bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of serum lipoproteins in rabbits fed a natural ingredient diet or low-fat, cholesterol-free, semipurified diets containing casein or isolated soy protein
Authors: Hrabek-Smith Jana M. , Carroll Kenneth K.
Published: December 2009

In rabbits fed a cholesterol-free, semipurified diet containing isolated soy protein, the average total serum cholesterol level was similar to that of rabbits fed a natural ingredient (chow) diet. However, the cholesterol and protein levels in very low density (VLDL) and low density lipoproteins (LDL) tended to increase, while the levels in high density lipoproteins (HDL) were reduced to about half of those on the chow diet, with little change in the cholesterol to protein ratio. Substitution of casein for soy protein in the semipurified diet caused a four- to five-fold increase in total serum cholesterol and a doubling of lipoprotein protein, with an increase of 1.4- to 3.0-fold in the cholesterol to protein ratio of the different lipoprotein fractions. Analysis of the apoproteins (apo) of the plasma lipoproteins indicated that apo B, E, and C all tended to increase in the VLDL and LDL of rabbits fed the soy protein diet compared with those fed chow diet. The levels of each of the apoproteins were increased further by substituting casein for soy protein in the semipurified diet. In this case, apo E showed the greatest relative increase (2.7-fold) in VLDL, while apo B and E were increased to a similar extent (about 4-fold) in LDL. Apo C was approximately doubled in each of these fractions. The apo A content in HDL of rabbits fed the semipurified diets was about half that of rabbits fed chow diet. No marked changes were noted in the apo E or C content of HDL. Separation of isoforms of the soluble apoproteins showed variations between individual animals, but these variations seemed largely unrelated to diet. The results of these studies indicate that semipurified diets produce changes in the serum lipoprotein patterns of rabbits that are only partly due to the protein component of these diets.
Cell growth and substrate effects on characteristics of a lysosomal enzyme (cathepsin C) in Duchenne muscular dystrophy fibroblasts
Authors: Doughty Michael J. , Gruenstein Eric I.
Published: December 2009

Skin fibroblasts from normal males and males suffering from Duchenne muscular dystrophy were studied in culture over a 10-week period. The lysosomal enzyme cathepsin C (dipeptidyl aminopeptidase I; EC 3.4.14.1), defined by the chloride-dependent hydrolysis of dipeptide-β-naphthylamide (dipeptide-β-NA) substrates at pH 5.1, was significantly lower in Duchenne cell sonicates and cell lysosomal preparations. The apparent difference in activity tended to increase with in vitro cell culture age, with the Duchenne cells being found also to grow faster and yield a greater number of cells at confluence. An analysis of all 10 cell lines as a group indicated that cathepsin C activity was related to growth rate. In addition, while analyses of cell homogenization and fractionation showed that the yield of cathepsin C was not different in Duchenne lysosomal preparations, the enzyme showed significantly lower latent activity in the Duchenne lysosomes with Gly-Phe-NA used as substrate. However, despite significant differences in specific activity compared with normal lysosomal preparations, no latency difference was observed if three other substrates were used (Gly-Arg-, Pro-Arg-, and Pro-Phe-NAs). The expression of this enzyme can thus be differentially influenced by cell growth and its latency characteristics can be influenced by the substrate used in assays.
The influence of lysophosphatidic acid on platelet protein phosphorylation
Authors: Gerrard Jon. M. , Beattie Laura L. , McCrae Jan M. , Singhroy Sandra
Published: December 2009

Lysophosphatidic acid (LPA) is a lysophospholipid that is produced during thrombin stimulation of platelets, which can promote platelet aggregation. The mechanism of the effect of LPA was explored in normal platelets and in platelets from a patient with a storage pool deficiency (SPD). A comparison with other lysophospholipids showed that only LPA exerted significant effects to cause or potentiate platelet aggregation. Aspirin, an inhibitor of prostaglandin endoperoxide synthetase, had little effect on LPA-induced aggregation, but completely blocked LPA-induced serotonin secretion. LPA also promoted phosphorylation of myosin light chain (MLC), a 47 kilodalton (kDa) protein, and actin-binding protein. Aspirin significantly inhibited the phosphorylation of the 47-kDa and actin-binding proteins at 3–8 min after the addition of LPA, but had no effect on protein phosphorylation within the 1st min and had no significant effect on MLC phosphorylation. In SPD platelets, aspirin partially inhibited both aggregation and phosphorylation of the 47-kDa protein (< 30% inhibition) and MLC (< 40% inhibition) at time points of 1 min or less. The addition of ADP to SPD platelets enhanced the LPA response in platelets either pretreated or not pretreated with aspirin. Studies with SPD platelets indicate that thromboxane and secreted ADP contribute to, but are not necessary for, LPA-induced aggregation and phosphorylation. A23187 (a calcium ionophore) and LPA showed some selectivity to promote MLC as opposed to the 47-kDa protein phosphorylation, particularly at low concentrations of agonists and at earlier time points. The protein phosphorylation changes seen are consistent with a role for MLC phosphorylation in the granule centralization promoted with LPA.
RNA design by in vitro RNA recombination and synthesis
Authors: Cedergren Robert , Grosjean Henri
Published: December 2009

The techniques of in vitro RNA synthesis and recombination are presented. These include the site-specific cleavage of RNA, the manipulation of terminal phosphates, and the ligation of RNA fragments. Areas of promising future research include the establishment of RNA cloning vectors and the use of in vitro transcription of natural or designed RNA genes. The chemical synthesis approach now offers the possibility of making large amounts of biologically active length RNAs and of incorporating modified or reporter nucleotides into RNA sequences for physical studies. The new RNA techniques taken with DNA technology will permit a new approach towards understanding the complexity of RNA metabolism and the relationship of structure to function in RNAs.
Repression of rRNA synthesis during slug reconstruction in Dictyostelium discoideum
Authors: Uchiyama Saburo
Published: December 2009

When cells dissociated from "slugs" are allowed to reaggregate, they reconstruct slugs. During this process, the cells showed a marked decrease in the ratio of labeled RNA lacking poly(A) to labeled total RNA as compared with that of cells in normal slugs. Irrespective of the change in total labeled RNA, the ratio remained low even after 6 h of reconstruction. Sucrose density gradient analysis of RNA showed that the synthesis of high molecular weight rRNA (26S and 17S) was considerably repressed in reconstructed slugs as compared with normal slugs. Polyacrylamide gel electrophoresis of low molecular weight rRNA revealed that the synthesis of 5S rRNA, but not 4S tRNA, was repressed.
Limb regeneration in vertebrates: regulatory factors
Authors: Liversage Richard A.
Published: December 2009

Various regulatory factors are required in epimorphic regeneration of an adult newt limb. These factors (namely, amputational injury, the wound and apical epithelium, nerves (mitogenic agents), hormones (the hormonal milieu), bioelectric fields, probably the immune system, and possibly cyclic nucleotides and heretofore unknown regulators) act in concert and contribute to the developing microenvironment of the regenerate in support of normal regrowth and differentiation.
Production of multiple forms of glucoamylase in Aspergillus awamori
Authors: Bhella Resham S. , Altosaar Illimar
Published: December 2009

The biosynthesis of glucoamylases in Aspergillus awamori was studied by in vivo protein labelling and analysis of glucoamylase-specific mRNAs. Two types of glucoamylases with molecular weights of 100 000 and 82 000 were shown to be synthesized de novo. Deglycosylation of the 100 000 molecular weight glucoamylase type resulted in the formation of another glucoamylase form with molecular weight of about 94 000. De novo synthesis of two types of glucoamylases was further confirmed by the existence of two types of glucoamylase-specific mRNAs, as demonstrated by in vitro translation and Northern blot analysis studies.
Differentiation defective mutants of skeletal myoblasts altered in a gelatin-binding glycoprotein
Authors: Cates George A. , Nandan Devki , Brickenden Anne M. , Sanwal Bishnu D.
Published: December 2009

We have previously described a myoblast cell surface glycoprotein of the molecular mass 46 000 (gp46), which is associated with myoblast differentiation. In this report we demonstrate that gp46 binds specifically to gelatin-Sepharose and in this respect is similar to a glycoprotein of the molecular mass 47 000, which has earlier been described as a cell surface localized protein in mouse parietal endoderm cells and in chick embryo fibroblasts. To ascertain the relationship of gp46 to myoblast differentiation, wild-type L6 myoblasts, as well as two concanavalin A (ConA) resistant, differentiation-negative, myoblast mutants (D-1 and C-8), were examined for gp46 expression. In the mutant designated D-1, which has a defect in dolichol mannosyl transferase, both mannose incorporation into gp46 and ConA binding to gp46 was reduced compared with L6, without markedly affecting the gelatin adhesion properties of gp46. Western blotting with a monoclonal antibody against gp46 was used to show that the expression of gp46 was normal in D-1 but was reduced in mutant C-8 compared with L6. Reduction occurred both in the plasma membrane and endoplasmic reticulum fractions of C-8 compared with wild-type L6. In L6 myoblasts, the expression of gp46 remained constant during myoblast replication and fusion but decreased markedly postfusion. In the nonfusing myoblast mutants D-1 and C-8 and in wild-type L6 cells that were prevented from fusing by treatment with 5-bromo-2′-deoxyuridine, the expression of gp46 remained invariant. We suggest that collagen interactions, mediated by gp46, are important for normal rat skeletal muscle differentiation.
DNA-dependent RNA polymerase from Pseudomonas aeruginosa
Authors: Allan Brenda , Kropinski Andrew M.
Published: December 2009

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having β′ (157 000), β (148 000), σ (87 000), and α2 (45 000) subunits as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+ produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5–9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 °C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.
Characterization of monoclonal antibodies to cytochrome c: analysis of the antigenic structure of holo- and apo-cytochromes, and CNBr-peptide fragments of horse cytochrome c
Authors: Kim In Cheol , Nolla Hector , Priola Suzette
Published: December 2009

Five mouse hybridoma cell lines secreting SA, SB, SC, SD, and SE monoclonal antibodies (McAb) to cytochrome c have been produced. From the cross-reactivities of these McAb with various vertebrate cytochromes c, the antigenic sites for SA and SB McAb were proposed to be at Thr(89)-Glu(92)-Ala(96) and Asn(103), respectively. The binding site for other McAb have not been determined. Cross-reactivity studies based on enzyme-linked immunosorbent assays and dot immunobinding assays indicated that SA, SB, and SC McAb did not bind to apo-cytochrome c nor to any of the three CNBr-peptide fragments. This observation suggests that (i) the antigenic specificity of these McAb is dependent on the conformatiuon of the antigenic site which is inherent to the native holoprotein molecule and (ii) the ordered conformation in the C-terminal regions of holo-cytochrome c is destroyed during CNBr-peptide fragmentation. On the other hand, the lack of binding of SD and SE McAb to apo-cytochrome c indicates that these McAb are also specific for conformational sites. The binding of SD and SE McAb to the heme-containing A-peptide fragment (residues 1–65) suggests that the conformation around the heme, as possible antigenic sites, are stable because of the thioether linkages by the Cys residues.
Regulation of carbonic anhydrase III by thyroid hormone: opposite modulation in slow- and fast-twitch skeletal muscle
Authors: Frémont Pierre , Lazure Claude , Tremblay Roland R. , Chrétien Michel , Rogers Peter A.
Published: December 2009

This laboratory previously reported that a major 30 kilodalton (kDa) protein of the soluble cytoplasmic fraction of the rat slow-twitch soleus muscle is modulated by thyroid hormone. This protein has been purified and a portion of the primary structure has been determined. The sequence analysis suggested that the 30-kDa protein is carbonic anhydrase III (CA III; EC 4.2.1.1). The reaction of the protein with a CA III specific antibody and the similar modulation of CA III by thyroid hormone also support this conclusion. Immunochemical quantification of CA III and measurement of CA activity were performed in skeletal muscles of defined fiber-type composition from rats that were rendered hyperthyroid by treatment with 3,3′,5-triiodo-L-thyronine. These experiments revealed that CA activity and CA III content are deinduced in the soleus muscle (primarily type I fibers) and induced in the superficial vastus lateralis muscle (primarily type IIb), whereas no changes were detected in the tibialis anterior muscle (primary type IIa). These results show that the modulation of CA III by thyroid hormone in rat skeletal muscle is not limited to the slow-twitch soleus muscle and that the amplitude and direction of this modulation are directly related to the initial fiber-type composition of the skeletal muscle.
Energetics of ligand and inhibitor interactions with acetylcholinesterase
Authors: Das Y. T. , Brown H. D. , Chattopadhyay S. K.
Published: December 2009

Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus, purified by affinity chromatography to a specific activity of 7000 – 10000 U/mg protein, was studied at 27 °C in conduction-type microcalorimeters for the heats of reaction, with the subsite-specific cationic ligands edrophonium and propidium and with the irreversible inhibitor diisopropylfluorophosphate (DFP), in an ion-free aqueous medium. Edrophonium and propidium, each at 0.5 × 10−5 M, yielded reaction heats of +3.2 and –1.5 kcal/mol (1 kcal = 4.184 J) respectively, with 1.3 × 10−5 M AChE active sites. DFP (1.3 × 10−5 M) reacted exothermically yielding −0.5 kcal/mol at stoichiometric level with AchE active sites. Circular dichroic spectra showed that a ternary complex of AChE (6.5 × 10−7 M active sites) and the two ligands (each at 1 × 10−3 M) in 1 mM Tris–HCl buffer (pH 8.0) had a positive Cotton effect at 235 nm. Neither DFP nor phosphoric acid 2,2-dichloroethenyl dimethyl ester (DDVP) caused any appreciable change. DFP–AChE, however, behaved like a normal enzyme in showing a positive Cotton effect in association with the two ligands. DDVP–AChE showed an increase in negative ellipticity at 287 nm in the presence of the two ligands. Another cationic ligand, d-tubocurarine, when present together with edrophonium, increased negative ellipticity at 302 nm and blue-shifted a 265-nm peak of the normal AChE. DFP interactions with AChE appear to be energetically different from those of edrophonium, the latter of which is believed to associate with the acetylcholine-binding subsite.
In vitro translation of human prostatic acid phosphatase mRNA and processing of the translation products by microsomal membranes and endoglycosidase H
Authors: Paradis Gilles , Dubé Jean Y. , Chapdelaine Pierre , Tremblay Roland R.
Published: December 2009

Poly(A)+ RNA was isolated from human prostatic tissue and translated in vitro in a rabbit reticulocyte lysate translation assay. Acid phosphatase labeled with [35S]methionine was immunoprecipitated with an antibody against seminal plasma acid phosphatase. Two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitate, followed by fluorography, revealed the presence of two spots (one major and one minor), both having a molecular mass of 43 kilodaltons (kDa) and an isoelectric point higher than mature acid phosphatase. Addition of canine pancreatic membranes to the translation assay resulted in the formation of four immunoprecipitable spots with molecular masses ranging from 43 to 49 kDa on one-dimensional gels. These spots probably represent acid phosphatases containing one to four core sugar groups, since after the addition of endoglycosidase H the molecular mass heterogeneity was abolished and we observed only one major band with a molecular mass (41 kDa) slightly lower than the ones of the primary translation product. These results suggest that human prostatic acid phosphatases are synthesized as two 43-kDa preproteins, which are further processed to 41-kDa proteins by removal of their signal peptide. Heterogeneity of the native protein arises mostly from glycosylation at four sites and not from differences in the amino acid sequence of the various forms.
Hydroxy[14C]urea uptake by normal and transformed human cells: evidence for a mechanism of passive diffusion
Authors: Tagger Aaron Y. , Boux Joan , Wright Jim A.
Published: December 2009

The antitumor agent hydroxyurea is a potent inhibitor of cell division and selectivity toxic for rapidly proliferating cells. This drug has been used in the treatment of human cancer and, since drug transport is an important aspect of drug action, we investigated the mechanism of hydroxy[14C]urea uptake by human diploid fibroblasts and their SV40-virus-transformed counterparts. Kinetic analysis of drug uptake, studies with metabolic inhibitors, and estimates of cell/medium distribution ratios and temperature coefficient (Q10) values indicated that hydroxyurea enters normal and SV40-virus-transformed human cells by a mechanism of diffusion.
The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin
Authors: Regoeczi Erwin , Chindemi Paul A. , Rudolph John R. , Spik Geneviève , Montreuil Jean
Published: December 2009

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92–94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60–63% of plasma transferrin, was bound by concanavalin A – Sepharose, while the smaller one (30–32% of plasma transferrin) resolved into an unbound (25–27% of plasma transferrin) and a retarded (4–5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20–29%) and an unbound (71–80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.
Structure of the capsular polysaccharide of Haemophilus pleuropneumoniae serotype 3
Authors: Altman Eleonora , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 3 (ATCC 27090) is composed of D-galactose (one part), 2-acetamido-2-deoxy-D-glucose (one part), glycerol (one part), and phosphate (one part). From hydrolysis, dephosphorylation, methylation, and 1H and 13C nuclear magnetic resonance studies, the polysaccharide was found to be a high molecular weight polymer of a repeating trisaccharide unit, joined through monophosphate diester linkages and having the following structure:[Formula: see text]
Calmodulin and troponin C: affinity chromatographic study of divalent cation requirements for troponin I inhibitory peptide (residues 104–115), mastoparan, and fluphenazine binding
Authors: Eyk Jennifer E. Van , Hodges Robert S.
Published: December 2009

The different conformations induced by the binding of Mg2+ or Ca2+ to troponin C (TnC) and calmodulin (CaM) results in the exposure of various interfaces with potential to bind target compounds. The interaction of TnC or CaM with three affinity columns with ligands of either the synthetic peptide of troponin I (TnI) inhibitory region (residues 104–115), mastoparan (a wasp venom peptide), or fluphenazine (a phenothiazine drug) were investigated in the presence of Mg2+ or Ca2+. TnC and CaM in the presence of either Ca2+ or Mg2+ bound to the TnI peptide 104–115. The cation specificity for this interaction firmly establishes that the TnI inhibitory region binds to the high affinity sites of TnC (most likely the N-terminal helix of site III) and presumably the homologous region of CaM. Mastoparan interacted strongly with both proteins in the presence of Ca2+ but, in the presence of Mg2+, did not bind to TnC and only bound weakly to CaM. Fluphenazine bound to TnC and CaM only in the presence of Ca2+. When the ligands interacted with either proteins there was an increase in cation affinity, such that TnC and CaM were eluted from the TnI peptide or mastoparan affinity column with 0.1 M EDTA compared with the 0.01 M EDTA required to elute the proteins from the fluphenazine column. The interaction of these ligands with their receptor sites on TnC and CaM require a specific and spatially correct alignment of hydrophobic and negatively charged residues on these proteins. In the case of the TnI peptide, which represents a naturally occurring target protein for TnC, Mg2+ and Ca2+ can induce the correct structure in TnC or CaM for interaction, while only Ca2+ can induce the correct structure for mastoparan or fluophenazine binding.
Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo
Authors: McCubbin William C. , Kay Cyril M. , Kennedy Theresa D. , Lane Byron G.
Published: December 2009

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.
Accumulation of D-arginine by rat liver mitochondria
Authors: Villalobos-Molina Rafael , Pardo J. Pablo , Saavedra-Molina Alfredo , Piña Enrique
Published: December 2009

The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate–malate as substrates.
Protein synthesis in perfused rat hearts after in vivo hyperthermia and in vitro cold ischemia
Authors: Currie R. William
Published: December 2009

Isolated and perfused rat hearts can be maintained for up to 2.5 h with minimal synthesis of a stress protein with a relative mass (Mr) of 71 kilodaltons (SP71). Isolated hearts, subjected to 17 h of cold (4 °C) ischemia, upon perfusion (37 °C) synthesize a large amount of SP71. In the present study, the effect of in vivo hyperthermia on protein synthesis in isolated and perfused hearts was examined. Hearts were excised from rats subjected to a 15-min episode of hyperthermia (42 °C), either immediately (no recovery) or after 24 h of recovery. The excised hearts were perfused either immediately or after 17 h of cold ischemia. Hyperthermia (no recovery) increased [3H]leucine incorporation into SP71, while hyperthermia with a 24-h recovery did not increase incorporation into SP71 during perfusion (no ischemia). Hyperthermia (no recovery) increased the incorporation of [3H]leucine into SP71 seen after cold ischemia. Hyperthermia with a 24-h recovery decreased the incorporation of [3H]leucine into SP71 seen after cold ischemia. This reduction in synthesis of SP71 after 24-h recovery from hyperthermia could be caused by the accumulation of SP71 suppressing its own synthesis or a measure of protection (tolerance) induced by the hyperthermia.
The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immmunologically distinct from the mouse NADP-dependent bifunctional enzyme
Authors: Gardam Michael A. , Mejia Narciso R. , MacKenzie Robert E.
Published: December 2009

Methylenetetrahydrofolate dehydrogenase – methenyltetrahydrofolate cyclohydrolase – formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2′,5′-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase–cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate – polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.
Germin. Molecular cloning of cDNA that selects germin mRNA from bulk wheat mRNA
Authors: Rahman Sadequr , Grzelczak Zbyszko , Kennedy Theresa , Lane Byron
Published: December 2009

(1) Bulk mRNA from germinated wheat embryos was denatured with methylmercury and subjected to electrophoresis in agarose gel to obtain a fraction of mRNA that was modestly enriched with respect to its complement of translatable germin mRNA. This fraction of mRNA was used as a source of primary templates for preparing a cDNA library.(2) Escherichia coli JM101 was transfected with recombinant pUC8 plasmids containing cDNA inserts. Colonies of transformed bacteria (ca. 4 × 103) were differentially screened by hybridizing them with cDNA probes that were prepared from RNA populations containing different proportions of translatable germin mRNA.(3) A 160 base pair (bp) cDNA, which hybridized more strongly to the probe made from the RNA population containing the greater proportion of translatable germin mRNA in colony hybridizations, also hybridized more strongly to the RNA population containing the greater proportion of translatable germin mRNA when it was used as a probe for Northern analysis.(4) As judged by peptide mapping of a protein made by cell-free translation, the 160-bp cDNA selected virtually pure germin mRNA from the bulk mRNA of germinated wheat embryos when it was used in "hybrid release" experiments. The same 160-bp cDNA was used to select a "full length" germin cDNA from a library prepared by the Gubler–Hoffman method.
Effect of the aggregation state of amphotericin B on its interaction with ergosterol
Authors: Gruda Ilona , Dussault Nathalie
Published: December 2009

The interaction of amphotericin B with ergosterol was studied in aqueous solutions of propanol. The mode of the interaction was found to be related to the aggregation state of amphotericin B. Ergosterol does not react (or reacts extremely slowly) with monomeric amphotericin B. Traces of a small aggregate, probably a dimer, enable a cooperative reaction. At high concentrations of the dimer, the reaction is immediate and the concentration of amphotericin B complexed with ergosterol is twice as high as the amount of added sterol. The interaction with ergosterol is hindered when the antibiotic is in micellar form. The pharmaceutical form, Fungizone, behaves similarly to the pure amphotericin B. Fungizone's greater solubility in water does not modify either the extent or the mode of interaction with ergosterol.
Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells
Authors: Sunga Paul S. , Pritchard P. Haydn , Rabkin Simon W.
Published: December 2009

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1–25 μM) extracellular choline concentrations, but significantly (p < 0.05) decreased choline uptake at higher (37.5–50 μM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.
Bovine thymus satellite I DNA sequences, which are highly methylated, are preferentially located in H1-rich chromatin
Authors: Davie James R. , Delcuve Genevieve P.
Published: December 2009

The distribution of 5-methylcytosine among H1-rich and -poor bovine thymus chromatin regions was determined. 5-Methylcytosine was enriched in H1-rich chromatin regions, with linker and nucleosomal DNA containing similar amounts of this modified base. Satellite I DNA sequences, which constitute 5–7% of the genome and are highly methylated, were preferentially localized among H1-rich chromatin regions, in accordance with the distribution of 5-methylcytosine. In contrast to the satellite I DNA sequences, prothrombin (a single copy DNA sequence) was localized among both H1-rich and -poor chromatin regions. The results of this study are consistent with the hypothesis that DNA methylation has a role in modulating the structure of chromatin.
Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture
Authors: Emerman Joanne T. , Jones Nancy , Fiedler Eveline E.
Published: December 2009

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs, and the increase in GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.
Maternal diabetes and its effect on biochemical and functional development of rabbit fetal lung
Authors: Bhavnani Bhagu R. , Enhorning Goran , Ekelund Laila , Wallace Duncan , Pan Chee Chung
Published: December 2009

The effect of maternal diabetes on functional and biochemical maturation of the fetal lung was studied in a rabbit model. Pregnancy was initiated only after diabetes had been established. Both the pregnant doe and its fetuses were hyperglycemic. For comparison, the fetal heart and liver were also studied. In the diabetic group, the DNA content was lower in the fetal heart and lung while the protein content was higher in all three tissues. The glycogen levels were higher only in the fetal lung. Glycogen synthase was higher in the fetal lung and heart while phosphorylase activity was higher in all three tissues from the diabetic group. The activities of key enzymes involved in glycolysis were not affected. No difference was observed in the concentration of total phospholipids or in the ability of the airway fluid to reduce surface tension. In contrast, fetal lungs from diabetic does did not expand as well as the controls and retained less air on deflation. These findings suggest that the utilization of glycogen in fetal lungs from the diabetic does was not complete and that the increased incidence of respiratory distress in infants of diabetic mothers may not be due to a lack of surfactant.
Preparative isolation and characterization of the B875 complex from Rhodobacter sphaeroides 2.4.1
Authors: Picorel Rafael , Gingras Gabriel
Published: December 2009

We have developed a simple and efficient method, using a mixed detergent system of sodium dodecyl sulfate and Triton X-100, for the preparative isolation of theB875 complex from Rhodobacter sphaeroides 2.4.1. As a bonus, the method allows the preparation of both the B875 and B800-850 complexes from the same batch of chromatophores. The preparations are spectrally pure, as indicated by absorption and circular dichroism spectroscopy. The latter method suggests that the Qy band of the B875 complex is due to weakly interacting bacteriochlorophyll molecules. Protein and pigment analysis shows that the B875 complex contains 2 mol of bacteriochlorophyll and 2 mol of sphaeroidene per mol of apoprotein (12 266 g), whereas the B800-850 complex contains 3 mol of bacteriochlorophyll and 1 mol of sphaeroidene per mol of apoprotein (11 497 g). While these stoichiometries are in accord with currently accepted models, they disagree with their published experimental basis. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol were found to be present in the B875 complex.
The regulation of mitotic spindle function
Authors: Wolniak Stephen M.
Published: December 2009

The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.
Cell-membrane hormone receptors: some perspectives on their structure and function relationship
Authors: Yip Cecil C.
Published: December 2009

There are three structural regions common to hormone receptors on the cell membrane: an external region containing the ligand-specific binding domain, a hydrophobic region transversing the membrane, and a cytoplasmic region involved in the expression of activity. The structural features of these regions are broadly reviewed in regard to their functional roles. It is suggested that the specificity of cellular response to hormonal stimulation lies not only in the binding of a specific ligand by the external domain but also in the cytoplasmic region of a receptor. The cytoplasmic region can either interact specifically with a regulatory system or express its own specific ligand-dependent catalytic activity. The fundamental question of how ligand binding can lead to the expression and regulation of receptor activity is considered.
DNA-mediated transfection as a model for oncogenesis
Authors: Moyer Mary Pat , Egan John W. , Aust J. Bradley , Moyer Rex C.
Published: December 2009

DNA transfer technology has greatly contributed to progress in understanding molecular biology and genetics. In recent years, great efforts have been expended to determine the oncogenic potential of single, defined genes or complex gene mixtures as a prelude to defining the role those genes may play in neoplastic transformation in vitro and tumor induction in vivo. This paper reviews the currently available DNA transfection techniques and their application toward understanding cancer initiation and progression, and how the in vitro and animal models may apply to human cancer.
Interaction of tumor cells and immune system in the metastatic process
Authors: Gorelik Elieser , Gunji Yoshio , Goldfarb Ronald H.
Published: December 2009

The metastatic process is rather complicated and relatively inefficient. Millions of tumor cells are constantly shedding from the primary tumor into the blood stream, but very few of them are able to form metastatic tumors in the different organs or tissues of the host. It is widely accepted that metastatic cells have to possess a complex array of various properties that allow them to complete the metastatic cascade. The realization of the metastatic potential largely depends on the ability of tumor cells to evade host defense mechanisms. The potential role of specific and nonspecific immune mechanisms in the control of metastatic spread and growth is the subject of the present review. A better understanding of the mechanisms of antimetastatic defense is of prime importance for development of efficient immunotherapeutic methods for the treatment and eradication of disseminated tumor metastases.
The production of tissue-specific histone complements during development
Authors: Lennox Ronald W. , Cohen Leonard H.
Published: December 2009

At least two mechanisms generate tissue differences in the histone subtype composition during development: subtype dilution and subtype replacement. Subtype dilution, which occurs when cells continue dividing after having ceased to synthesize one or more histone subtypes, allows the elimination of stable subtypes. It is the major mechanism generating cell differences in histone composition in sea urchin embryogenesis. Subtype replacement has been observed in mammalian tissues, both in the intact animal and in cultured cells. It is most evident in nondividing cells but occurs to some extent in dividing cells as well. Examples of the two mechanisms are presented and their possible biological significance, as well as that of the differences they produce, is discussed.
The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines
Authors: Aujame L.
Published: December 2009

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS – one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89 000, 70 000, and 68 000 Da. When the heat-shock response of the mouse erythroleukemia cell line DIB, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 °C, or 1 h at 45 °C, no protein band corresponding to hsp 68 was observed. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 °C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0–72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5′ ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 μM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.
Purification and characterization of spore-specific catalase-2 from Bacillus subtilis
Authors: Loewen Peter C. , Switala Jacek
Published: December 2009

Catalase-2, the catalase found in spores of Bacillus subtilis, has been purified to homogeneity from a nonsporulating strain. The apparent native molecular weight is 504 000. The enzyme appears to be composed of six identical protomers with a molecular weight of 81 000 each. The amino acid composition is similar to the composition of other catalases. Like most catalases, catalase-2 exhibits a broad pH optimum from pH 4 to pH 12 and is sensitive to cyanide, azide, thiol reagents, and amino triazole. The apparent Km for H2O2 is 78 mM. The enzyme exhibits extreme stability, losing activity only slowly at 93 °C and remaining active in 1% SDS – 7 M urea. The green-colored enzyme exhibits a spectrum like heme d with a Soret absorption at 403 nm and a molar absorptivity consistent with one heme per subunit. The heme cannot be extracted with acetone–HCl or ether, suggesting that it is covalently bound to the protein.
Enzymatic oxidation of all-trans retinal to retinoic acid in rat tissues
Authors: Bhat Pangala V. , Poissant Lyne , Falardeau Pierre , Lacroix André
Published: December 2009

The 100 000 × g supernatant (cytosolic) fraction of rat tissue homogenates catalyzes the oxidation of all-trans retinal to retinoic acid. Kidney, testis, and lung were the most active of the tissues examined. The presence of enzyme activity in liver and intestine could be detected only when a substrate concentration beyond the saturation point for retinal reductase was used. Spleen, brain, and plasma had no activity. Boiled supernatants did not catalyze the reaction. The enzymatic product was chemically and physically identified as retinoic acid. The cytosol of kidney tissue also catalyzed the conversion of retinol to retinoic acid. These data indicate that kidney tissue has the highest retinal oxidase activity and suggest that it may play a major role in the oxidative metabolism of retinol in the body.
Structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 23F (American type 23)
Authors: Richards James C. , Perry Malcolm B.
Published: December 2009

The specific capsular polysaccharide of Streptococcus pneumoniae serotype 23F (American type 23) is composed of a repeating tetrasaccharide unit containing D-glucose (one part), D-galactose (one part), L-rhamnose (two parts), glycerol (one part), and phosphate (one part). By composition analysis, optical rotation, partial hydrolysis, periodate oxidation, methylation, and high-resolution 1H and 13C nuclear magnetic resonance studies, the elucidated unambiguous structure was in agreement with our earlier proposal but is at variance with structures proposed later by other authors. The structure of the type 23F pneumococcal polysaccharide is[Formula: see text]