyme, including its substrate specificity, cofactor requirement, and sensitivity to inhibitors. We are currently purifying DOPA decarboxylase from line 7E10 which should enable us to provide a molecular description of the novel form.
Thermodynamic profiles for alcohol dehydrogenase action in free solution
Authors: Laidler Keith J. , Banerjee D. K.
Published: December 2009

Stopped-flow equipment was used to study the kinetics of the reaction between nicotinamide adenine dinucleotide (NAD) and ethanol, catalyzed by yeast alcohol dehydrogenase. By measuring rates over a range of concentrations of NAD and ethanol and of temperatures, thermodynamic profiles were obtained for the reaction, which occurs by an ordered ternary complex mechanism with NAD adding first. There are significant negative entropies of activation and negative entropy changes for the additions of NAD and of ethanol; the breakdown of the ternary complex is, however, accompanied by a positive entropy of activation. The results are consistent with structural constraints associated with the binding of the substrates, these restraints being to some extent removed when the ternary complex undergoes reaction. The system follows a similar pattern to that found with three different varieties of lactate dehydrogenase.
Kinetic equations and mechanisms for activation and inhibition in enzyme systems
Authors: Laidler Keith J.
Published: December 2009

The rates of enzyme reactions that are activated or inhibited by added modifiers can in some cases be expressed as a rational function of the first degree, v = (α0 + α1[Q])/(β0 + β1[Q] where [Q] is the concentration of the modifier and α0, α1, β0, and β1 are functions of rate constants and sometimes of the enzyme and substrate concentrations; the behaviour is then said to be linear. Three simple mechanisms that give rise to linear kinetics are examined, and the conditions under which there is activation or inhibition are determined. Sometimes there is a transition from activation to inhibition as the substrate concentration is varied. Definitions of competitive, uncompetitive, and noncompetitive activation are suggested, by analogy with the generally accepted definitions for inhibition. In second-degree activation or inhibition the rate can be expressed as the ratio of two quadratic polynomials with positive coefficients. Ten patterns are then possible for plots of v against [Q], and they may be classified with respect to (i) overall activation or inhibition, (ii) initial (at [Q] → 0) activation or inhibition, (iii) terminal (at [Q] → ∞) activation or inhibition, and (iv) whether there is an initial inflexion. The general case of an n:n rational function is also discussed.
Comparative inhibition of hepatic hydroxymethylbilane synthase by both hard and soft metal cations
Authors: Farmer D. J. , Hollebone B. R.
Published: December 2009

The in vitro inhibition of hydroxymethylbilane synthase (EC 4.3.1.8, uroporphyrinogen I synthetase) obtained from livers of Sprague–Dawley rats has been studied with a wide range of di- and tri-valent metal ions. After purification by cell lysis, heat treatment, and centrifugation, the stable, soluble enzyme yielded sigmoidal inhibition curves with increasing concentrations of each of the 16 test ions. Using the negative logarithm of metal concentration for 50% inhibition (the pM50 value), the metal ions could be classified according to their Klopman hardness values. Very soft ions including Hg2+, intermediate ions including Cr3+, and very hard ions including Al3+ all yielded large pM50 values indicating strong inhibition. In comparison to known metal-ion chemical behaviour, these three ions could indicate three different types of inhibitory binding sites at or near the active site: Hg2+ corresponding to sulfur in cysteine, Cr3+ corresponding to nitrogen in histidine, and Al3+ corresponding to oxygen in carboxyl groups. The presence of the first two sites is also indicated by the pH dependence of activity.
Retinitis pigmentosa and retinal degeneration in animals: a review
Authors: Matuk Yousef
Published: December 2009

Recent biochemical findings in the human disease, retinitis pigmentosa, and related retinal degenerative diseases in animals were reviewed and discussed. While the biochemical etiology of the human disease is not known, there are indications that retinal degeneration in the rd mouse and the Irish Setter dog are related to a deficiency in cGMP phosphodiesterase and the accumulation of cGMP in the photoreceptor outer segments. The biochemical basis of retinal degeneration in the Royal College of Surgeons (RCS) rat does not seem to be related to a defect in the metabolism of cGMP, but there are suggestions that a defect in retinoid metabolism may be involved. The possibility that the defect in RCS rats may involve receptors on the membranes of the cells of the retinal pigment epithelium or phagocytic markers on those of the rod outer segment disks was discussed.
Adipocyte insulin-binding species: the 40 Å Stoke's radius protein
Authors: Elliott C. , Goren H. Joseph
Published: December 2009

Several laboratories have demonstrated the presence of large (70 Å) (1 Å = 0.1 nm) and small (40 Å) insulin receptors. This report provides evidence that the 40 Å insulin receptor migrates on dodecyl sulfate – acrylamide gel electrophoresis as a 90 000 dalton protein and that this protein is a single polypeptide chain. 125I-labeled insulin was bound to plasma membranes from isolated rat adipocytes. Following removal of unbound 125I-labeled insulin, the mixture was exposed to disuccinimidyl suberate. Proteins tagged with 125I-labeled insulin were separated by dodecyl sulfate gel electrophoresis or Sepharose 6B chromatography. Autoradiography of the gels demonstrated several large (relative mass (Mr) > 300 000) and one small (Mr ~ 90 000) labeled protein in nonreduced membrane proteins. Dithiothreitol reduction decreased the large insulin-binding species to its known subunits, but the 90 000 dalton protein did not decrease in size. Triton X-100 solubilized plasma membranes were separated by Sepharose 6B chromatography. One labeled protein, with Kav = 0.57 elution position, on dodecyl sulfate gel electrophoresis migrated as a 90 000 dalton protein. Thus, rat adipocyte plasma membranes contain both an oligomeric insulin-binding species and a monomeric insulin-binding species. The relationship of the monomeric to the oligomeric insulin receptor is discussed.
Catabolism of 6-keto prostaglandin E1 by rat kidney homogenates
Authors: Pace-Asciak C. R. , Micallef S.
Published: December 2009

6-Keto prostaglandin E1 (PGE1) was catabolized into two major products by homogenates of rat kidney in the presence of NAD. Rat brain was mostly deficient in this catabolic activity. A total profile of products from these experiments could be obtained using the pentafluorobenzyl esters, O-methyloxime, and trimethylsilyl ether derivatives and analysis by capillary gas chromatography with electron capture detection. The same samples could be effectively analyzed by mass spectrometry using negative ion chemical ionization (NICI) detection. While 6-keto PGE1 showed spasmogenic activity on the rat stomach fundus as well as inhibition of the ADP-induced platelet aggregation, its catabolites lacked such activity. Analysis of rat whole blood by mass spectrometry using the NICI technique showed the presence of small amounts of 6-keto PGE1 and its 15-keto and 15-keto 13, 14-dihydro catabolites in two of eight blood samples.
On the role of lipid-bilayer elasticity for the lipid–protein interaction and the indirect protein–protein coupling
Authors: Sackmann E. , Kotulla R. , Heiszler Franz-Josef
Published: December 2009

The present paper deals with the curvature (or splay) elasticity of lipid bilayers and its possible consequences for the microscopic organization of membranes. Experimental studies of the microstructure of lipid–lipid and lipid–protein mixtures are reported and discussed in terms of this type of membrane elasticity. In particular, evidence is provided that the elastic distortion of lipid bilayers caused by the incorporation of proteins can lead to mechanisms of the indirect protein–protein interaction. Finally the question is discussed whether these mechanisms could also play a role for the formation of enzyme complexes in biological membranes.
Heat and nutritional shock-induced proteins of the fungus Achlya are different and under independent transcriptional control
Authors: LéJohn Herb B. , Braithwaite Cleantis E.
Published: December 2009

When the temperature of exponentially growing cells of the coenocytic fungus Achlya klebsiana strain 1969 was suddenly elevated from 24 to 37 °C (thermal stress), synthesis of at least 12 preexisting proteins (heat-shock proteins, HSPs) was vigorously induced while synthesis of most other cell proteins declined transiently. After 2–3 h of thermal stress, the cells recovered and resumed normal protein synthesis. If the cells were first starved of nutrients (nutritional stress) before the temperature was raised to 37 °C, the same 12 HSPs were induced, but synthesis of both heat-shock-inducible and nonheat-shock proteins declined to trace levels after 4 h of thermal stress. Molecular weights (MW) of the HSPs were approximately 96 000a, 96 000b, 85 000, 72 000, 70 000, 69 000a, 69 000b, 68 000, 60 000, 52 000, 26 000a, and 26 000b, and they had similar isoelectric points (5.8–6.2). Nutritionally stressed cells showed an induced synthesis of some 28 proteins (nutritional stress proteins, NSPs), when they were not heat shocked, and an induced synthesis of 20 NSPs when heat shocked. In the presence of glutamine, nutritionally stressed cells induced the synthesis of 15 NSPs when they were not heat shocked and 17 NSPs when they were heat shocked. The NSPs and HSPs were electrophoretically different proteins. Glutamine did not affect the induction pattern of the HSPs, but it arrested reproductive development of starving cells while altering the pattern of NSP synthesis. Since actinomycin D inhibited the induced synthesis of HSPs and some NSPs, they may be under transcriptional control. In vitro translation of poly(A)+ RNAs from heat-shocked cells showed that these cells were rich in HSP mRNAs and poor in NSP mRNAs. We speculate that NSPs, but not HSPs, may play a role in reproductive development and sporulation in this fungus.
Effect of cholesterol upon the conformation of band 3 and its transmembrane fragment
Authors: Klugerman Abbey H. , Gaarn Alex , Parkes Joel G.
Published: December 2009

Vesicles enriched in the anion transport protein band 3 and its transmembrane domain were prepared, and the cysteine residues were labelled with an extrinsic fluorescent probe, monobromobimane. Fluorescence energy transfer was demonstrated between intrinsic tryptophans and monobromobimane, and an average interchromophoric distance, Rav, was defined. Rav values and fluorescence emission wavelengths were used to monitor the conformation of band 3 and its transmembrane domain as a function of cholesterol content. The vesicles were treated with ovolecithin liposomes to reduce the cholesterol concentration, and there was an increase in Rav from 17.25 to 20.70 Å (1 Å = 0.1 nm) in intact band 3. A somewhat smaller increase in Rav for the transmembrane domain was observed (18.03–19.04). The tryptophan fluorescence emission wavelength was also blue shifted in the cholesterol-depleted preparations relative to the untreated samples. Combining the effects of cholesterol depletion upon Rav and the fluorescence emission maxima, it is suggested that the conformation of band 3 is influenced by the level of cholesterol in the bilayer.
Production of somatomedin-like activity by human adult tumor-derived, transformed, and normal cell cultures and by cultured rat hepatocytes: effects of culture conditions and of epidermal growth factor (urogastrone)
Authors: Atkison Paul R. , Hayden L. Joseph , Bala R. Marvin , Hollenberg Morley D.
Published: December 2009

We have measured the production of a basic-somatomedin-like activity (SLA) by a variety of human tumor-derived, transformed, and normal postnatal cell cultures; and we have compared the production of SLA by these cell types with the production of SLA by adult rat hepatocytes cultured in serum-free medium. Cells derived from a human epidermoid carcinoma (KB), a pancreatic carcinoma (Panc-1), a Simian virus 40 transformed adult human skin-derived cell line (SV40 fibroblasts), and a normal adult human skin-derived fibroblast line released SLA when cultured in a serum-free growth medium. No SLA was recovered from the culture medium of human choriocarcinoma-derived cells (BeWo) or of a human lymphoblastoid cell line (IM-9). The production of SLA by rat hepatocytes cultured in serum-free medium appeared to exceed the production of SLA by the other cell cultures. In cultures of KB cells, SV40 fibroblasts, and rat hepatocytes, the production of SLA depended on the frequency with which the growth medium was renewed; in general, the highest rates of SLA production were observed when the medium was renewed every 48–72 h. The presence of mouse epidermal growth factor (urogastrone) (EGF-URO) in the serum-free culture medium stimulated the production of SLA by KB cells and by rat hepatocytes, but did not increase SLA production by normal or by SV40-transfonned human skin-derived fibroblasts. We conclude that tumor-derived cells are capable of producing somatomedin-like activity and that the production of SLA by such cells can be subject to controls (nutrient availability, EGF-URO stimulation) that regulate SLA production, either by normal adult tissues, like liver, or by a variety of normal embryonic tissues.
Evaluation of possible mechanisms of phorbol ester stimulation of phosphatidylcholine synthesis in HeLa cells
Authors: Cook H. W. , Vance D. E.
Published: December 2009

Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12,13-dibutyrylphorbol acetate, markedly stimulate the synthesis of phosphatidylcholine (PC) and the activity of CTP:phosphocholine cytidylyltransferase (CT) in cultured HeLa cells. Two possible mechanisms whereby the phorbol esters stimulated PC biosynthesis were investigated. One consideration was that phorbol esters may induce the release of fatty acyl chains from endogenous complex lipids; increased fatty acids or fatty acyl-CoAs could cause the translocation of CT from cytosol to microsomes and thereby increase the activity of the rate-limiting enzyme in PC synthesis. In HeLa cells prelabeled with [3H]oleate or [3H]arachidonate, phorbol ester treatment increased the redistribution of arachidonate in phospholipids and neutral lipids and release of label to the medium, but there was little effect on the cellular fatty acid pools with either of the labeled fatty acids or of the phorbol esters. A second possibility was that protein kinase C (PKC), a receptor for phorbol esters, might be involved in activation of CT activity. TPA stimulated the phosphorylation of cytosolic proteins of HeLa cells more than twofold during a 10- or 60-min incubation with 32Pi. However, an approximate sixfold purified preparation of PKC from rat brain did not stimulate the activity of partially purified (12- to 15-fold) CT; a slight inhibition, dependent on ATP but independent of Ca2+ and diolein, was observed. Our results suggest that intracellular release of free fatty acids or direct phosphorylation of CT by PKC probably do not account for the observed levels of stimulation by phorbol esters. Other possible mechanisms are discussed.
Kinetic comparison of the 3β-hydroxysteroid dehydrogenase activity in human placenta, chorion laeve, and ovary
Authors: Gibb W. , Lavoie J. C. , Morin-Gonthier M.
Published: December 2009

Recent studies from our laboratory and others have shown that Km values for steroid substrates of the 3β-hydroxysteroid dehydrogenase in the human placenta were in the nanomolar range compared with micromolar values previously described. The purpose of the present study was to measure the kinetic parameters of the 3β -hydroxysteroid dehydrogenase in other human tissues, namely the ovary and chorion laeve, and to determine whether they were similar to those of the placental enzyme. In chorion laeve microsomes the 3β-hydroxysteroid dehydrogenase had Km values for dehydroepiandrosterone and pregnenolone similar to those found in placenta. Microsomes from human ovaries, on the other hand, had Km values for both substrates 10- to 20-fold higher. However, the ability of various steroids to inhibit the ovarian enzyme was similar to that previously described from the placenta and the chorion laeve.
In vitro concatemerization of bacteriophage T7 DNA: Role of DNA synthesis and gene 6 exonuclease
Authors: Lee Donald D. , Sadowski Paul D.
Published: December 2009

The replication of bacteriophage T7 DNA in vivo proceeds via the synthesis of complex concatemeric intermediates which are joined via the 160 base pair terminal redundancies at either end of the phage chromosome. To gain some insight into the mode of generation of these structures, we have examined the role of DNA synthesis in the formation of concatemeric bacteriophage T7 DNA in vitro. Incubation of mature T7 DNA with T7-infected cell extracts and a deoxynucleoside [32P]triphosphate resulted in the incorporation of significant radioactivity into the DNA. Highest levels of incorporation were at the termini of the DNA and decreased toward the middle of the molecule. Incorporation was dependent upon the presence of the activity of the gene 6 exonuclease and correlated with the generation of concatemeric DNA. A model explaining the role of exonucleolytic degradation and DNA synthesis in the generation of concatemeric DNA is presented.
Isolation, purification, and partial characterization of stable forms of monomeric bacteriorhodopsin in lauryl sucrose
Authors: Baribeau Johanne , Boucher François
Published: December 2009

The purple membrane of Halobacterium halobium was solubilized by treatment with lauryl sucrose and bacteriorhodopsin was found to be more stable in that detergent than in any other. While bacteriorhodopsin could not be completely delipidated by chromatography in lauryl sucrose alone, the purified bacteriorhodopsin monomers previously delipidated by treatment with Triton X-100 could be stabilized for a long time once Triton was exchanged for lauryl sucrose. In that state, an acid–base equilibrium exists between two spectral forms of the pigment absorbing, respectively, at 540 and 480 nm. The pKa of the equilibrium is 6.8. Consideration of the pKa value together with the amplitude of the spectral shift and the fact that both bacteriorhodopsin species, upon illumination, form different bathointermediates but share the same metaintermediate suggests that the acid–base equilibrium corresponds to the titration of the retinal Schiff base of the pigment. Since this equilibrium is only observable in completely delipidated bacteriorhodopsin, we propose that some specific lipid–protein interaction is responsible for the prevention of its occurrence in the membrane and for the maintenance of structural and functional integrity of bacteriorhodopsin.
1α, 25-Dihydroxyvitamin D3 enables regenerating liver cells to make functional ribonucleotide reductase subunits and replicate DNA in thyroparathyroidectomized rats
Authors: Youdale T. , Whitfield J. F. , Rixon R. H.
Published: December 2009

Between 16 and 20 h after partial (70%) hepatectomy (HPX) in normal rats, the remaining liver cells accumulate ribonucleotide reductase subunits, assemble these into active holoenzyme, and initiate DNA replication. These late prereplicative activities did not occur in most of the liver cells remaining after HPX in rats which had been thyroparathyroidectomized (TPTX) 72 h previously. However, one intraperitoneal injection of 400 or 600 ng 1α, 25-dihydroxyvitamin D3/100 g body weight at the time of HPX enabled the remaining liver cells in such TPTX rats to make functional ribonucleotide reductase subunits, assemble these subunits into active CDP-reducing holoenzymes, and replicate their DNA, though they started to do so 4 to 16 h later than in normal animals.
Development and characterization of monoclonal antibodies to pregnancy-specific β1-glycoprotein
Authors: Gidney M. A. J. , Bundle D. R. , Godard A. , Griffiths B. W.
Published: December 2009

Six monoclonal antibodies were developed to pregnancy-specific β1-glycoprotein (PSβ1G). Studies of ascitic fluid antibodies by a double-antibody radioimmunoassay (RIA) included an evaluation of titers, dose–response parameters, and mass action properties. Four of the antibodies demonstrated moderate to high titers ranging from 1/40 000 to > 1/120 000, as determined by the specific binding of 125I-labeled PSβ1G. In inhibition studies utilizing a standard containing known quantities of placental PSβ1G, two of the antibodies (AR#11 and B#2) were highly sensitive and only slightly lower in this regard than a high affinity polyclonal antiserum. The binding affinities of AR#11 and B#2 monoclonals were greater than 109 mol−1 which underline their importance as potential clinical reagents for the RIA of PSβ1G. The Scatchard plots, for several of the antibodies, were linear and in full agreement with a single order of binding sites predicted for specific monoclonal reagents. Immunodiffusion results provide preliminary evidence that at least three distinct determinants on PSβ1G are recognized by a number of the monoclonal antibodies. Further studies on the fine specificities of the antibodies by solid-phase RIA, as well as a detailed evaluation of their clinical applications, are in progress.
Germin. Compartmentation of the protein, its translatable mRNA, and its biosynthesis among roots, stems, and leaves of wheat seedlings
Authors: Grzelczak Zbyszko. F. , Rahman Sadequr , Kennedy Theresa D. , Lane Byron G.
Published: December 2009

(1) Onset of growth during germination of the isolated wheat embryo is allied with the emergence of a protein we have called germin. This study was undertaken to learn if germin is present and synthesized in the root, stem, and leaf during postgerminative growth of the wheat seedling.(2) Seedlings were grown from mature wheat grains on water-soaked filter paper and organs excised at various times were pulse labeled with [35S]methionine. Germin is synthesized in all organs at all times between 1.5 and 7 days.(3) During early seedling development (1.5 days), the fraction of [35S]methionine incorporated into germin, relative to other proteins, is much greater in the case of stem than other organs, either in vivo or when cell-free protein synthesis is directed by bulk RNA from different organs.(4) During late seedling development (4–7 days), when root growth is greater than stem growth, total isotope incorporation into germin is much greater in roots than stem.(5) Quasi-quantitative estimates of germin in pepsin-treated soluble fractions of homogenates were made by visual comparison of dye binding when gels were stained by Coomassie blue. The quantity of germin (ca. 1 mg/100 g) keeps pace with growth in spite of a 100-fold increase in fresh weight during transformation of the germinating embryo into a 7-day seedling.(6) During early seedling growth (1.5 days), germin is concentrated in the stem, but later (7 days), after extensive growth of the root but not the stem, the amount of germin in the root is about 1/2 and in the leaf is about 1/10 as great as in stem, on a fresh-weight basis, but more nearly equal on a per-organ basis owing to high mass proportions of the leaf and root relative to stem.(7) The dye-staining technique following pepsin treatment of soluble proteins has been used to detect germin in the stems of other cereal (rye, barley, oat) seedlings (3–8 days).
Reconstitution of lymphocyte 5′-nucleotidase in lipid bilayers: behaviour and interaction with concanavalin A
Authors: Sharom Frances J. , Lorimer Ian , Lamb Mary P.
Published: December 2009

Pure 5′-nucleotidase (EC 3.1.3.5) and a membrane glycoprotein fraction (partially purified 5′-nucleotidase) were isolated from pig lymphocyte plasma membrane by affinity chromatography techniques, using the cationic detergent dodecyltrimethylammonium bromide as a solubilizing agent. A detergent-dialysis technique was used to reconstitute both partially purified and pure enzyme into large unilamellar phospholipid vesicles, where it remains functional. 5′-Nucleotidase is relatively unstable in detergent solutions, but is highly stable once reconstituted into lipid vesicles. Arrhenius plots of the enzyme in bilayers of dimyristoyl phosphatidylcholine show a break point at 22–23 °C, with a different activation energy above and below the phospholipid gel-to-liquid crystalline phase transition. 5′-Nucleotidase in intact plasma membrane is inhibited more than 95% by concanavalin A in a positively cooperative fashion (Hill coefficient = 2.1), as is partially purified reconstituted enzyme. Purification of the enzyme before reconstitution results in less than 50% inhibition by concanavalin A and a complete loss of positive cooperativity (Hill coefficient < 1.0). The inhibition properties of the enzyme can be fully restored by co-reconstituting pure 5′-nucleotidase with the remaining lymphocyte glycoproteins.
Thermodynamic characterization of the partially denatured states of ribonuclease A in calcium chloride and lithium chloride
Authors: Ahmad Faizan
Published: December 2009

The denaturations of ribonuclease A by calcium chloride and lithium chloride were studied by circular dichroism measurements in the far-ultraviolet region. The temperature dependence of the equilibrium constant for the unfolding of the protein by calcium chloride and lithium chloride gave values of 46 and 52 kcal mol−1 (1 cal = 4.1868 J) for the enthalpy of denaturation at 25 °C and pH 7.0, respectively. Thermodynamic parameters for the denaturation by calcium chloride and lithium chloride are compared with those for the heat and guanidine hydrochloride denaturation. It has been observed that the thermodynamic quantity, be it free energy, entropy, or enthalpy, cannot be related quantitatively to the extent of unfolding measured by various conformational properties of the protein.
Thermotropic phase behavior of the platelet-activating factor: an infrared spectroscopic study
Authors: Mushayakarara Ernest C. , Mantsch Henry H.
Published: December 2009

Thermotropic phase characteristics of the platelet-activating factor 1-O-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine in excess water were studied by Fourier transform infrared spectroscopic techniques. Infrared bands characteristic of the interface and alkyl chain regions were monitored between −4 and 40 °C. Spectral parameters derived from these infrared bands demonstrate that in an aqueous environment this highly bioreactive lipid can assume three different structures: a micellar form at temperatures above 21 °C, a crystalline lamellar coagel form at temperatures below 12 °C, and a lamellar gel form that exists at temperatures between 12 and 21 °C. The three structures are separated by two phase transitions: a lower temperature (Tl) phase transition at 12 °C, that involves a structural rearrangement in the region of the interface, and an upper temperature (Tu) phase transition at 21 °C, that involves the "conformational melting" of the alkyl chains.
Influence of mercury (II), cadmium (II), methylmercury, and phenylmercury on the kinetic properties of rat liver glutathione peroxidase
Authors: Bem E. M. , Mailer K. , Elson C. M.
Published: December 2009

The effect of phenylmercury and methylmercury on rat liver glutathione peroxidase (GSH Px) is investigated and compared with that of Hg(II) and with some previously reported results for Cd(II). Analysis of the kinetics of metal binding to the enzyme gives apparent inhibition rate constants: kc = 9.7 mM−1 min−1 for all three mercury compounds and 75 mM−1 min−1 for CdCl2. Glutathione (0.2 mM) protects the enzyme from metal inhibition, decreasing the apparent inhibition rate constants (kc) by 3.6 times for mercury compounds and 4.4 times for CdCl2. K1 for the three mercury compounds is found to be 53 μM. It is unexpected that the same value of K1 exists for all three forms of mercury studied and that inhibition of the enzyme by the metals is a relatively slow process. For Cd(II) the value of K1 is 8.5 μM. It is suggested that inhibition of GSH·Px enzyme activity by cadmium, mercury, and organic mercury salts may not be due to simple complexation of the active site selenium moiety but may be due to a slower process, e.g., an alteration of the enzyme tertiary or quaternary structure.
Effects of some inhibitors of DNA synthesis and repair upon the cycle of serotonin N-acetyltransferase activity in cultured chick pineal glands
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: December 2009

When chick pineal glands were cultured in the dark with aphidicolin from midphotoperiod, the increase of serotonin N-acetyltranferase (NAT) activity was stimulated and the time of peak NAT activity was advanced. The peak level of NAT activity was also reached sooner on the 2nd day of culture. The increase of NAT activity was also stimulated in glands cultured under diurnal illumination, but the time of peak activity was not advanced. Effects with glands explanted into culture in the dark at other times were smaller and the time of peak NAT activity was not changed. Cytosine arabinoside and dideoxythymidine also stimulated the increase of NAT activity and advanced the time of peak activity with glands cultured in the dark from midphotoperiod. 3-Aminobenzamide markedly stimulated the increase of NAT activity both in the dark and under diurnal lighting when pineal glands were explanted into culture at mid- or late photoperiod. In contrast, with glands in culture from earlier in the photoperiod, aminobenzamide had no effect upon the increase of NAT activity up to the peak level found with control glands. Thereafter results were variable. Effects of cordycepin upon development of NAT activity were similar to those of 3-aminobenzamide but less marked. Incorporation of thymidine into acid-insoluble material in the dark was very markedly inhibited by aphidicolin, cytosine arabinoside, and dideoxythymidine, but only slightly by cordycepin. Aminobenzamide strongly inhibited incorporation by glands cultured from midphotoperiod, but had little effect with glands in culture from near the end of the photoperiod. We adopt the working hypothesis that excision repair of DNA may be a major component in the mechanism of the chick pineal clock.
Production of an antibody against rat liver nuclear T3 receptor
Authors: Faure Robert , Ruel Jean , Dussault Jean H.
Published: December 2009

Rabbits were immunized with rat liver nuclear L-triiodothyronine (T3) receptor purified by preparative sodium dodecyl sulfate – polyacrylamide (SDS–PAGE) gel electrophoresis using bromoacetyl[125I]T3 as an affinity label. SDS–PAGE confirmed the presence of two receptor forms of the apparent molecular weights 57 000 and 45 000. We describe here a specific antibody, raised against the 57 000 receptor type, which reacts with both receptor forms as assessed by electroimmunoblotting and immunoprecipitation in liquid medium.
Purification par séparation de phase et caractérisation du plasmalemme de l'épicotyle de pois
Authors: Rembur Jacques , Landré Pierre , Nougarède Arlette
Published: December 2009

The validity of phase partition to obtain a substantial proportion of vesicles of plasmalemma origin from the microsomal fraction of pea epicotyl has been demonstrated. In the fractions enriched with plasma membranes, N-naphthyl phtalamic acid binding and β-glucan synthetase II activity, showed a yield of about 60% and an enrichment of 2.3 and 2.2, respectively, in comparison with the microsomal fraction. When such plasmalemmic vesicles are permabilized by Triton X-100, an intense Mg2+-ATPase activity is obtained in presence of K+ at acid as well as alkaline pH. Inhibition of Mg2+-ATPase by vanadate in presence of K+ and its variations in relation to pH were shown. Dicyclohexylcarbodiimide and diethylstilbestrol inhibit 40–55% of this enzymatic activity, both at acid and neutral pH. The data show a slight contamination of the plasmalemmic fraction by endomembranes and suggest an asymmetry of the two sides of the plasmalemma.
Characterization of opioid peptides in the rat stomach
Authors: Nishimura Erica , Kwok Yin Nam , McIntosh Christopher H. S.
Published: December 2009

A combination of several chromatographic and assay systems was used to characterize the opioid peptides in rat stomach extracts. Partial purification of opioid material in acetic acid extracts of the corpus plus antrum regions of the rat stomach was carried out by gel filtration chromatography on Sephadex G-50, followed by adsorption onto Amberlite XAD-2 resin. A single peak in opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography, this peak was resolved into five distinct components, characterized by RRA and (or) radioimmunoassay, with retention times corresponding to methionine enkephalin (met-enk), leucine enkephalin, met-enk-arg6-gly7-leu8, met-enk-arg6-phe7, and dynorphin 1–13. Closer examination of the dynorphin component revealed the presence of dynorphins 1–17, 1–13, and 1–8. Trypsin digestion of the partially purified (Sephadex G-50 and Amberlite XAD-2 chromatographed) extract resulted in an overall increase in opioid activity, suggesting the presence of larger, possibly precursor forms.
Cytolocalization artifacts with immunofluorescent probes
Authors: Friedman Susan J. , Dewar Catharine L. , Skehan Philip
Published: December 2009

Formaldehyde fixation, nonionic detergent extraction, and ligand binding are commonly used in conjunction with immunofluorescence microscopy to visualize antigens and lectin-reactive molecules in cytoskeletal preparations. These procedures have the potential to produce serious artifacts in cytolocalization studies, as is shown in the present investigation of wheat-germ agglutinin (WGA) binding and localization in BeWo choriocarcinoma cells. Formaldehyde fixation of intact cells reduced the binding of 125I-labeled WGA by 30% and altered the pattern of staining with fluorescein isothyocyanate (FITC)–WGA. Except for perinuclear sites which were brightly stained, the binding of FITC–WGA to other cell surface regions was significantly decreased. Nonionic detergent extractions had two different effects on lectin binding activity depending on whether or not the cells had been pretreated with lectin. In lectin-pretreated cells, 50% of bound lectin was solubilized by detergent but all of the surface binding sites appeared to be retained in active form in the detergent-insoluble residue. Nuclear-cytoskeletal monolayers prepared from cells that were not lectin pretreated lost considerable binding activity, however. These results suggest that a number of erroneous conclusions can be drawn from studies on cytoskeletal associations with membrane components using immunofluorescence microscopy on fixed and detergent-extracted cells.
Site-directed protection of RNA during nuclease digestion
Authors: Beauchemin N. , Paquette J. , Cedergren Robert
Published: December 2009

We report the use of oligodeoxynucleotides to block the nucleolytic hydrolysis of single-stranded regions of RNA. Using complementary oligomers, the hydrolysis of the CCA terminus of methionine initiator tRNA could be prevented. This method can be useful in the production of specific single-stranded fragments of RNA, which are necessary in recombinant RNA technology.
Restriction of single-stranded M13 DNA using synthetic oligonucleotides: the structural requirement of restriction enzymes
Authors: Qi Guo-Rong , Wong Pierre , Cedergren Robert
Published: December 2009

A targeted ss (single stranded) DNA cleavage technique is reported which involves the use of synthetic oligomers complementary to the ss M13 DNA polylinker. BamHI, SmaI, and KpnI restriction enzymes were tested with a partial duplex DNA formed from ss M13 DNA and a nested series of fragments derived from a synthetic 21 -mer which were complementary to the polylinker region. These enzymes require up to two flanking nucleotides in addition to the hexameric recognition site for efficient cleavage. This technique could be useful for effecting unique cleavages of DNA with enzymes which generally give a large number of fragments and for strategies of ss DNA manipulation.
Rapid assay of poly(ADP-ribose) glycohydrolase
Authors: Ménard Luc , Poirier Guy G.
Published: December 2009

We have developed a rapid, highly reproducible assay to determine poly(ADP-ribose) glycohydrolase activity which measures directly the appearance of the reaction product. We also analysed the majority of different techniques which are used to determine poly(ADP-ribose) glycohydrolase activity and found that the apparent activity can vary extensively depending on the method used. Thin-layer chromatography using PEI-F–cellulose was the only method which evaluated directly the specific release of ADP-ribose; by comparison with this method, the other procedures gave an over- or under-estimation of 2- to 10-fold of the enzymatic activity. A rapid method of affinity chromatography has also been developed to synthesize and purify in high yield poly(ADP-ribose) (35% conversion of 1 mM NAD to poly (ADP-ribose)).
Identification of the phosphomonoesterases that hydrolyze lysopolyphosphoinositides in rat brain and liver
Authors: Palmer Frederick B. St. C.
Published: December 2009

The phosphatase activities responsible for the sequential dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate (lysoPtdIns(4,5)P2) to lysophosphatidylinositol that precedes reacylation in rat brain and liver microsomes were characterized. LysoPtdIns(4,5)P2 and the intermediate lysophosphatidylinositol 4-phosphate (lysoPtdIns4P) were hydrolyzed by two distinct phosphatase activities which were distinguishable by their substrate and cation requirements. The lysoPtdIns(4,5)P2 phosphatase activity was Mg2+ dependent and partially inhibited by Ca2+, excess Mg2+, and cationic detergent (cetyltrimethylammonium bromide). Activity was maximal at neutral (brain) or slightly alkaline (liver) pH when the Mg2+/lysoPtdIns(4,5)P2 molar ratio was 1.0 in the presence of bovine serum albumin (1 mg∙mL−1). LysoPtdIns4P phosphatase activity did not require divalent cations (not inhibited by EDTA). This activity was inhibited by Ca2+, Mg2+, and substrate concentrations above 0.2 mM. Maximum activity was observed over a broad pH range (6.0–8.5). Both activities were inhibited by lysophosphatidylinositol and lysophosphatidylcholine, but not other lysophospholipids. The lysopolyphosphoinositides are most likely hydrolyzed by the same phosphatases that act on the diacylpolyphosphoinositides, since PtdIns(4,5)P2 and PtdIns4P were also hydrolysed by Mg2+-dependent and cation-independent phosphatases, respectively. Activities with the diacylpolyphosphoinositides differed only in their requirement of detergents for maximum activity in vitro. Specific activities for the diacyl and "lyso" forms of each substrate were very similar when suitably optimized reaction mixtures were used. The subcellular distributions of the two phosphatase activities in both brain and liver were the same when acting on diacyl- or lyso-polyphosphoinositides, as was their response to inhibitors. Alkaline, acid, phosphoprotein, and inositol-1-phosphate phosphatases did not contribute substantially to the hydrolysis of either lysoPtdIns4P or lysoPtdIns(4,5)P2, since the activities were not significantly inhibited by cysteine, dithiothreitol, NaF, or LiCl. Lack of inhibition by 2,3-bisphosphoglycerate and absence of stimulation by cysteine or dithioerythritol, as well as a different subcellular distribution in liver, excluded inositol-1,4,5-trisphosphate and inositol-1,4-bisphosphate phosphatases as sources of the lysoPtdIns(4,5)P2 and lysoPtdIns4P phosphatase activities.
The limited photochemical activity of solid aggregated forms of bovine rhodopsin
Authors: Guérette Louise , Tessier Michel , Boucher François
Published: December 2009

The visual pigment rhodopsin has been purified and depleted of detergent. Under these conditions, the pigment strongly aggregates. When dried, a significant fraction of these aggregates appear insensitive to light. We have characterized them by means of absorption and photoacoustic spectroscopies and we find that their photochemical behavior is best explained by a limited activity that does not reach photointermediates beyond the lumirhodopsin step in the bleaching sequence of rhodopsin. We interpret this result as an indication of a significant conformational change of the protein during the transition from lumi- to meta-rhodopsin.
Structure and function of nonhistone phosphoproteins
Authors: Halikowski M. J. , Liew C. C.
Published: December 2009

Many nonhistone nuclear proteins have been shown to be phosphorylated and associated with a diverse range of cellular activities. The aim of this present review is to update the most recent developments in this field with respect to the traditional roles that these proteins have been postulated to play as enzymes, DNA-binding proteins, hormonal receptors, nucleosome associated proteins, and nucleolar associated proteins. In addition, evidence is presented suggesting that these proteins may also function as nuclear oncogene protein products, structural constituents of nuclear organization (e.g., lamina-nuclear matrix associated proteins), and RNA processing factors.
Effect of pepsin on partially purified pig gastric mucus and purified mucin
Authors: Lee Sum P. , Nicholls Jane F. , Roberton Anthony M. , Park Han Z.
Published: December 2009

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid – Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (<10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.