bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences
Authors: Fliegel Larry , Burns Kimberly , Wlasichuk Ken , Michalak Marek
Published: December 2009

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa)) appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.Key words: sarcoplasmic reticulum, endoplasmic reticulum, amino acid sequences, peripheral membrane proteins, KDEL retention sequence.
Lipid polymorphism and hydrocarbon order
Authors: Lafleur Michel , Bloom Myer , Cullis Pieter R.
Published: December 2009

The use of 2H nuclear magnetic resonance for the characterization of the polymorphic behavior of lipids is illustrated. Different lipid phase preferences may be expected to influence the orientational order and its variation along the acyl chains. Several results are presented to support that view. An increase of motional freedom and a redistribution of the order along the acyl chains are observed during the lamellar-to-hexagonal phase transition, showing that the order profile is sensitive to the lipid phase symmetry. In addition, if the preferences for nonlamellar phases are not expressed explicitly, the presence of "nonbilayer" lipids constrained in bilayer environment induces increased hydrocarbon order. This suggests that order parameters of the acyl chains and lipid polymorphic tendencies are intimately related.Key words: lipid, polymorphism, 2H nuclear magnetic resonance, hydrocarbon order.
Wheat germ agglutinin chromatography of GlcNacβ1-3(GlcNAcβl-6)Gal and GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactosaminoglycans
Authors: Seppo Antti , Penttilä Leena , Makkonen Anne , Leppänen Anne , Niemelä Ritva , Jäntti Jussi , Helin Jari , Renkonen Ossi
Published: December 2009

GlcNAcβ1-3(GlcNAcβ1-6)[14C(U)]Gal and GlcNAcβ1-3(GlcNAcβ1-6)[14C(U)]Galβ1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N- acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.Key words: GlcNAcβ1-3(GlcNAcβ1-6)Gal, GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc, wheat germ agglutinin – agarose chromatography, in vitro biosynthesis, teratocarcinoma cell.
Antibodies to liposomal phosphatidylcholine and phosphatidylsulfocholine
Authors: Wassef Nabila M. , Swartz Jr. Glenn M. , Alving Carl R. , Kates Morris
Published: December 2009

Antibodies against dimyristoyl phosphatidylsulfocholine or dimyristoyl phosphatidylcholine were raised in rabbits after injection of liposomes containing phosphatidylsulfocholine or phosphatidylcholine, cholesterol, and lipid A. The antibody activities were assayed by complement-dependent immune damage to liposomes and by a solid-phase, enzyme-linked immunosorbent assay using purified dimyristoyl phosphatidylcholine or dimyristoyl phosphatidylsulfocholine as antigen. Each antiserum raised against phosphatidylsulfocholine reacted with liposomes containing phosphatidylcholine, and each antiserum raised against phosphatidylcholine reacted with liposomes containing phosphatidylsulfocholine. However, adsorption of dimyristoyl phosphatidylsulfocholine antiserum with liposomes containing dimyristoyl phosphatidylcholine removed all activity against dimyristoyl phosphatidylcholine, but did not eliminate antibody activity against dimyristoyl phosphatidylsulfocholine. These results indicate that the antiserum against phosphatidylsulfocholine contained mixed populations of antibodies. Polyclonal antisera that have been appropriately adsorbed can therefore be obtained with a high degree of specificity for phosphatidylsulfocholine and such antisera can distinguish between phosphatidylsulfocholine and phosphatidylcholine.Key words: liposomes, antibodies, phosphatidylsulfocholine, phosphatidylcholine.
Thymidine incorporation by cultured chick pineal glands is not subject to adrenergic regulation
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: December 2009

Norepinephrine is known to play a role in regulating the circadian rhythms of serotonin N-acetyltransferase activity and melatonin formation in the chick pineal gland. We have recently demonstrated that the cultured chick pineal exhibits a circadian rhythm in the incorporation of thymidine. In this study we show that this latter rhythm is not subject to adrenergic control.Key words: chick, pineal gland, thymidine incorporation, adrenergic regulation.
Study of the function of Escherichia coli ribosomal RNA through site-directed mutagenesis
Authors: Leclerc Daniel , Brakier-Gingras Léa
Published: December 2009

Various approaches have been developed to study how mutations in Escherichia coli ribosomal RNA affect the function of the ribosome. Most of them are in vivo approaches, where mutations are introduced in a specialized plasmid harboring the ribosomal RNA genes. The mutated plasmids are then expressed in an appropriate host, where they can confer resistance to antibiotics whose target is the ribosome. Conditions can be used where the host ribosomal RNA genes or the host ribosomes are selectively inactivated, and the effect of the mutations on ribosome assembly and function can be studied. Another approach, which has been developed mainly with 16S ribosomal RNA, can be used entirely in vitro. In this approach, a plasmid has been constructed which contains the 16S ribosomal RNA gene under control of a T7 promoter. Mutations can be introduced in the 16S ribosomal RNA sequence and the mutated 16S ribosomal RNAs are produced by in vitro transcription. It is then possible to investigate how the mutations affect the assembly of the 16S ribosomal RNA into 30S subunits and the activity of the reconstituted 30S subunits in cell-free protein synthesis assays. Although these approaches are recent, they have already provided a large body of interesting information, relating specific RNA sequences to interactions with ribosomal proteins, to ribosome function, and to its response to antibiotics.Key words: ribosomal RNA, ribosome, site-directed mutagenesis, antibiotic resistance.
Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells
Authors: Hogue Douglas L. , Hodgson Kevin C. , Cass Carol E.
Published: December 2009

Membrane polypeptides (relative mass (Mr) 48 000 – 55 000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42 000 – 47 000) during sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS–polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41 000 – 48 000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by > 95% had little, if any, effect on either the affinity (Kd values, 0.1–0.2 nM) or abundance (Bmax values, 200 000 – 220 000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 °C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.Key words: nucleoside transport, L1210 cells, nitrobenzylthioinosine, glycoproteins, tunicamycin.
The effect of salinity on growth and lipid composition of a moderately halophilic Gram-negative bacterium HX
Authors: Adams Rachel L. , Kogut Margot , Russell Nicholas J.
Published: December 2009

The effect of NaCl concentration on the growth rate and phospholipid composition of a moderately halophilic Gram-negative rod-shaped bacterium has been studied. This bacterium, designated HX, grows between 0.5 and 4.25 M NaCl at 20, 30, and 37 °C. It has a broad optimum salt concentration for growth of 1.0–2.0 M NaCl at 20 and 30 °C and 1.5–2.8 M NaCl at 37 °C. Its major lipids are phosphatidylethanolamine and phosphatidylglycerol, which constitutes >90% of the total lipid, together with smaller amounts of diphosphatidylglycerol; there are traces of an unidentified glycolipid, lysophosphatidylethanolamine, and phosphatidylserine. Lipid composition depends on NaCl concentration of the culture medium: at higher salinities the proportion of anionic phospholipids, mainly phosphatidylglycerol, is greater relative to the proportion of zwitterionic phospholipids, mainly phosphatidylethanolamine. The major fatty acids are 16:0, 16:1, 18:0, 18:1, 17:0cyc, and 19:0cyc; the proportions of the cyclopropane fatty acids are greater in cultures grown at higher salinities and this increase is due to NaCl concentration, not growth rate. Following a sudden increase in NaCl concentration of the culture medium (shift up), there are concomitant changes in growth rate and phospholipid composition. A lag period in growth is observed only when the magnitude of the salinity shift up is at least threefold. After a salinity shift up, there is a rapid decrease in the ratio of phosphatidylethanolamine to phosphatidylglycerol. It appears that the alterations in growth rate and lipid composition are a physiological response for adaptation to a change in salinity.Key words: halophilic bacteria, haloadaptation, membranes, lipids, salinity.
Intra- and inter-molecular hydrogen bonding in diphytanylglycerol phospholipids: an infrared spectroscopic investigation
Authors: Stewart L. C. , Kates M. , Yang P. W. , Mantsch H. H.
Published: December 2009

A series of diphytanylglycerol phospholipids, i.e., diphytanylglycerol phosphate (PA), diphytanylglycerol phosphoglycerophosphate (PGP), the tri- and tetra-methyl derivatives of PGP, and the 2-deoxyglycerol analogue of PGP (dPGP) were studied by Fourier transform infrared spectroscopy. The use of the "deoxy" and methylated analogues of PGP, as well as that of PA and PGP of varying degrees of ionization, allowed the assignment of characteristic infrared bands associated with the phosphate groups. Analysis of these phosphate bands showed that at neutral pH, each of the two phosphate moieties in PGP is singly ionized, whereas in dPGP the phosphomonoester is doubly ionized. This is a consequence of the marked increase in the pK of one of the P-OH groups on the terminal phosphate of PGP (pK > 11), owing to the formation of an intramolecular hydrogen bond between the head group glycerol hydroxyl and the phosphate groups of PGP. Such an intramolecular hydrogen bond can not be formed by the dPGP analogue, and thus both negative charges in dPGP are located at the terminal phosphomonoester group. The O=P—OH groups of PGP also forms a network of intermolecular hydrogen bonds, the exact nature of which depends on concentration and degree of ionization. The possibility of a complex network of hydrogen bonds within (intramolecular) and between (intermolecular) anionic membrane lipids such as that found in PGP, is consistent with the hypothesis that these lipids function as proton-conducting pathways in membranes.Key words: phospholipids, infrared, hydrogen bonding, phosphatidylglycerophosphate, 2-deoxyphosphatidylglycerophosphate.
Ovarian uptake of vitellogenin and another very high density lipoprotein in winter flounder (Pseudopleuronectes americanus) and their relationship with yolk proteins
Authors: Nagler James J. , Idler David R.
Published: December 2009

Ultracentrifugation analysis of female winter flounder plasma confirmed that in addition to vitellogenin another different lipoprotein is found as very high density lipoprotein (VHDL). Accordingly this protein (formerly known as peak A protein) has been named VHDL II, with respect to vitellogenin which we designate VHDL I. Both these proteins were shown to be taken up in vivo by the ovary of vitellogenic females. Based on total mass, vitellogenin and VHDL II could potentially contribute in similar amounts to yolk protein accumulated by developing oocytes. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of vitellogenic oocyte yolk proteins revealed five major protein subunits of 101.4, 94.4, 68.7, 25.5, and 22.5 kilodaltons (kDa). Western blots of these yolk protein subunits established three of them (104.4, 94.4, and 22.5 kDa) as originating from vitellogenin. Similar Western blots utilizing a VHDL II antisera identified the 68.7-kDa yolk protein subunit as arising from incorporated serum VHDL II.Key words: VHDL II, vitellogenin, oocyte yolk proteins, in vivo uptake, Western blot.
A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1
Authors: Kamekura Masahiro , Seno Yukio
Published: December 2009

An unidentified halophilic archaebacterium strain 172 P1 produced three extracellular proteases in media containing 15–27% salts. One component, F-II, was purified to homogeneity. It is a serine protease that can be inhibited by phenylmethylsulfonyl fluoride and chymostatin. A high concentration of NaCl was required for its stability; in the presence of 25% NaCl, only 4% of the activity was lost by incubating at 60 °C for 30 min, while complete inactivation occurred in the presence of 5% NaCl. F-II is a thermophilic and halophilic protease. High activity was obtained at 75–80 °C when F-II was assayed in the presence of 25% NaCl. The optimal concentration of NaCl required was 10–14% when assayed at 70 °C with azocasein as substrate, though a halophilic characteristic was not distinct at lower temperatures. Hydrolyses of the synthetic substrates succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-methylcoumaryl-7-amide or succinyl-alanyl-alanyl-alanyl-p-nitroanilide at 26 °C were maximal at 25 and 30% NaCl, respectively. F-II was most stable at pH 6–7, and its optimal pH was 10.7. Its molecular weight was estimated as 44 000 – 46 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and by gel filtration – high-pressure liquid chromatography. The sequence of the 35 N-terminal amino acid residues was determined and compared with that of other serine proteases.Key words: halophilic, archaebacteria, serine protease, thermophilic.
Formation of acylphosphatidylglycerol by a lysosomal phosphatidylcholine: bis(monoacylglycero)phosphate acyl transferase
Authors: Huterer Srebrenka J. , Wherrett John R.
Published: December 2009

A delipidated soluble fraction prepared from a mitochondrial–lysosomal fraction of rabbit alveolar macrophages that catalyzes transacylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate was also found to transfer oleic acid from [14C]dioleoyl phosphatidylcholine to form acylphosphatidylglycerol. The reaction was dependent on the presence of bis(monoacylglycero)phosphate and was maximal at a concentration of 44 μM when the ratio of fatty acid transferred to fatty acid released was 0.28. Addition of phosphatidylglycerol had only a small effect. Homogenates of rat liver also catalyzed the reaction and after subcellular fractionation the activity was localized to lysosomes. The lysosomal activity was solubilized by delipidation with butanol to give a preparation with a specific activity 2462 times that of the homogenate. Optimal activity of soluble preparations from both macrophages and liver was at pH 4.5, with little activity above 6.0. Release of free fatty acid was also stimulated under conditions of optimal acyl transfer. Both acyl transfer and release of fatty acid were inhibited by Ca2+, detergents, chlorpromazine, lysophosphatidylcholine, and oleic acid. Where there was disproportional inhibition, acyl transfer was always more affected. These results suggest that sequential acylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate and then acylphosphatidylglycerol constitute a mechanism in the lysosome for the transport and partition of fatty acids released by the lysosomal phospholipases.Key words: acylphosphatidylglycerol, bis(monoacylglycero)phosphate, transacylase, lysosomes, macrophages.
Studies on the activity of phospholipase D with alkyl phosphorylcholines
Authors: Ohno Yoshimi , Okazaki Mitsuyo , Hara Ichiro
Published: December 2009

The activity of phospholipase D from the Savoy cabbage on alkyl phosphorylcholines containing alkyl groups of C14 to C22 was examined. Apparent Km and maximal velocity values in the presence of diethyl ether were 3.6–8.3 mM and 0.14–0.29 μmol/(min∙mg protein), respectively. The maximal velocities for alkyl phosphorylcholine homologues were lower than the value for pig phosphatidylcholines, but higher than those for lysophosphatidylcholines and synthetic phosphatidylcholine with saturated acyl chains. Results suggest that solubilities of the substrates in the organic solvent play an important role in activity observed with phospholipase D.Key words: phospholipase D, alkylphosphorylcholines, apparent Km values, Vmax values, diethyl ether.
Regulation of proliferation by the budding yeast Saccharomyces cerevisiae
Authors: Johnston Gerald C. , Singer Richard A.
Published: December 2009

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.Key words: Saccharomyces cerevisiae, cell cycle, growth, cAMP, stationary phase.
Kinetics of reconstitution of apotyrosinase by copper
Authors: Wigfield Donald C. , Goltz Douglas M.
Published: December 2009

The kinetics of the reconstitution reaction of apotyrosinase with copper (II) ions are reported. The reaction is pseudo first order with respect to apoenzyme and the values of these pseudo first order rate constants are reported as a function of copper (II) concentration. Two copper ions bind to apoenzyme, and if the second one is rate limiting, the kinetically relevant copper concentration is the copper originally added minus the amount used in binding the first copper ion to enzyme. This modified copper concentration is linearly related to the magnitude of the pseudo first order rate constant, up to a copper concentration of 1.25 × 10−4 M (10-fold excess), giving a second order rate constant of 7.67 × 102 ± 0.93 × 102 M−1∙s−1.Key words: apotyrosinase, copper, tyrosinase.
Intestinal absorption of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat
Authors: Yang L.-Y. , Kuksis A. , Myher J. J.
Published: December 2009

The relative cellular uptake and incorporation into prechylomicrons and chylomicrons was investigated for the menhaden and rapeseed oil fatty acids, when given by stomach tube as the original oils or the corresponding methyl and ethyl esters. The intermediates and final products of cellular acylation were determined by chromatographic methods at various times over a period of 1–24 h. There was little selectivity in the uptake among the oligo- and poly-unsaturated fatty acids of menhaden oil, when either oil or esters were fed. In contrast, the long-chain saturated and monounsaturated fatty acids of rapeseed oil were discriminated against during both cellular uptake and reacylation (60% overall reduction in utilization). Also, there was detectable discrimination against the long-chain polyunsaturated monoacylglycerols of menhaden oil and against the long-chain saturated and monounsatured monoacylglycerols of rapeseed oil during both cellular uptake and reacylation (30% overall reduction in utilization). Evidence was obtained for an indiscriminate cellular uptake of variable amounts (4–22%) of intact dietary methyl and ethyl esters of fatty acids, which, however, appeared in the chylomicrons only to a very limited extent (0.1–1.0% of total lipid). During peak absorption the cellular and lymphatic appearance of fatty acids from the digestion and absorption of the alkyl esters was nearly 50% lower than that from the corresponding triacylglycerols. The slower absorption of the fatty acids from the alkyl ester feeding is hypothetically attributed to a lower efficiency of the phosphatidic acid pathway, which is required in the absence of dietary 2-monoacylglycerols, but other mechanisms cannot be excluded.Key words: villus cells, prechylomicrons, chylomicrons, triacylglycerol biosynthesis, monoacylglycerol pathway, phosphatidic acid pathway.
Levels of sulfogalactosylglycerolipid in capacitated motile and immotile mouse spermatozoa
Authors: Tanphaichitr Nongnuj , Smith Jacqueline , Kates Morris
Published: December 2009

The purpose of this study was to determine whether sulfogalactosylglycerolipid (SGG) was desulfated during mouse sperm capacitation. Levels of [35S]SGG were determined in freshly retrieved caudal epididymal sperm, motile capacitated sperm, and immotile sperm, after feeding mature male mice with [35S]sulfate-laced chow for 32 days. Caudal epididymal sperm and coisolated epididymal cells were separated into pellet and interphase fractions by centrifugation through a two-step Percoll gradient (45 and 90%). Upon resuspension in Krebs–Ringer bicarbonate medium supplemented with 0.4% bovine serum albumin, the Percoll-gradient pellet fraction consisted mainly of motile capacitated sperm, whereas the interphase fraction comprised largely immotile sperm and fragmented epididymal epithelial cells. The level of [35S]SGG in the Percoll-gradient-pelleted sperm appeared to be much higher than that in the Percoll-gradient interphase sperm. Percoll-gradient-pelleted sperm were further incubated in the culture medium for 2 h. The level of [35S]SGG showed little or no change after 1 h, but was reduced appreciably after 2 h. At this time point, sperm motility was also decreased. Reduction of sperm SGG is correlated with sperm immotility and (or) senescence and may have no direct relation to the capacitation process.Key words: sulfogalactosylglycerolipid, sperm motility, sperm capacitation.
Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium
Authors: Jois Markandeya , Hall Beatrice , Collett Vaughn M. , Brosnan John T.
Published: December 2009

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10−7 M), forskolin (10−4 M), and dibutyryl cAMP (10−4 M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 ± 1.1 and 8.5 ± 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by gluagon.Key words: amino acid, metabolism, liver, mitochondria, hormones.
Effect of thiocarbamate, urea, and uracil herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves
Authors: Rajasekharan R. , Sastry P. S.
Published: December 2009

The effect of thiocarbamates (S-ethyldipropylthiocarbamate and diallate), substituted ureas (monuron and diuron), and uracils (bromacil and terbacil) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. The uptake of [1-14C]acetate by leaf disks was inhibited by the thiocarbamates and marginally by the substituted ureas, but not by the uracil herbicides. The uptake of [methyl-14C]choline was inhibited to a lesser extent by thiocarbamates, while the other herbicides showed a slight stimulation. The thiocarbamates almost completely inhibited uptake of [32P]orthophosphate at 1.0 mM concentration, while diuron and terbacil showed significant inhibition. [1-14C]Acetate incorporation into lipids was inhibited only by diallate. [methyl-14C]Choline incorporation into the choline phosphoglycerides was inhibited by diallate, diuron, and bromacil. The incorporation of [32P]orthophosphate into phospholipids was substantially inhibited (over 90% at 1.0 mM) by the thiocarbamates, but not by the other herbicides. [35S]Sulfate incorporation into sulfoquinovosyl diglycerides was markedly inhibited only by the thiocarbamates. Fatty acid synthesis by isolated chloroplasts was inhibited 40–85% by thiocarbamates, substituted ureas, and bromacil, but not by terbacil. The inhibitory effect of the urea derivatives was reversible, but that of thiocarbamates was irreversible. sn-Glycerol-3-phosphate acyltransferase(s) of the chloroplast and microsomal fractions were profoundly inhibited by thiocarbamates, but not by the other two groups of herbicides. Phosphatidic acid phosphatase was insensitive to all the herbicides tested.Key words: herbicides, thiocarbamates, substituted ureas, uracils, effects on plant lipids.
Effect of elevated temperatures on heat shock protein and ribulose-1,5-bisphosphate carboxylase gene expression in Brassica napus
Authors: Halle J. R. , Ghosh S. , Dumbroff E. B. , Heikkila J. J.
Published: December 2009

Leaf segments of Brassica napus were exposed to 22, 35, 38, or 40 °C for up to 4 h. Analysis of radiolabeled proteins by two-dimensional sodium dodecyl sulfate – polyacrylamide gel electrophoresis and fluorography revealed two major groups of heat shock proteins (HSPs). One group comprised HSPs 70, 76, and 87, with (pIs) isoelectric points ranging from 5.7 to 6.1, whereas the second group had molecular masses ranging from 23 to 16 kilodaltons (kDa) and pIs from 5.6 to 6.9. Immunoblot analysis using antibodies directed against the large (RLSU) and small (RSSU) subunits of ribulose-1,5-bisphosphate carboxylase (RUBISCO) showed that increasing temperatures from 35 to 38° or 40 °C for the duration of thermal stress (i.e., from 1 to 5 h) did not affect levels of the RSSU (15 kDa), whereas levels of the RLSU (52 kDa) fell sharply. Nevertheless, the activity of RUBISCO was not adversely affected at 38 °C for periods of up to 5 h. Northern blot analysis revealed that the increase observed in HSP 70 synthesis during heat shock may be transcriptionally regulated, but the decrease in the RLSU was not accompanied by a corresponding reduction in levels of its mRNA.Key words: Brassica, heat shock, ribulose-1,5-bisphosphate carboxylase, gene expression.
Isolation and characterization of a developmentally regulated homeobox sequence in the Mexican axolotl Ambystoma mexicanum
Authors: Whiteley Mary , Armstrong John B.
Published: December 2009

A homeobox-containing genomic DNA fragment was isolated from the Mexican axolotl. This clone was obtained from a partial genomic library enriched for sequences that cross-hybridized with the Drosophila Antp homeobox under low stringency hybridization conditions. DNA sequence analysis revealed that this sequence (Ahox1) was 66% homologous to the Antp homeobox sequence and was most closely related to the mouse Hox-1.6 (84% identity) and Drosophila lab (79% identity) homeobox sequences. Several cross-hybridizing fragments to Ahox1 were detected in both mouse and axolotl genomic DNA. This sequence was also shown to be conserved in other Ambystoma species. Northern blot analysis revealed that genes containing this sequence are developmentally regulated. Transcripts hybridizing to the Ahox1 homeobox probe were detected during the neurula and tail bud stages of development.Key words: axolotl, homeobox, mouse, Drosophila, gene expression.
Intracellular proteolytic processing of proopiomelanocortin in heterologous COS-1 cells by the yeast KEX2 endoprotease
Authors: Zollinger L. , Racine C. , Crine P. , Boileau G. , Germain D. , Thomas D. Y. , Gossard F.
Published: December 2009

We have transiently expressed the yeast KEX2 gene together with the proopiomelanocortin (POMC) cDNA in COS-1 cells. Characterization of the POMC-related immunoreactive peptides by gel permeation and reversed-phase high pressure liquid chromatography showed that the KEX2 enzyme was active and capable of carrying out cleavage of POMC to release the authentic maturation product β-endorphin(1–31). Peptides resembling β-lipotropin, the amino terminal glycopeptide, and ACTH(1–39) were also detected as major products in the cell extracts. Our results indicate that the KEX2 enzyme can proteolytically release from POMC a set of peptides similar to that normally found in interior pituitary.Key words: prohormone proteolytic processing, KEX2 expression, COS-1 cells.
A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk
Authors: Kubohara Yuzuru , Okamoto Koji
Published: December 2009

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5–6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.Key words: Dictyostelium discoideum, Dictyostelium mucoroides, wheat germ agglutinin.
The effect of methyl lidocaine on lysophospholipid metabolism in hamster heart
Authors: Tardi Paul G. , Man Ricky Y. K. , McMaster Christopher R. , Choy Patrick C.
Published: December 2009

An important feature in the remodelling of fatty acyl chains in cellular phospholipids is the acylation of lysophospholipids. Since lysophospholipids are cytolytic al high concern rat ions, the acylation reaction may provide an alternate pathway for the removal of cellular lysophospholipids. However, the physiological role of the acylation process in the maintenance of lysophospholipid levels in mammalian tissues has not been clearly defined. In this study, methyl lidocaine was found to inhibit both lysophosphatidylcholine:acyl-CoA and lysophosphatidylethanolamine:acyl-CoA acyltransferase activities in the hamster heart, but the drug had no effect on the other lysophospholipid metabolic enzymes. When the heart was perfused with 0.5 mg methyl lidocaine/mL, acyltransferase activities were attenuated, but there was no change in the activities of phospholipase A or lysophospholipase. The levels of the major lysophospholipids in the heart were not altered by methyl lidocaine perfusion. When the hearts were perfused with labelled lysophospholipid in the presence of methyl lidocaine, there was a reduction in the formation of the phospholipid and an increase in the release of the free fatty acid. However, the labelling of lysophospholipid in the heart was not altered by methyl lidocaine. We postulate that the acylation reaction has no direct contribution to the maintenance of the lysophospholipid levels in the heart.Key words: lysophosphatidylcholine, lysophosphatidylethanolamine, acyltransferase, methyl lidocaine, hamster heart.
S-Adenosylmethionine and S-adenosylhomocysteine transitions in encysting Physarum flavicomum amoebae
Authors: Cumaraswamy Arunthathi , Henney Jr. Henry
Published: December 2009

Previous studies from this laboratory suggested that in Physarum flavicomum an S-adenosylmethionine (SAM) related metabolic flux is involved in the developmental control of microcyst formation. In the present study, this phenomenon was further analyzed by comparing the metabolic effects of compounds found to favor, stimulate, or inhibit the normal encystment process. The compounds utilized were adenine, cycloleucine, L-ethionine, DL-selenomethionine, cadaverine, cyclohexylammonium sulfate, papaverine, mycophenolic acid, decoyinine, putrescine, spermidine, and spermine. Following incubation with the compounds or combinations of the compounds, the intracellular levels of SAM and SAM metabolites were determined using high performance liquid chromatography. The actual level of the intracellular SAM pool varied considerably (notably after 15 h of incubation), depending on the compound(s) in the incubation solution, but was always highest during inhibition of encystment. SAM was undetectable or barely detectable during stimulation of encystment or under conditions favoring encystment. In cells exposed to L-ethionine, we detected intracellular S-adenosylethionine rather than SAM. The intracellular methionine pool in cells encysting normally for 15 h was found to be 1.3 μmol/pg DNA. Both SAM and S-adenosylhomocysteine (SAH) were present in growing amoebae at concentrations greater than that of cells undergoing normal encystment. The developmental transition from growing amoebae to dormant cysts in P. flavicomum is metabolically characterized by the adjustment of the intracellular concentration of SAM and SAH to a minimal critical level of 0.31 and 0.70 fmol/pg DNA, respectively.Key words: S-adenosylmethionine, S-adenosylhomocysteine, encystment, Physarum, amoebae.
Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype
Authors: Leitch Robert A. , Richards James C.
Published: December 2009

The specific capsular polysaccharide produced by Rhodococcus equi serotype 1 was found to be a high molecular weight acidic polymer composed of D-glucose, D-mannose, and D-glucuronic acid. Structural analysis of the polysaccharide employed a combination of chemical and nuclear magnetic resonance techniques, from which it was determined that the polysaccharide possessed a linear repeating tetrasaccharide unit containing a single O-acetyl substituent and an acetal-linked pyruvic acid moiety:[Formula: see text]The 1H and 13C nuclear magnetic resonances of O-deacetylated and pyruvic-free serotype 1 polysaccharides were fully assigned by homo- and hetero-nuclear chemical shift correlation methods.Key words: capsular polysaccharide, structural analysis, Rhodococcus equi.
Structural analysis of the lipopolysaccharide of Actinobacillus (Haemophilus) pleuropneumoniae serotype 10
Authors: Perry Malcolm B.
Published: December 2009

The antigenic O-chain of the lipopolysaccharide produced by Actinobacillus (Haemophilus) pleuropneumoniae serotype 10 was shown by chemical and magnetic resonance methods to be a unique linear unbranched homopolymer of exclusively 1,2-linked β-D-galactofuranose residues.Key words: Actinobacillus, pleuropneumoniae, lipopolysaccharide, magnetic resonance, galactan.
ΔLys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12
Authors: Laughrea Michael , Higgins Eric
Published: December 2009

Five-residue-long deletions centered on A1a63, A1a75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118–Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12ΔLys120. L7/L12ΔLys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28–35% ethanol in 0.25–0.5 M NH4Cl. It follows that ΔLys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.Key words: ribosome, ribosomal protein L7/L12, site-directed mutagenesis.
Subcellular distribution of ribosomal proteins in Dictyostelium discoideum
Authors: Ramagopal S.
Published: December 2009

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cystosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two- to four-fold lower than that of cytoplasmic ribosomes.Key words: cellular slime mold, cell-specific ribosomal proteins, nucleus, cytoplasm, two-dimensional gel electrophoresis.
Variations in c-fos mRNA expression during serum induction and the synchronous cell cycle
Authors: Bird R. Curtis , Kung Tzyy-Yun Tzeng , Wu Gin , Young-White Randy R.
Published: December 2009

Induction of c-fos mRNA levels associated with the stimulation of growth by fetal bovine serum following quiescence was examined in three cell types following brief (24 h) serum starvation. Starved NIH-3T3 and HeLa S3 cells experienced c-fos mRNA induction 20–30 min after addition of serum. In contrast, Swiss-3T3 cells expressed c-fos constitutively following serum starvation. The pattern of oncogene expression coincided with the level of quiescence of each cell line prior to induction. Serum induction of c-fos expression was dependent upon the response of each cell line to serum starvation, c-fos expression was also examined in HeLa S3 cells that had been separated into sequential cell cycle phases by centrifugal elutriation. c-fos expression peaked during the earliest part of the synchronous G1 phase. The amount of c-fos mRNA measured was approximately twice that found during other cell cycle phases. This suggests that, in addition to its role during the transition from quiescence, the c-fos gene product may play a regulatory role during the earliest part of G1 phase of the continuous cell cycle.Key words: c-fos, cell cycle, mRNA, 3T3, HeLa.
Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue
Authors: Parkes Joel G. , Angel Aubie
Published: December 2009

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose–response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.Key words: cholesteryl ester transport, high density lipoprotein receptor, cholesterol storage.
Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein
Authors: Sikorska Marianna , Brewer Linda M. , Youdale Tony , Richards Robert , Whitfield James F. , Houghten Richard A. , Walker P. Roy
Published: December 2009

Epitope-specific antibodies to the M1 and M2 subunits of mammalian ribonucleotide reductase were prepared using peptides predicted to have a high antigenic index. Western blotting demonstrated that the anti-M1 antibody was specific for the 89-kilodalton M1 subunit (and its degradation fragments) and the anti-M2 antibody specifically recognized the 45-kilodalton M2 subunit. Both antibodies inhibited the CDP-reductase activity of the holoenzyme. Using these antibodies, both the M1 and M2 subunits were shown to be localized in the cytoplasm and in the nuclear regions of a number of cell types, including B77 avian sarcoma virus transformed NRK cells, T51B rat liver cells, 5123tc hepatoma cells, and rat liver cells in vivo. In addition, the M1 subunit was found to be localized as a halo around isolated rat liver nuclei. Biochemical analysis of the cytoplasmic fraction of liver cells and a Triton X-100 wash of nuclei from these cells confirmed the location of the enzyme activity in these cellular compartments. The M1 subunit appears to be glycosylated, as indicated by its retention on a Affi-Gel – concanavalin A affinity column. Therefore, in mammalian cells ribonucleotide reductase appears to be not only in the cytoplasm, but is also associated with the nuclear membrane or nuclear lamina. The activity of the enzyme in the membrane fraction changes dynamically during the cell cycle.Key words: replication, DNA, synthesis, glycosylation, liver.
Studies of NADP+-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii
Authors: Al-Kassim Loola S. , Tsai C. Stan
Published: December 2009

Alcohol dehydrogenase has been purified from the cell-free preparation of Thermoanaerobium brockii to homogeneity, employing combined DEAE, Sephadex, and affinity chromatographic procedures. The enzyme is tetrameric having subunit molecular weight of 40.4 × 103. The purified alcohol dehydrogenase is capable of utilizing either NAD+ or NADP+ to oxidize primary and secondary alcohols, although it prefers NADP+ as the coenzyme and secondary alcohols as substrates. Inactivation of the enzymic activity by sensitized photooxidation and carboxymethylation implicates the presence of catalytically important histidine and cysteine residues. Kinetic studies indicate that Thermoanaerobium alcohol dehydrogenase catalyzes NADP+-linked oxidations of secondary alcohols by an ordered bi-bi mechanism with NADP+ as the leading reactant. The preference of the Thermoanaerobium enzyme for NADP+ is correlated with its low dissociation constants (KA and KiA) and high turnover rate (V/Et). The corresponding kinetic parameters also contribute to the preference of this enzyme for secondary alcohols.Key words: NADP+-preferred secondary alcohol dehydrogenase.