bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective loss of uncoupling protein from mitochondria of surgically denervated brown adipose tissue of cold-acclimated mice
Authors: Desautels M. , Dulos R. A. , Mozaffari B.
Published: December 2009

The effects of unilateral surgical denervation on brown adipose tissue (BAT) composition were evaluated to assess the importance of the sympathetic innervation in the maintenance of a high concentration of the uncoupling protein thermogenin in cold-acclimated (CA) mice and to assess whether suppression of neural activity could account for BAT atrophy observed during fasting or when CA mice are returned to a thermoneutral environment (33 °C). Denervation-induced BAT atrophy was characterized by protein and thermogenin losses in absence of changes in the tissue cellularity (DNA content). There was a marked reduction in the concentration of thermogenin in mitochondria isolated from denervated BAT, but the concentration of the adenine nucleotide translocator was unchanged. Fasting or exposure of CA mice to 33 °C induced a rapid and extensive loss of tissue protein from both innervated and denervated BAT. In CA mice exposed to 33 °C, there was also reduction in tissue cellularity and loss of thermogenin from BAT mitochondria. Since surgical denervation suppressed BAT hyperplasia and the increase in the mitochondrial concentration of thermogenin observed during cold exposure, these results indicate that an intact innervation is required for both synthesis and maintenance of a high mitochondrial content of thermogenin in CA mice. In addition, the lesser changes in tissue composition caused by denervation compared with those caused by fasting or exposure of CA mice to 33 °C question the importance of the suppression of neural activity as the exclusive cause of rapid BAT atrophy in mice.
Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue
Authors: Guertin Denise , Grisé-Miron Lucie , Riendeau Denis
Published: December 2009

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogenous bands corresponding to relative masses (Mrs) of 42 000–51 000 under nonreducing conditions or as a single polypeptide of Mr 51 000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.
The measurement of the production of during erythroid differentiation of the Friend erythroleukemia cell
Authors: Schmedt E. , Kleiman L.
Published: December 2009

The production of [Formula: see text] during Friend cell erythroid differentiation has been studied. In vitro measurements of total nuclear RNA synthesis in nuclei isolated from Friend cells at different stages of differentiation show the total RNA synthesis increases 1.5-fold at day 1 of induction and then decreases through days 2 and 3 to approximately 75% of its rate of synthesis in the nuclei of uninduced cells. The synthesis of RNA polymerase III transcripts undergoes a similar fluctuation through day 2 of induction, but increases again at day 3. The specific synthesis of [Formula: see text] was measured by hybridization of labelled nuclear RNA to a [Formula: see text] gene probe. During erythroid differentiation the percentage of nuclear RNA represented by [Formula: see text] remains constant (0.065%), so that the absolute synthesis of [Formula: see text] fluctuates during differentiation, in parallel with the fluctuations in the synthesis of total nuclear RNA. The relative synthesis of [Formula: see text]in vivo was studied by labelling cells with 35Pi, isolating the resulting radioactive tRNA – 5S RNA population, and hybridizing this population to a [Formula: see text] gene probe. The ratio of [Formula: see text] in newly synthesized cytoplasmic RNA remains similar throughout differentiation (averaging 0.0171), implying that the fluctuations observed in the nuclear synthesis of [Formula: see text] during differentiation probably also occur for the nuclear synthesis of most tRNA and 5S RNA species. Attempts were made to measure the relative steady-state concentration of [Formula: see text] using both aminoacylation and in vitro end labelling of tRNA followed by hybridization to a [Formula: see text] gene probe. These two methods gave different results and we discuss the possible pitfalls of using enzymatic methods for quantitating tRNA concentrations in the cell.
31P nuclear magnetic resonance study of trout spermatozoa at rest, after motility, and during short-term storage
Authors: Robitaille Pierre-Marie L. , Mumford Karen G. , Brown George G.
Published: December 2009

31P nuclear magnetic resonance was utilized to study respiration in spermatozoa isolated from the rainbow trout. The spectrum obtained under aerobic conditions revealed the presence of phosphomonoesters, intracellular inorganic phosphate, phosphodiesters, phosphocreatine, and free nucleotide triphosphates. The anaerobic spectrum was characterized by an increase in the inorganic phosphate resonance and by the disappearance of the phosphagen and free nucleotide triphosphate peaks. Upon reaeration, the aerobic spectrum was once again observed. Initiation of motility using fish Ringer or freshwater resulted in a decrease in phosphocreatine and nucleotide di- and tri-phosphate levels along with an increase in inorganic phosphate. The presence or absence of a phosphagen peak seemed to be correlated with motility. Preliminary studies on short-term storage of spermatozoa were also performed. These findings are analyzed in terms of the maintenance of motility in the spermatozoa of fish and some invertebrates.
Vertebrate liver cytokeratins: a comparative study
Authors: Pankov R. G. , Uschewa A.A. , Tasheva B. T. , Markov G. G.
Published: December 2009

The structure and composition of intermediate filaments isolated from liver of representatives of different vertebrate classes have been studied by electron microscopy and biochemical and immunochemical methods. It has been shown that the methodological approach for isolation of rat liver intermediate filaments can be efficiently applied to all other classes of vertebrates. The intermediate filaments studied have the same electron microscopic morphology and are species undistinguishable. The molecular weight of intermediate filament proteins varies from 40 000 to 60 000 and their isoelectric point varies from 5.0 to 6.45. Immunological investigations show that in all animals studied the intermediate filaments are built up of cytokeratins belonging to both types of keratins: type I and type II. Only one protein of the type II cytokeratins is present in all vertebrate classes, whereas in lower vertebrates two or even three type I cytokeratins contribute to the structure of liver intermediate filaments. The biochemical and immunochemical results are discussed with regard to the evolution of liver cytokeratins.
Epidermal growth factor (EGF) affects sulphydryl and disulphide levels in cultured mouse skin : possible relationship between effects of EGF and of the tabby gene on thiols
Authors: Robertson Nancy Weeks , Blecher Stan R.
Published: December 2009

Studies from our laboratory have previously shown that the syndrome produced in the mouse by the X-linked gene tabby (Ta) has many features in common with human X-linked hypohidrotic ectodermal dysplasia. We have also demonstrated that tabby has abnormally elevated epidermal sulphydryl (SH) : disulphide (SS) ratios, in common with an autosomal form of ectodermal dysplasia. The organs and tissues affected in many of the traits of these syndromes are targets of epidermal growth factor (EGF) and we have shown that the EGF-producing cells are deficient in tabby. In the present study we examined whether EGF affects SH and SS levels in normal mouse skin in tissue culture, and we report here that it does. EGF at a concentration of 25 ng/mL tissue culture medium lowers SH levels as compared with controls (0 ng/mL) in the epidermal layers examined (stratum malpighii of the tail and stratum malpighii and stratum corneum of flank skin). In general, other concentrations of EGF increase epidermal SH levels, although very high doses also reduce them. EGF at 25 ng/mL also lowers total SH + SS concentrations in the epidermal layers. Fetuses hemizygous for the Ta gene appear to have higher total SH + SS epidermal concentrations than their wild-type control littermates. These data, taken together with some of our previous findings, suggest the possibility that a relationship may exist between Ta, EGF, and thiol concentrations. Further study is required to elucidate this relationship.
Cell cycle controls and the role of nerves and the regenerate epithelium in urodele forelimb regeneration: possible modifications of basic concepts
Authors: Tassava Roy A. , Goldhamer David J. , Tomlinson Bruce L.
Published: December 2009

Data from pulse and continuous labeling with [3H]thymidine and from studies with monoclonal antibody WE3 have led to the modification of existing models and established concepts pertinent to understanding limb regeneration. Not all cells of the adult newt blastema are randomly distributed and actively progressing through the cell cycle. Instead, many cells are in a position that we have designated transient quiescence (TQ) and are not actively cycling. We postulate that cells regularly leave the TQ population and enter the actively cycling population and vice versa. The size of the TQ population may be at least partly determined by the quantity of limb innervation. Larval Ambystoma may have only a small or nonexisting TQ, thus accounting for their rapid rate of regeneration. Examination of reactivity of monoclonal antibody WE3 suggests that the early wound epithelium, which is derived from skin epidermis, is later replaced by cells from skin glands concomitant with blastema formation. WE3 provides a useful tool to further investigate the regenerate epithelium.
Characteristics of membrane transport losses during reticulocyte maturation
Authors: Blostein Rhoda , Grafova Eva
Published: December 2009

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+–glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+–glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.
Expression and base sequence of the citrate synthase gene of Acinetobacter anitratum
Authors: Donald Lynda J. , Duckworth Harry W.
Published: December 2009

The sequence of 1895 base pairs of Acinetobacter anitratum genomic DNA, containing the structural gene for the allosteric citrate synthase of that Gram-negative bacterium, is presented. The sequence contains an open reading frame of 424 codons, the 5′ end of which is the same as the N-terminal sequence of A. anitratum citrate synthase, less the initiator methionine. The inferred amino acid sequence of the enzyme is about 70% identical with that of citrate synthase from Escherichia coli, which like the A. anitratum enzyme is sensitive to allosteric inhibition by NADH. There is also a more distant homology with the nonallosteric citrate synthases of pig heart and yeast. The gene contains sequences that strongly resemble those found in E. coli promoters, an E. coli type of ribosomal binding site, and a hyphenated dyad sequence at the 3′ end of the gene which resembles the rho-independent terminators found in some E. coli genes. The plasmid clone containing the A. anitratum citrate synthase gene pLJD1, originally isolated because it hybridized with the cloned E. coli citrate synthase gene under conditions of reduced stringency, produces large amounts of A. anitratum citrate synthase in an E. coli host which lacks citrate synthase. This work completes proof of the hypothesis that the three major kinds of citrate synthases are formed of similar subunits, although their functional properties are different.
The presence of 1-radyl-glycerophosphoethanolamine acyltransferase activity in guinea pig heart mitochondria
Authors: Arthur Gilbert , Zaborniak Cheryl L. , Choy Patrick C.
Published: December 2009

Ethanolamine glycerophospholipids of mammalian heart mitochondria have a high content of arachidonic acid. Since the presence of acyltransferases that acylate 1-radyl glycerophosphoethanolamine had not been reported in the organelle, it was not known whether this high arachidonate content could be attained by the deacylation–reacylation pathway. In this study we have detected the presence of acyl-CoA:1-acyl-glycerophosphoethanolamine acyltransferase and acyl-CoA:1-alkenyl-glycerophosphoethanolamine acyltransferase activities in the guinea pig heart mitochondria. Both acyltransferases were active with palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, and arachidonoyl-CoAs, but the highest activities were obtained with arachidonoyl-CoA. The acyl-CoA specificities of the enzyme(s) did not reflect the fatty acid composition of the ethanolamine glycerophospholipids. The utilization of arachidonoyl-CoA by these acyltransferases in the guinea pig heart mitochondria suggests that these enzymes may play a significant role in contributing to the high arachidonate content of the ethanolamine glycerophospholipids. However, mechanisms beyond the acyl specificity of the reacylation reactions are also involved in the maintenance of the overall acyl composition of the ethanolamine glycerophospholipid in the cardiac mitochondria.
Effet du vieillissement sur la défense rapide contre la polyspermie chez l'ovule d'oursin
Authors: Sullivan Robert , Couillard Pierre
Published: December 2009

Aging of unfertilized ova of Echinoids is marked by a lowered fertilizability and an increased susceptibility to polyspermy. Since the fast block to polyspermy is electrically mediated in these species, we have studied the electrophysiological events triggered by fertilization in aging ova of Strongylocentrotus purpuratus. Aging of unfertilized ova is accompanied by a decrease in the resting potential. This could be explained by a decreased activity of membrane ionic pumps and could account for the gradual unfertilizability of aged eggs. Similarly, the depolarization plateau, which follows fertilization in freshly shed eggs, is abolished in eggs older than 80 min. Such aged eggs become vulnerable to polyspermy until the definitive, complete block is established by the cortical reaction. Finally, irrespective of age, fertilized ova repolarize at the same average membrane potential of −72 mV. This could reflect the general metabolic activation that follows fertilization.
Externalization of membrane-bound activities during sheep reticulocyte maturation is temperature and ATP dependent
Authors: Orr Linda , Adam M. , Johnstone R. M.
Published: December 2009

During the maturation of sheep reticulocytes in vitro, there is release of material that can be pelleted from the cell-free incubation medium by centrifugation at 100 000 × g. This pellet contains activities that are derived from both the plasma membrane and lysosomes. No evidence was obtained for the presence of mitochondrial activities or cytosolic enzyme activities. The release of these activities is ATP and temperature dependent, since reduction of either results in a greater retention of the activities by the cells and a lesser amount in the 100 000 × g pellet. The pelleted material is vesicular in nature, and the production and (or) release of the material are reduced upon ATP depletion or lowering of the temperature. It is concluded that the externalization of specific membrane components is a normal metabolic process that occurs during reticulocyte maturation and represents a means by which reticulocytes shed specific types of membrane-associated functions that are known to decrease during reticulocytes maturation.
Changes in structure and protein composition of bovine lymphocyte nuclear matrix during concanavalin-A-induced mitogenesis
Authors: Bladon Trevor , Brasch Klaus , Brown David L. , Setterfield George
Published: December 2009

A major component of nuclear change in concanavalin-A-stimulated bovine lymphocytes is a severalfold increase in interchromatinic volume, which coincides with nuclear swelling and extensive structural remodelling. Large-scale ultrastructural changes in isolated nuclei and nuclear matrices (NM) reflect those occurring within nuclei in situ during mitogenesis. While nonchromatinic nuclear material embedded within nuclease- and salt-extracted whole cells closely resembled in situ interchromatinic matrices, large NM isolated in solution shrank after chromatin was extracted. Numerous perinuclear filaments persisted throughout NM isolation and cytoskeletal proteins were identified in two-dimensional (2-D) gels of such preparations. Taken together these data indicated that the lymphocyte cytoskeleton is likely continuous with the nuclear matrix and could play a role in maintaining nuclear organization. A wide range of lymphocyte NM proteins were resolved in 2-D gels. Significant changes in protein composition coincided with nuclear structural remodelling. Lamin B was prominent at each stage of nuclear development, whereas lamins A anc C were only found in stimulated lymphocyte matrices. Lymphoblast NM contained more large basic proteins. Progressively increasing polypeptide complexity of these NM arose by de novo protein synthesis and posttranslational modifications throughout concanavalin A stimulation. NM from stimulated lymphocytes also contained more ribonucleoproteins, possibly indicating the presence of significant amounts of transcriptional material.
Quantitative assays for uracil-DNA glycosylase of high sensitivity
Authors: Morgan A. Richard , Chlebek Janusz
Published: December 2009

We have developed a sensitive fluorometric assay using bisulfite deaminated (C → U), covalently-closed circular PM2 DNA as the substrate. We describe a reliable way to prepare this substrate without nicking the PM2 DNA. Methods, which depend on toluenization of the cells, are described for reproducibly and quantitatively assaying uracil-DNA glycosylase. The sensitivity is such that only 200 EL4 mouse thymoma cells or 30 000 Escherichia coli cells are needed for each point in a kinetic assay.
Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis
Authors: Otsuka K. , Pitaru S. , Overall C. M. , Aubin J. E. , Sodek J.
Published: December 2009

To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse–chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. Collagen constituted 24–26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3–8% RGF, 8–18% RPL) and type V (~1%) collagens. Procollagen processing in the culture medium of RPL cells was more rapid than for RGF cells, but was increased in multilayered cultures of both RPL and RGF. In multilayered cultures, collagen TCA fragments, indicative of tissue collagenase activity, were also identified. Active and latent tissue collagenases and a latent form of a novel collagenolytic enzyme (matrix metalloendoproteinase-V) that cleaves native TCA fragments were demonstrated in these cultures. Addition of either concanavalin A (10−6 M) or retinoic acid (10−5 M) to the culture medium stimulated the secretion of the latent collagenolytic enzymes. Collagenase inhibitor was also synthesized by both RGF and RPL cells. SPARC/osteonectin, a 40-kilodalton glycoprotein, represented 0.5–1.0% of the secreted radiolabelled proteins of both cell types.
Attenuated cold-induced increase in mRNA for uncoupling protein in brown adipose tissue of obese (ob/ob) mice
Authors: Reichling Susanna , Patel Hasmukh V. , Freeman Karl B. , Kates Anna-Lisa , Himms-Hagen Jean
Published: December 2009

The level of mRNA for uncoupling protein was measured in brown adipose tissue of young (8–10 weeks) and old (11 months) lean and ob/ob mice using a cDNA clone constructed previously. The level of poly(A)+ RNA was also measured using an oligo(dT)18 probe. Mice were kept at 28 °C or exposed to 14 °C for 12 h. The level of mRNA for uncoupling protein was normal in brown adipose tissue of younger obese mice but reduced in brown adipose tissue of old obese mice. The cold-induced absolute increase in uncoupling protein mRNA was smaller in obese mice, regardless of age. It is concluded that the known attenuation of the acute thermogenic response of brown adipose tissue of the ob/ob mouse to cold is accompanied by a similar attenuation of the initiation of the trophic response. It is likely, however, that these defects are secondary to the chronic reduction in sympathetic nervous system activity in brown adipose tissue of the ob/ob mouse, which results in a functional atrophy of the tissue.
The change of cadystin components in Cd-binding peptides from the fission yeast during their induction by cadmium
Authors: Hayashi Yukimasa , Nakagawa Chiaki W. , Uyakul Duangchan , Imai Kunio , Isobe Minoru , Goto Toshio
Published: December 2009

Previously we reported that, upon exposure to Cd, Cd-bindimg peptides (Cd-BP1 and Cd-BP2) were induced in the fission yeast and that these Cd-BPs were composed of Cd atoms and multiples of the unit peptide cadystin. In this paper, the changes of relative amounts of cadystin species in each Cd-BP were determined during their induction by Cd in the logarithmically growing yeast. The preferential syntheses of the smaller cadystin species, in the early stages of induction by Cd and of the larger cadystin species at the late stage of induction suggested the biosynthetic pathway of cadystins in the fission yeast. Binding affinities of Cd to glutathione and cadystins indicate that the larger peptides bind Cd more firmly than du the smaller peptides, coinciding with the preferential synthesis of the larger peptides with the higher concentration of Cd in the cell at the late stage of Cd induction.
Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart
Authors: Hatch Grant M. , Stevens Willem K. , Choy Patrick C.
Published: December 2009

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (≥0.1 mM), choline uptake was enhanced 20–38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.
Differentiation, cancer, and anticancer activity
Authors: Tzen Chin-Yuan , Estervig David N. , Minoo Parviz , Filipak Michiko , Maercklein Peter B. , Hoerl Bryan J. , Scott Robert E.
Published: December 2009

Carcinogenesis is a multistep process that results from the development of a variety of defects in the control of differentiation and proliferation. To investigate this concept further, 3T3 T mesenchymal stems cells were employed to establish that a distinct sequence of biological processes is involved in the control of differentiation and proliferation, and that these processes are integrally regulated. Specific defects in these regulatory processes were next established as being involved in carcinogenesis. These defects, however, were found not to be absolute; rather, they appear to involve changes in the stringency by which differentiation and proliferation are integrally regulated. Finally, it was established that when normal or transformed stem cells are induced to undergo nonterminal differentiation (which is one step in the integrated control of proliferation and differentiation), they can be made resistant to carcinogenesis or to revert to a nontransformed state. These data provide strong evidence that a critically important requirement for normal homeostasis is maintenance of intact cellular mechanisms to integrally regulate differentiation and proliferation.
The diversity of mitosis: the value of evolutionary experiments
Authors: Wise Dwayne
Published: December 2009

The diversity of genome distribution systems in protists, algae, fungi, and insects is reviewed. Ultrastructural and experimental details are cited that illustrate this diversity. It is argued that an orthodox mode of daughter genome distribution does not exist; as long as the necessary precision was maintained, different components of the primordial mitotic system were modified to perform the same functions. This premise is applied to homologous genome distribution systems, and examples of meiotic diversity are cited. It is suggested that these "evolutionary experiments" provide unique opportunities for dissection of functions common to all genome distribution systems.
Integration of signal-transduction processes
Authors: Mooibroek Marilyn J. , Wang Jerry H.
Published: December 2009

The adenylate cyclase – cAMP, phospholipase C – IP3 (inositol 1,4,5-triphosphate), and DAG (diacylglycerol) signal transduction systems are used to illustrate general principles underlying the process of information transfer during cell stimulation. Both systems consist of reaction cascades that convert the external signal to an intracellular messenger, translate the messenger to regulatory activities, and then modulate the activities of appropriate cellular proteins to result in specific cell responses. Almost all of these reactions are under second-messenger-dependent regulation, with many being regulated by multiple messengers. Such complex regulation provides ample opportunities for the fine-tuning of the signal cascades and for coordination between cascades during cell stimulation. Specific examples are used to illustrate how the cell uses different intrasystem and intersystem regulatory reactions to achieve specific responses.
Rabbit intestinal and colonic mucins: isolation, partial characterization, and measurement of secretion using an enzyme-linked immunoassay
Authors: Mantle M. , Thakore E.
Published: December 2009

Mucin was purified separately from the upper, mid, and distal small intestine and the proximal colon of the rabbit. The carbohydrate profiles of the three intestinal mucins were the same but differed from that of the colonic mucin, which contained less N-acetylgalactosamine but more sialic acid. All four mucins had similar polymeric structures composed of large, heterogeneous glycoprotein monomers and a smaller protein of Mr ~ 120 000, held together by disulphide bonds. The three intestinal mucins, however, were more resistant to dissociation by thiol reduction or degradation by proteolysis than colonic mucin. An antibody against a pool of the purified mucins was developed in guinea pigs and used to establish an enzyme-linked immunosorbent assay (ELISA). The antibody was highly specific for rabbit intestinal and colonic mucins, showing no cross-reactivity with nonmucin components of rabbit intestinal and colonic tissue, and very little or no reactivity with purified intestinal mucins from other species. The four purified rabbit mucins had the same affinity for the antibody and similar numbers of antigenic determinants. Antigenicity was apparently associated with the protein moeity of the mucins and was critically dependent on three-dimensional conformation, since proteolysis decreased the number of antigenic determinants while thiol reduction abolished antigen–antibody affinity. Using the ELISA, the tissue mucin content and the rate of mucin secretion were found to be significantly higher in the proximal colon that in the three regions of the intestine, which were the same.
The structures of the two lipopolysaccharide O-chains produced by Salmonella boecker
Authors: Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

Salmonella boecker, which belongs to group 0:6, 14(H) and shows the antigenic factors 6, 14, [1], and [25], defined by the Kauffmann–White system, produces two lipopolysaccharides differing from each other in the structures of their 0-polysaccharide moieties. By glycose composition, partial hydrolysis, nitrous acid deamination, methylation, optical rotation, and 1H and 13C nuclear magnetic resonance studies, the O-polysaccharides were demonstrated to be high-molecular-weight polymers (I and II) composed of either structurally related repeating tetrasaccharide or repeating pentasaccharide units having the structuresand[Formula: see text][Formula: see text]
Water of hydration in the intra- and extra-cellular environment of human erythrocytes
Authors: Cameron Ivan L. , Ord Virginia A. , Fullerton Gary D.
Published: December 2009

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocyte ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1ρ) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.
Redistribution of cholesterol and β-sitosterol between Intralipid and rat plasma lipoproteins and red blood cells in vivo
Authors: Kakis G. , Kuksis A. , Breckenridge W. C.
Published: December 2009

Male Wistar rats were injected intravenously with 2 mL of Intralipid containing 7.5 × 105 counts per minute (cpm) [14C]cholesterol and 7.5 × 105 cpm β-[3H]sitosterol. Blood was withdrawn immediately and at 5, 10, 20, 60,120, and 1440 min after injection from different animals. Plasma and red cells were separated and washed by conventional centrifugation, while lipoprotein density classes corresponding to chylomicrons, very low (VLDL), low (LDL), and high density lipoproteins (HDL) were isolated by ultracentrifugation. Total lipid and sterol compositions were determined by thin-layer chromatography in combination with gas–liquid chromatography, whereas radioactivity was measured by scintillation counting. The ratio of [14C]cholesterol/β-[3H]sitosterol rose from 1 to 3.65 in the plasma VLDL fraction, whereas that in the LDL and HDL fractions were equilibrated at about 2, following an initial transient increase in favour of cholesterol. The appearance and disappearance of the radioactivity from LDL and HDL fractions exhibited precursor–product relationship owing probably to the conversion of the Intralipid into an intermediate lipoprotein-X-like particle, which possesses a density similar to that of LDL. The radioactive cholesterol and β-sitosterol were incorporated into the red blood cell membranes at nearly similar initial rates, while at later times the incorporation of cholesterol was much preferred.
Tightly bound NADPH in Proteus mirabilis PR catalase
Authors: Jouve Hélène M. , Beaumont Frédéric , Léger Isabelle , Foray Jacques , Pelmont Jean
Published: December 2009

Previous work with catalase from Proteus mirabilis PR, a mutant with high resistance to H2O2, had shown that two different forms of this enzyme, named A and B, could be resolved by anion exchange chromatography. The present results showed that catalase B differed from A by the presence of bound NADPH. Direct evidence for the presence of this dinucleotide within the bacterial catalase was obtained using high performance liquid chromatography. The obtention of pure catalase B without any addition of external NADPH showed that this dinucleotide was bound tightly to the protein as to be carried along with the protein during purification. Catalase complex II was formed from either-enzyme A or B by the continuous supply of low amounts of hydrogen peroxide as in the case of bovine liver catalase. The reversal of complex II formation from catalase B was obtained using a NADPH-regenerating system (isocitrate plus isocitrate dehydrogenase) in the absence of any added dinucleotide, confirming that this cofactor was present in the enzyme molecule. The addition of dinucleotide was required when performing the same experiment with catalase A. This was the first observation of NADPH binding by a bacterial catalase, and the results agreed with the former theory where NADPH is considered as a rescuer of the enzyme from inactivation in the form of complex II.Key words: catalase, bovine liver catalase, NADPH, Proteus mirabilis.
Evidence of direct interaction between actin and membrane lipids
Authors: St-Onge Dominique , Gicquaud Claude
Published: December 2009

Actin is a protein component of the cystoskeleton and is involved in cell motility. It is believed generally that actin filaments are attached to the cell membrane through an interaction with membranous actin-binding proteins. By using an in vitro system composed of liposomes and actin, we have shown that actin may also interact directly with the phospholipids of the membrane. Actin deposited at the surface of the liposome is organized in two regular patterns: a paracrystalline sheet of parallel filaments in register, or a netlike organization. These interactions of actin with membrane lipids occur only in the presence of millimolar concentrations of Mg2+. These results suggest that the interaction of the cytoskeleton with the membrane involves, at least in part, a direct association of actin with phospholipids.Key words: actin, membrane, liposome.
Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions
Authors: MacDonald David , Dunford H. Brian
Published: December 2009

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.Key words: prostaglandin H synthase, cyclooxygenase activity, peroxidase activity, arachidonic acid, compounds I and II of PGH synthase.
Proteolytic degradation of HDL1 on the fat cell surface is nutritionally regulated
Authors: Zsigmond E. , Fong B. , Angel A.
Published: December 2009

The importance of plasma HDL apolipoprotein concentration as a predictor of atherosclerotic risk is well recognized, yet the processes of HDL modification and degradation in various cells are not clearly understood. We examined the characteristics of HDL1 apolipoprotein degradation and cellular uptake by rat adipocytes and determined the effects of fasting on these processes. Epididymal and perirenal adipocytes were isolated from male Wistar rats (310 ± 4 g) fed ad libidum and incubated with 5 μg of rat 125I-labeled HDL, (d: 1.07–1.10 g/mL) mL−1 for 2 h at 37 °C. Cellular uptake of HDL1 was calculated as the trichloroacetic acid precipitable radioactivity associated with adipocytes following incubation. Intracellular and medium degradation of HDL1 were determined as trichloroacetic acid soluble 125I counts associated with cells and measured in the postincubation medium, respectively. Fifty to sixty percent of cellular uptake and degradation of HDL1 was inhibited by the addition of 25-fold excess unlabeled HDL. HDL1 degradation measured in the medium was 10- to 12-fold greater than cellular uptake of HDL1 apolipoproteins. Intracellular degradation of HDL1 was negligible. The presence of EDTA in the incubation medium reduced HDL1 degradation measured in the medium, but enhanced HDL1 cellular uptake. Conditioned medium separated from cells after 2 h of incubation at 37 °C in the absence of HDL and subsequently incubated with 125I-labeled HDL1 for an additional 2 h at 37 °C, degraded less than 5% of HDL compared with degradation in the presence of cells. These results suggest that rat adipocytes degrade, or modify, HDL1 particles, possibly by interactions with cell surface proteases. Compared with the fed state, fasting rats for 24 h significantly decreased cell-dependent, surface degradation of HDL1 by perirenal adipocytes and, to a lesser extent, HDL1 degradation by epididymal cells. In contrast to HDL1 degradation, cellular uptake of HDL1 increased as a result of fasting, but this increment was not statistically significant. Therefore, the cellular uptake and surface degradation of HDL1 by adipocytes appear to be dissociated processes. The significant decrease in HDL1 degradation as a result of fasting indicates that this process, presumably mediated by a lipoprotein protease, is nutritionally regulated.Key words: HDL metabolism, lipoprotein protease, adipocyte HDL receptor, lipoprotein degradation.
Altered expression of leucocyte sialoglycoprotein in Wiskott-Aldrich syndrome is associated with a specific defect in O-glycosylation
Authors: Greer Wenda L. , Higgins Elizabeth , Sutherland D. Robert , Novogrodsky Abraham , Brockhausen Inka , Peacocke Monica , Rubin Laurence A. , Baker Michael , Dennis James W. , Siminovitch Katherine A.
Published: December 2009

The Wiskott-Aldrich syndrome (WAS) is an X-linked immune deficiency disorder characterized clinically by both lymphocyte and platelet dysfunction. Studies of WAS T lymphocytes have revealed deficient or defective cell surface expression of the highly O-glycosylated leucocyte sialoglycoprotein CD43. To further elucidate the basis for, and functional relevance of, CD43 modifications on WAS lymphocytes, we have studied lymphocytes from two WAS patients with regard to membrane glycoprotein profile and mitogen-induced proliferative responses. CD43 was found to be either absent or altered in size on peripheral blood lymphocytes and lectin-stimulated T cells from both patients. Compared with control cells, the WAS lymphocytes displayed reduced, but measurable proliferative responses to lectins and neuraminidase/galactose oxidase, and virtually no response to periodate, a mitogenic agent which targets sialic acid residues on membrane glycoproteins such as CD43. Analysis of activities of three glycosyltransferases involved in O-glycosylation revealed marked reduction in the level of activity of UDP-N-acetylglucosamine: Galβ1-3GalNAc-R β-1,6-N-acetylglucosamine (β-1,6-GlcNAc) transferase in one WAS patient and no detectable activity of this enzyme in a second. β-1,6-GlcNAc transferase activity has recently been shown to increase during T cell activation coincident with changes in the O-linked glycans on CD43. A selective reduction of this glycosyltransferase in WAS lymphocytes suggests that O-linked oligosaccharides may be important to the structure of membrane glycoproteins involved in lymphocyte activation.Key words: Wiskott-Aldrich syndrome, immune deficiency, O-glycosylation, glycosyltransferase, lymphocyte activation.
Microtubule organization during the cell cycle of cultured Vicia hajastana Grossh.
Authors: Simmonds D. H. , Conibear E. , Setterfield G.
Published: December 2009

Microtubule (MT) organization was examined at each stage of the cell cycle in cell suspension cultures of Vicia hajastana Grossh. Simultaneous staining of MTs by immunofluorescence and DNA by Hoechst 33258, and microfluorimetric quantitation of DNA in interphase allowed direct correlation of MT configuration with mitotic stage and with G1, S, and G2 of interphase. The results indicated that in the majority of the cells the cortical MTs were disorganized in early G1, but then organized rapidly into parallel transverse arrays, remained ordered throughout S, and lost order in G2, probably near the onset of mitosis. A wide range of cell sizes were found in S, indicating that entry into S was loosely controlled. Preprophase bands, present in 55–80% of cells in prophase were observed in both disorganized cell clumps and regular cell files, indicating they were not exclusively associated with organized patterns of growth and division. In many cells, short MTs or MT clusters were observed in the cell cortex during all stages of mitosis. These MT remnants may serve as nucleating centres for new cortical MTs, which appear in late telophase as a disordered network. Ordering of MT occurs later in G1, indicating that MT nucleation and organization are two different processes.Key words: interphase, microfluorimetry, microtubules, mitosis, plant cell culture.
Neurospora endo-exonuclease in heat-shocked mycelia: evidence for a novel heat shock induced function
Authors: Ramotar Dindial , Fraser Murray J.
Published: December 2009

Shifting rapidly growing mycelial cultures of Neurospora crassa from 30 to 45 °C for 2 h resulted in 75% of the active endo-exonuclease, an enzyme implicated previously in recombinational DNA repair, being released from nuclei and vacuoles into the cytosol, where it was inactivated by a heat shock induced inhibitor. Cycloheximide, at a level which inhibited [35S]methionine incorporation into the protein of unshocked cells by 95%, did not block the release of endo-exonuclease from the organelles during heat shock, but rather allowed a 6- to 15-fold accumulation of the nuclease activity in the cytosol. The heat shock also resulted in depletion of about 65% of nuclear, and over 35% of cytosolic, trypsin-activable endo-exonuclease (comprised of both endo-exonuclease–inhibitor complex and endo-exonuclease precursor?). Heat shock also caused the release of about 40% of the total trypsin-activable endo-exonuclease plus active enzyme from mitochondria. During a subsequent 1-h recovery at 30 °C, trypsin-activable endo-exonuclease, but not active enzyme, was restored in full to the nuclei. Some restoration of vacuolar endo-exonuclease was also observed. In the cytosol, neither the level of active enzyme nor the level of trypsin-activable endo-exonuclease was appreciably restored during this recovery period. The accumulation of the new inhibitor in the cytosol of heat-shocked mycelia was estimated to be 20-fold. Although cycloheximide prevented this accumulation, it is not clear whether the inhibitor itself is synthesized in response to heat shock or whether it is generated from a preexisting form by some other heat shock induced function. The high level of inhibitor that accumulated during heat shock persisted throughout the 1-h recovery period. The properties of this inducible inhibitor showed both similarities to and differences from a highly specific constitutive inhibitor of endo-exonuclease purified from unshocked mycelia.Key words: Neurospora crassa, endo-exonuclease, heat shock, inducible, inhibitor.