bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein–glycolipid interactions during spermatogenesis. Binding of specific germ cell proteins to sulfatoxygalactosylacylalkylglycerol, the major glycolipid of mammalian male germ cells
Authors: Lingwood C. A.
Published: December 2009

Specific binding of membrane proteins extracted from rat spermatogenic cells to the major glycolipid of the male germ cell has been demonstrated by affinity chromatography. A new method for the production of affinity matrices, using photoactivatable heterobifunctional cross-linking agents, has been used to immobilize sulfatoxygalactosylacylalkylglycerol. Three proteins of apparent molecular weights 68 000, 34 000, and 24 000 from spermatogenic cells have been shown to selectively and reversibly bind to this affinity matrix. Antiserum raised against the major of these species (68 000) was demonstrated to be specific for this protein by immunoblotting. A new technique for the reduction of background nonspecific antibody staining using this method is described. The immune serum has been used to localize the antigen in frozen testicular sections. The protein is present in the plasma membranes of all germ cells, but the expression is elevated for testicular spermatozoa and cells in or near the basal compartment of the seminiferous epithelium. The relevance of these findings to intercellular communication and spermatogenesis is discussed.
Cytoplasmic malate dehydrogenase from Phycomyces blakesleeanus: Kinetics and mechanism
Authors: Teixido Francisco , Arriaga Dolores De , Busto Félix , Soler Joaquin
Published: December 2009

The kinetics and reaction mechanism of cytoplasmic malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) in 0.1 M potassium phosphate buffer (pH 7.5) at 30 °C have been investigated. The initial rate and product inhibition studies were consistent with an ordered bi-bi mechanism that involved more than one kinetically significant ternary complex and also with the coenzyme binding first. The dissociation of the coenzyme from the enzyme–coenzyme complex appeared to be the slowest step in either direction of the reaction. The kinetic and rate constants for the individual steps of the reaction were determined.
Cell sonicates used in the analysis of how measles and herpes simplex type 1 virus infections influence Vero cell mitochondrial calcium uptake
Authors: Lund Karen , Ziola Barry
Published: December 2009

Brief sonication was used to rapidly prepare an in situ mitochondrial Ca2+ uptake system from Vero cells. This method yielded intact, functional mitochondria capable of accumulating and retaining approximately 50% of Ca2+ available in the reaction mix. Mitochondria in cells infected with herpes simplex type 1 virus exhibited a gradual decline in the initial Ca2+ uptake rate, dropping to 65% of the control rate at the end of the 12-h lytic cycle. Influence of a lytic measles virus infection on the initial Ca2+ upake rate was different. The rate initially dropped to 75% of the control rate by 9 h postinfection, then recovered to slightly above the control rate at 21 h postinfection, and finally declined to 75% of the control rate at 30 h postinfection. Neither viral infection altered the total amount of Ca2+ accumulated by Vero cell mitochondria.
Immunological analyses of selected eukaryotic RNA polymerases II
Authors: Bettiol Michael F. , Irvin Randall T. , Horgen Paul A.
Published: December 2009

Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.
Mechanisms of cholesterol synthesis inhibition by D-glucosamine
Authors: Friedman Susan J. , Cheng Stanley , Skehan Philip
Published: December 2009

The amino sugar D-glucosamine possesses antitumor activity which is thought to depend in part upon its ability to impair cholesterol biosynthesis and damage cellular membranes. The present study examined the effect of glucosamine on acetate utilization for lipid and sterol synthesis in rat C6 glial tumor cells. At cytotoxic concentrations, the amino sugar inhibited [14C]acetate incorporation into nonesterified sterols and lipids but increased the flow of label into cholesteryl esters. A comparison of the rates of acetate utilization for glucosamine metabolism (N-acetylation) and sterol and lipid synthesis suggested that glucosamine might act by competing for a common cytosolic pool of acetyl CoA. The inhibition of lipid and sterol synthesis, however, remained constant over a wide range of extracellular acetate concentrations. These results suggest that, if glucosamine acts by restricting the supply of acetate for these biosynthetic processes, it probably inhibits a step prior to the formation of acetyl CoA. Alternative mechanisms are discussed.
A 105 000 dalton antigen of transformed mouse cells is a stress protein
Authors: Rose T. M. , Khandjian E. W.
Published: December 2009

Antisera prepared in mice against syngeneic spontaneously transformed AL/N cells (anti-TAL/N serum) identified a number of protein antigens synthesized by simian virus 40 (SV40) transformed cells, among which was a protein with a molecular mass of 105 000 daltons (p105). Of these transformed cell antigens which were immunogenic in a syngeneic system, only p105 was detected in primary mouse kidney cell cultures, p105 isolated from normal and transformed mouse cells was demonstrated to be identical by two-dimensional gel analysis. Relatively small amounts of p105 were synthesized in quiescent primary cultures, while the protein was actively synthesized in SV40-infected as well as in proliferating mouse kidney cells, and its synthesis in quiescent cells could be induced by subjecting the cultures to glucose starvation or heat-shock treatment. Immunofluorescent staining and cellular fractionation showed that p105 is normally localized to cytoplasmic structures. The results suggest that the expression of p105 is intimately associated with the metabolic state of the cell.
A comparison of the acylation of 1-acyl-sn-glycero-3-phosphoinositol and 1-acyl-sn-glycero-3-phosphocholine in neuronal nuclei in vitro using radioactive arachidonate and oleate
Authors: Baker R. Roy , Chang Huu-Yi
Published: December 2009

A neuronal nuclear fraction (N1) was isolated from cerebral cortices of 15-day-old rabbits. Samples of N1 were incubated with a radioactive fatty acid ([3H]arachidonate or [14C]oleate), acylation cofactors, and 1-acyl-sn-glycero-3-phosphoinositol (1-acyl-GPI) or 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC). In competition studies, both radioactive fatty acids were incubated with one lysophospholipid or the two lysophospholipids were incubated with one radioactive fatty acid. Using [3H]arachidonate and one lysophosphoglyceride, a maximal rate of incorporation into phosphatidylinositol (PI) was found at a relatively low concentration of 1-acyl-GPI (10 μM), while increasing rates of incorporation into phosphatidylcholine (PC) were seen with increasing concentrations of 1-acyl-GPC (to 65 μM). At low concentrations of lysophosphoglyceride (≤ 25 μM) the rate of arachidonate incorporation into PI greatly exceeded rates of arachidonate incorporation into PC. This higher rate of arachidonate incorporation into PI was also seen in incubations where both lysophospholipids were present. For oleate, greater rates of incorporation into PC were found in comparison with rates of labelling of PI in assays using relatively high concentrations of one or both lysophospholipids. When comparing arachidonate and oleate, in assays with one or both fatty acids, the polyunsaturate showed at least threefold higher rates of incorporation into PI. For PC labelling higher rates of arachidonate incorporation were evident at the higher concentrations of 1-acyl-GPC and the superiority over oleate was not as marked as that seen in PI labelling.
Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7
Authors: Perry Malcolm B. , MacLean Leann , Griffith Douglas W.
Published: December 2009

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.
The effect of developing hypertension on the synthesis and accumulation of elastin in the aorta of the rat
Authors: Keeley F. W. , Johnson D. J.
Published: December 2009

This report describes an investigation of the effects of developing hypertension on the synthesis and accumulation of insoluble elastin in the thoracic aorta of young rats. Uninephrectomized male rats were made hypertensive by administration of deoxycorticosterone acetate and addition of 1% NaCl to their drinking water. Divergence of systolic blood pressures between treated and control animals and hypertrophy of the vessel began after about 2 weeks of treatment. Coincident with the appearance of hypertrophy, there was an increased accumulation of insoluble elastin in the aorta and a large increase in the capacity of the aortic tissue to synthesize elastin. However, in spite of continued increases in blood pressure and vessel hypertrophy, this effect on elastin synthesis and accumulation was transient. The results of this study suggest that synthesis of elastin in aortic tissue of young rats is highly sensitive to alterations in blood pressure.
Effect of saturated phosphatidylcholines on the functional properties of reconstituted cytochrome oxidase
Authors: Singer Michael A. , Dinda Maria , Young Marlene , Finegold Leonard
Published: December 2009

Cytochrome oxidase was incorporated into liposomes, at various protein/lipid ratios, composed of either a phosphatidylcholine of varying chain length and symmetry or asolectin. Catalytic activity and respiratory control were assayed at two temperatures. All preparations showed higher activity at low protein/lipid ratios, but only asolectin showed respiratory control. A spectroscopic determination of the vectorial orientation of oxidase molecules showed that, for proteoliposomes with saturated lipids, 100% of oxidase molecules could be reduced by external substrate as compared with 75% for asolectin proteoliposomes. Freeze-fracture electron microscopy confirmed that oxidase was incorporated into these proteoliposomes and differential scanning calorimetry indicated that the protein induces significant disruption in the long range packing of the saturated phospholipids. We propose that the oxidase molecules in proteoliposomes formed from saturated phosphatidylcholines do not display respiratory control because they are unable to assume the transmembrane orientation necessary for full vectorial activity.
Freeze-etch visualization of the outer surface of myoblasts in culture
Authors: Barber Kathryn R. , Mehlhorn Ingrid E. , Párraga Grace , Grant Chris W. M.
Published: December 2009

The Pfenninger device is one of several types of specimen holders designed to permit freeze-fracture electron microscopy of cultured cells growing attached to solid substrates. It achieves this by directing a fracture plane horizontally through a monolayer of cells frozen with overlying medium (without need for prior disruption of cell attachments or relationships). The end result is a platinum-shadowed replica of the cell membrane hydrophobic interior. Here we describe the features seen when this traditional fracture step is followed by a lengthy etching step, making possible views of the cell membrane outer surface with a resolution 100 × better than that of fluorescence microscopy. Because of the technical difficulties involved, such views have in past been restricted to samples which may be handled in suspension, particularly blood cells and model membranes. Thus we have been able to examine extensive regions of the myoblast outer surface (the so-called etch face) at a magnification that permits visualization of details on the order of individual macromolecules. Prominent clumps of glycocalyx material occupying some 50% of the surface can be readily resolved as a closely spaced network of uniformly distributed 20- to 60-nm irregular granular patches. Receptors for wheat germ agglutinin were found to be associated almost exclusively with these surface prominences, so that bound lectin tended to exist in a uniform distribution of small clusters corresponding to the patches described above. When cells were not fixed until 15 min after lectin addition there was visibly more binding, but in a similar distribution. The details of cell surface architecture recorded here at a resolution of 2–4 nm are well below the limit of resolution of light microscopy and complement existing studies by fluorescence techniques. The presence of surface receptors in small patches reinforces the possibility that some literature observations of receptor interaction may be explained on the basis of direct receptor–receptor contact.
Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties
Authors: Squires E. James , Haard N. F. , Feltham L. A. W.
Published: December 2009

Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 °C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychrotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36 000–38 000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.
Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking
Authors: Sone Nobuhito , Hou Cynthia , Bragg Philip D.
Published: December 2009

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.
Inhibition of protein synthesis enhances transformation of primary cells by viral DNA
Authors: Mak I. , Fatt R. B. Lai , Mak S.
Published: December 2009

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK−) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.
The structure of myosin and its role in energy transduction in muscle
Authors: Shriver John W.
Published: December 2009

The present understanding of the relationship between the structure of the myosin ATPase and its role in force production for muscle contraction is reviewed. Emphasis is placed on structural transitions in myosin that occur during ATP hydrolysis which may be correlated with force production. Although detailed structural information is presently lacking, numerous spectroscopic and kinetic experiments have indicated that myosin exists in two structural states for each chemical intermediate in the hydrolysis of ATP. Models are discussed which view a transition between these two states as the energy transduction "event" (i.e., force production).
UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase from Bacillus sphaericus: activation by potassium phosphate
Authors: Anwar Rashid A. , Vlaovic Mariana
Published: December 2009

During the course of purification of UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase, we observed a marked stimulation of the enzymatic activity in the presence of phosphate ions. This activation effect was studied with enzyme purified 979-fold from Bacillus sphaericus. Each salt tested stimulated the activity of the synthetase. The order of activation by different anions was [Formula: see text]. In every case, the potassium salt gave higher activity than the corresponding sodium salt. The activation in the presence of phosphate was quite pronounced (almost sevenfold with K2HPO4) and occurred at a relatively low concentration. The Ka for K2HPO4 was found to be 3.4 mM and the Hill coefficient was calculated to be 1.0. This would suggest that there is one phosphate-binding site per active centre. The presence of phosphate did not affect either the pH optimum of this enzyme or the optimum concentration of Mg2+ required. The presence of phosphate has little or no effect on the Km of any of the substrates. Thus, it appears that the presence of phosphate changes the enzyme conformation to a catalytically more active form. The activation of this enzyme in the presence of phosphate anion is all the more interesting because phosphate is a product of the reaction catalyzed by this enzyme.
The so-called of Escherichia coli is a stable denatured conformer of the major isoacceptor
Authors: Tremblay Thomas-Louis , Lapointe Jacques
Published: December 2009

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This [Formula: see text] (or [Formula: see text] when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.
Proton motive force in a photodenitrifier, Rhodopseudomonas sphaeroides forma specialis denitrificans
Authors: Kundu Balaram , Nicholas D. J. D.
Published: December 2009

The washed cells of Rhodopseudomonas sphaeroides f.sp. denitrificans developed a Δp of about −175 to −200 mV during denitrification in the dark and −200 to −245 mV in the light. With NO2− as the terminal acceptor, Δp was less than with NO3−, N2O, or O2. The values of Δψ in the dark were about −150 mV for NO3− and N2O and −140 mV for NO2−. During photodenitrification with NO3−, NO2−, or N2O or respiration to O2 in light, Δψ ranged between −152 and −167 mV. Like Δψ, the ΔpH was higher in light than in the dark, resulting in a 20- to 30-mV increase in Δp during illumination with NO3−, NO2−, or N2O as the acceptor. Both ΔpH and Δψ were reduced at higher pH values (≥ 7.5). Changes in pH in response to O2 in the light were less than those in the dark, indicating light inhibition of O2 respiration. The cells maintained a reasonably high Δp without addition of a substrate or when inhibitors were used; the cells retained a fairly high Δψ even in the presence of an inhibitor. However, ΔpH was appreciably lowered and in some cases it was almost abolished when either KCN, rotenone, NaN3, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-dinitrophenol, N,N′-dicyclohexylcarbodiimide, antimycin A, or 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) was used. The combination of an uncoupler (e.g., CCCP) and an electron transport effector (e.g., antimycin A) further reduced the ΔpH. Antimycin A and HOQNO were more effective in inhibiting photosynthetic electron transport to NO3−, NO2−, N2O, or O2 than the dark respiration to these substrates. Dibromomethylisopropyl-p-benzoquinone, a quinone antagonist, markedly reduced ΔpH in light with NO3−, NO2−, N2O, or O2 as the terminal acceptor, indicating that photosynthetically generated electrons are used for denitrification in this bacterium.
Effects of growth rate and methotrexate on folate polyglutamates and folylpolyglutamate hydrolase activity in Krebs ascites cells
Authors: Sur Pratima , Priest David G. , Doig Marion T.
Published: December 2009

The activity of folylpolyglutamate hydrolase was measured throughout intraperitoneal growth of Krebs ascites cells in mice and after exposure to methotrexate. Hydrolase activity was lowest during the log phase of growth. Methotrexate administered intraperitoneally during log growth caused a dose- and time-dependent increase in hydrolase activity. Modest changes were observed in endogenous folate polyglutamate chain length distributions throughout growth and upon exposure to methotrexate, but these changes coud not be correlated with hydrolase activity.
Purification and characterization of a gelatinase produced by fibroblasts from human gingiva
Authors: Nakano Takuo , Scott Paul G.
Published: December 2009

An endopeptidase which digests denatured collagen to small, dialysable fragments was purified 2675-fold from medium that had been conditioned by the culture of fibroblasts grown from explants of human gingiva. This enzyme was inhibited by chelating agents, but not by phenylmethylsulphonyl fluoride nor by N-ethylmaleimide, and is therefore probably a metalloproteinase. It showed no demonstrable activity against native collagen or ovalbumin, while α-casein was digested slowly, if at all. It therefore belongs to the group of enzymes which have been called tissue gelatinases. This gelatinase was secreted in a latent form or forms and could be activated by proteolysis with trypsin. The active enzyme had an apparent molecular weight of 69 000 (gel chromatography) or 72 000 (gel electrophoresis in sodium dodecyl sulphate) and an apparent isoelectric point of 4.15.
Purification of mRNA coding for the enzyme deficient in hereditary tyrosinemia, fumarylacetoacetate hydrolase
Authors: Nicole Louis M. , Valet Jean Paul , Laberge Claude , Tanguay Robert M.
Published: December 2009

As a step towards the cloning of the gene for fumarylacetoacetate hydrolase (FAH), we have purified the FAH mRNA from rat liver by specific immunoadsorption of polysomes. The relative abundance of this mRNA has been estimated to be 0.14%. The major in vitro translation product of the purified mRNA preparation is specifically precipitated by a rabbit anti-rat FAH antiserum and it is, furthermore, undistinguishable by criteria of mass and charge from purified rat FAH.
Heating RNA before cell-free translation gives no selective increases among the translation products of RNA from rat heart and mammary gland, rabbit reticulocyte membranes, or trout liver
Authors: Mezl Vasek A. , Clark Jane , Garber Pascale M.
Published: December 2009

A recent study has reported that the heating of a population of guanidinium-extracted mRNAs prior to translation causes a selective increase in the translation of certain mRNAs. To determine if this phenomenon is a general property of mRNAs, we carried out a comparison of the translation products obtained when phenol-extracted rat heart and mammary gland RNAs, rabbit reticulocyte membrane RNAs, and trout liver RNAs were translated in the reticulocyte translation system, with and without a prior heat treatment. Our results show that no selective increase in the translation of mRNAs was observed for any of these samples. Among the 14 RNA preparations examined, one total mammary RNA preparation did display a twofold increase in the translation of all mRNAs after heat treatment. It is shown that the heat enhancement of translational activity observed for this sample was due to the reversible formation of intermolecular aggregates with a contaminant that can be removed by chromatography on oligo(dT)-cellulose. Since heat treatment did not selectively enhance the activity of any mRNA in these samples, our results show that the current practice of translating phenol-extracted RNAs without a prior heat treatment should be satisfactory for the translation of most mRNA populations.
A detailed examination of the iodination of β-galactosidase: stoichiometric inactivation by nonspecific iodination
Authors: Edwards L. A. , Huber R. E.
Published: December 2009

The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of β-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail. The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides. The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253. This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15–18% of the total. To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out. In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5–7% of the incorporation). In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin. No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253. Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously. The competitive inhibitor isopropyl-β-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.
Relationship of the cGMP-binding activity to the cGMP-specific phosphodiesterase in Dictyostelium discoideum
Authors: Parissenti A. M. , Coukell M. B.
Published: December 2009

Optimal conditions for assaying and stabilizing the soluble cGMP-binding activity in Dictyostelium discoideum were established. Using these procedures, we investigated the relationship between the cGMP-binding activity and the cGMP-specific phosphodiesterase in this organism. In wild-type strains, the binding and phosphodiesterase activities were found to be regulated differently during development. Also, stmF mutants, which possess very low levels of cGMP-specific phosphodiesterase activity, exhibited normal levels of cGMP-binding activity. Fractionation studies revealed that the binding and phosphodiesterase activities could be resolved by DEAE-cellulose chromatography. Finally, the effect of pH on cGMP binding was different from that reported for cGMP-mediated activation of the phosphodiesterase. Taken together, these results indicate that the cGMP-binding protein and the cGMP-specific phosphodiesterase are probably unrelated. In addition, the cGMP-binding activity is not associated with cGMP-stimulated kinase activity and it does not elute from DEAE-cellulose like the highly conserved cGMP-dependent protein kinases found in other systems.
Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures
Authors: Orlowski J. , Clark A. F.
Published: December 2009

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6–7 days) actively metabolized 3H-labelled testosterone (T), 5α-dihydrotestosterone (5α-DHT), 5α-androstane-3α,17β-diol, and 5α-androstane-3β,17β-diol. The epithelial cells actively reduced T to 5α-DHT and formed significant amounts of 5α-androstane-3,17-dione from T, 5α-DHT, and 5α-androstane-3α,17β-diol. All substrates were converted to significant amounts of C19O3metabolites. The stromal cells also metabolized all substrates, but very little 5α-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have Δ4-5α-reductase, 3α- and 3β-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17β-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.
The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase
Authors: Gonzalez Jose E. , Somerville Ronald L.
Published: December 2009

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme–anthranilate–pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accomodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 μM) and phosphoribosylpyrophosphate (Km = 50 μM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 μM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod–Wyman–Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregrate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.
Structural studies of the capsular polysaccharide from Haemophilus pleuropneumoniae serotype l
Authors: Altman Eleonora , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 1 (ATCC 27088) was found to be a teichoic acid type polysaccharide of a repeating disaccharide unit composed of 2-acetamido-2-deoxy-D-glucose and D-galactose units. By composition analysis, methylation, partial hydrolysis, dephosphorylation, and one- and two-dimensional 500-MHz proton nuclear magnetic resonance experiments, together with 13C nuclear magnetic resonance studies, it was concluded that the capsular polysaccharide is a high molecular weight linear polymer having the structure:[Formula: see text]
Chloride-insensitive, glycine–phenylalanine–naphthylamide hydrolysis at neutral pH in human skin fibroblasts
Authors: Doughty Michael J. , Gruenstein Eric I.
Published: December 2009

Crude lysosomal preparations from a cultured human skin fibroblast line were found to contain significant levels of a neutral pH hydrolase activity towards glycine – phenylalanine – β-naphthylamide (NA), a substrate normally used for the assay of lysosomal dipeptidyl aminopeptidase I. However, the activity was chloride ion insensitive, nonlatent, and inhibitable by cationic detergents and amino acids. Assays of substrate selectivity, relative substrate affinity, pH, and anion and cation sensitivity indicated the activity to be distinct from dipeptidyl aminopeptidases I (chloride-dependent hydrolysis of Pro-Phe-, Gly-Phe-, Gly-Arg-, and Pro-Arg-NA's at acid pH), II (Lys-Ala-NA hydrolysis), III (Arg-Arg-NA hydrolysis), and IV (Gly-Pro-NA hydrolysis). The lysosomal preparations also contained significant activity towards several amino acid – naphthylamides, notably Arg-NA. Only dipepidyl aminopeptidase I activity showed sensitivity to chloride anions, both dipeptidyl aminopeptidases I and II showed substantial latency, and none of the activities displayed a significant metal cation dependence.
The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells
Authors: Burres Neal S. , Cass Carol E.
Published: December 2009

When cultured human choriocarcinoma (BeWo) cells arc exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast. Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines. This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype. When methotrexate exposures (1 μM, 48 h) were conducted in a purine-frec basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur. Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis. In contrast, morphological differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.
Enzymatic dephosphorylation of retinyl monophosphate by rat liver
Authors: Rodríguez Providencia , Bello Olga
Published: December 2009

Rat liver has been shown to contain an enzyme that catalyzes the dephosphorylation of retinyl monophosphate. This activity was extracted with 0.1 M Tris buffer (pH 7.5). Maximum reaction rate was observed at a pH range of 7.0–7.5. It did not require metal ions for activity and was sensitive to fluoride ion. The retinyl monophosphate phosphatase activity was proportional to time and protein and substrate concentration. Triton X-100 (range of 0.05–0.10%) increased the activity 100%, whereas other detergents (Tween 80, cholate, and deoxycholate) did not activate the enzyme. A number of phosphorylated compounds tested as inhibitors of retinyl monophosphatase activity, such as glucose 6-phosphate (20 mM), glycerophosphate (20 mM), phosphatidic acid (8 mM), and dolichyl phosphate (3 mM), did not compete with retinyl monophosphate as substrate. However, at 20 mM concentration, ATP, ADP, 5′-AMP, and pyrophosphate were inhibitors of the enzyme. It is not possible at present to give further details about the specificity of the phosphatase activity. The enzyme described could play a regulatory role in retinol-mediated glycosylations, by altering the endogenous level of retinyl monophosphate.
Use of dialysis fibers for the study of aerobic cells
Authors: Boulanger Yvan , Vinay Patrick
Published: December 2009

The suitability of a perifusion system for the study of renal tubules trapped in different types of dialysis fibers has been investigated. First, the equilibration times across the fiber membranes were estimated by following the penetration of the dysprosium tripolyphosphate shift reagent inside the fibers using 23Na nuclear magnetic resonance. These times ranged from less than 1 min for microfibers to 30 min for some large diameter fibers. When cortical tubules were introduced into cuprophan dialysis microfibers (200 μm internal diameter) and perifused by rapid recirculation of cell culture medium, metabolic viability, as determined by a linear glucose and ammonia production from glutamine and lactate, was found to exceed 10 (dog) or 3 h (rat), an unprecedented time period for such structures. As evidenced by the sensitivity to furosemide, the lumen of thick ascending limb tubules was kept open by including 50 mM mannitol in the perifusate. Fibers with large internal diameters (1100 μm) were found to be less suitable owing to the slow equilibration rates with the intrafiber compartment. All types of fibers showed minimal line-broadening effects on the 31P nuclear magnetic resonance signals.
The substrate specificity of pollen extracellular nuclease
Authors: Matoušek Jaroslav
Published: December 2009

A detailed study of substrate specificity of tobacco pollen extracellular nuclease showed that the nuclease degraded, in order of decreasing rate, poly(U), RNA, heat-denatured DNA (ssDNA), poly(dA), poly(dA)∙poly(dT) duplex, and native or undenatured DNA (dsDNA). Poly(dC) was cleaved at a very low rate, while the poly(dC)∙poly(dG) duplex was degraded only at a negligible rate (0.8% of that with dsDNA). Poly(dG) was not detectably cleaved. The nuclease exhibited lower activity towards AG and GC copolymers than with AC and AU copolymers. Preference of the nuclease for ssDNA over dsDNA strongly depended on the ionic strength. The nuclease exhibited no preference for alkylated native DNA versus native DNA and degraded apurinated DNA at a lower rate. It also cleaved ultraviolet-irradiated DNA, but the cleavage did not lead to the specific excision of pyrimidine photodimers. Two molecular forms of the tobacco pollen nuclease isolated from polyacrylamide gels showed the same substrate specificity. The extracellular nucleases of five other pollen species examined also cleaved the following substrates in order of decreasing rate: poly(U) > ssDNA > poly(dA) > dsDNA. In each case poly(dG) was resistant to cleavage.
Unsaturated fatty acids inhibit ADP- arachidonate-induced platelet aggregation without affecting thromboxane synthesis
Authors: Dratewka-Kos Ella , Tinker D. O. , Kindl Brigitte
Published: December 2009

The inhibitory effects of three cis-unsaturated C18 fatty acids (oleic, linoleic, and linolenic acids, sodium salts) on ADP- and sodium-arachidonate-induced aggregation of washed rabbit platelets were investigated. When the platelets were suspended in protein-free medium containing dextran, it was found that these fatty acids at very low concentrations (2–45 μM) were potent inhibitors of platelet responsiveness and the inhibitory effect occurred within seconds. The inhibition of ADP-induced aggregation was not affected by abolishing the activity of platelet cyclooxygenase using aspirin. Human serum albumin relieved the inhibition caused by fatty acids for both ADP- and arachidonate-induced aggregation. The inhibitory effect of fatty acids does not seem to be due to decreased thromboxane formation (except possibly in the case of linolenate), and the relief of fatty acid inhibition by albumin does not potentiate thromboxane B2 formation from exogenous arachidonate. It is suggested that the inhibitory effect of polyunsaturated fatty acids on platelet aggregation is specific and not related to a general surfactant effect, since inhibition occurs far below the critical micelle concentration of fatty acid soaps.
A soluble biotinyl-α-amanitin affinity probe for the isolation of RNA polymerase B
Authors: Vaisius A. C. , Faulstich H.
Published: December 2009

Utilizing the 6′-hydroxyindole moiety of α-amanitin for substitution, biotinyl-α-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-α-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 × 10−8 M as compared with a Ki of 5 × 10−9 M for natural α-amanitin. RNA polymerase B complexed with biotinyl-α-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the amatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-α-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.
Na+ and K+ transport in a photodenitrifier, Rhodopseudomonas sphaeroides f.sp. denitrificans
Authors: Kundu Balaram , Nicholas D. J. D.
Published: December 2009

Washed cells of Rhodopseudomonas sphaeroides f.sp. denitrificans depleted of cellular K+ by treatment with diethanolamine contained less than 5 mM K+. The K+-depleted cells accumulated 22Na+ only when there was no diethanolamine in the external buffer (50 mM Tris–HCl, pH 7.5). Studies with 22Na+-loaded cells indicate that this photodenitrifier had antiporters for Na+–H+, K+–Na+, and K+–H+ and lacked a respiration (denitrification) dependent Na+ pump. The K+–Na+ antiporter was electrogenic and required proton-motive force for its operation. Thus the addition of either NaCl or KCl to K+-depleted cells resulted in a depolarization of Δψ, both in light and in the dark, by 10–35 mV, which was partially compensated for by a concomitant increase (5–13 mV) in ΔpH.