bluuebunny/crossref_metadata_2025 huggingface dataset:
Further characterization of isolated presumptive euchromatin fractions of mouse hepatoma cells utilizing a cDNA probe complementary to the homologous poly(A)+ mRNA
Authors: Duerksen Jacob D. , Chan Julia Y. , Robichaud Brenda
Published: December 2009

Poly(A)+ mRNA from mouse hepatoma ascites cell cytoplasm is characterized by three frequency classes: an abundant frequency class of a limited number of different nucleotide sequences, a less abundant frequency class of a larger number of different nucleotide sequences, and a rare frequency class containing a high number of different nucleotide sequences. [3H]cDNA synthesized on this poly(A)+ mRNA template hybridizes with some of the DNAs of the putative transcribable euchromatin fraction at a significantly faster rate than with total DNA if residual contaminating RNA is not removed. Following NaOH incubation to remove such RNA, the cDNA probe hybridized with essentially the same rate to the euchromatin fractions and total DNA. Nick translation of the nuclease-sensitive sequences of chromatin demonstrated that, even with limited nuclease digestion, the excised sequences rapidly converted to small oligonucleotides. The nick-translatable, small chromatin segments showed no enrichment for transcribable sequences. Chromatin segments, which distribute to the 50S–70S glycerol gradient fractions and which satisfy several of the presumptive criteria for enrichment for transcribable sequences, therefore show no enrichment for sequences complementary to the cDNA for poly(A)+ mRNA.
Stability of acetylcholinesterase in guanidine hydrochloride solution
Authors: Ahmad Faizan
Published: December 2009

The irreversible unfolding of acetylcholinesterase (acetylcholine hydrolyase, EC 3.1.1.7) by guanidine hydrochloride was studied by difference spectral, circular dichroic, and enzyme activity measurements. At pH 7.0 and in 1.1 M denaturant solution, a conformational state in which enzyme is completely inactive was detected. It is identical to the native enzyme as far as sedimentation coefficient and molecular weight are concerned, but differs from the native molecule by a slight loss in secondary structure and by a small perturbation of aromatic residues. Acetylcholinesterase in concentrated guanidine hydrochloride solution containing β-mercaptoethanol dissociates and exists as a random coil of molecular weight 68 000.
Brown adipose tissue mitochondria of cold-acclimated rats: change in characteristics of purine nucleotide control of the proton electrochemical gradient
Authors: Desautels Michel , Himms-Hagen Jean
Published: December 2009

Growth of brown adipose tissue in cold-acclimated rats, known to result in a large increase in capacity for noradrenaline-induced thermogenesis, is accompanied by changes in mitochondria (increase in concentration of binding sites for GDP with no change in affinity and increase in proportion of a 32 000 polypeptide in the inner membrane) which suggest an increased concentration of thermogenic proton conductance pathways. The objective of this study was to find out whether respiratory properties of brown adipose tissue mitochondria of cold-acclimated rats also change in a way which is in keeping with an increased thermogenic function in the cold-acclimated state. Mitochondria from both warm-acclimated and cold-acclimated rats were completely uncoupled when isolated and both albumin and guanosine diphosphate (GDP) were necessary for maintenance of a maximum proton electrochemical gradient. The maximum rate of loosely coupled respiration was, however, the same in the two types of mitochondria and appeared to be limited by the activity of substrate dehydrogenases rather than by the proton permeability of the mitochondria. The relation between proton electrochemical gradient and respiration was the same in the two types of mitochondria. However, mitochondria from cold-acclimated rats required three to four times more GDP to maintain a given proton electrochemical gradient, a difference due to altered sensitivity of the transmembrane pH gradient and not the membrane potential component. Because of this increased need for purine nucleotides, mitochondria of brown adipose tissue of the cold-acclimated rat in vivo may be in a more loosely coupled state and hence more susceptible to regulation by substrate availability.
Differential distribution of sphingomyelins among plasma lipoprotein classes
Authors: Myher J. J. , Kuksis A. , Breckenridge W. C. , Little J. A.
Published: December 2009

Individual and pooled samples of plasma from normolipemic and hyperlipemic subjects were separated into very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein fractions (HDL2 and HDL3) by conventional ultracentrifugation and total lipid extracts prepared by standard methods. The composition of the molecular species of the sphingomyelins in each lipoprotein class was determined by packed column and capillary gas–liquid chromatography of the trimethylsilyl (TMS) and t-butyldimethylsilyl (t-BDMS) ethers and by gas chromatography – mass spectrometry of t-BDMS ethers of ceramides derived by phospholipase C hydrolysis of the corresponding parent compounds. It was demonstrated that the molecular weight of the species of the sphingomyelins increases with the density of the lipoprotein fraction in normolipemic subjects, and that this increase is due to an increase in the chain length of the fatty acids in the ceramide molecules. In contrast, patients with type IV hyperlipoproteinemia possessed similar species in the LDL and HDL fractions, while maintaining normal differences between HDL and VLDL. Type III patients possessed normal HDL and VLDL differences, but had variable LDL. Type II patients had ceramide profiles for VLDL, LDL, and HDL fractions that were very similar to those of normals. The differential distribution of the molecular species of the sphingomyelins is rationalized on the basis of a lateral phase separation of the short and long chain sphingomyelins during the shedding of the excess VLDL or chylomicron surface material and a subsequent preferential transformation of the long chain species into HDL. The LDL sphingomyelins in type III hyperlipemia are variable and approximate either the VLDL or HDL composition.
Heterogeneity of VLDL triglyceride production by the liver and intestine
Authors: Steiner G. , Ilse W. K.
Published: December 2009

These studies examine the mechanisms responsible for the heterogeneous nature of very low density lipoprotein (VLDL) triglyceride production. The fasting dog has been used as a model to follow the incorporation of [2-3H]glycerol and [1-14C]palmitate into the triglyceride of VLDL isolated from plasma and from mesenteric lymph. The former represents mainly hepatic VLDL, and the latter, intestinal VLDL. VLDL from each source has been subfractionated into Sf 100–400, Sf 60–100, and Sf 20–60 components. The plateau in triglyceride specific activity achieved during constant infusions of the labelled precursors was higher in small VLDL than in large VLDL. This indicates that the small VLDL triglyceride is not derived exclusively from that in large VLDL. This applies to both hepatic and intestinal VLDL. This contrasts with apolipoprotein B in small VLDL, which other studies show to be entirely derived from large VLDL. Thus both the liver and the intestine incorporate triglyceride into particles whose density extends through the entire VLDL spectrum. Unless triglyceride-rich lipoproteins with the density of VLDL but with no apolipoprotein B are produced, these data raise the possibility that triglyceride may enter VLDL directly.
Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells
Authors: Stein O. , Coetzee G. A. , Stein Y.
Published: December 2009

The turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 μM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein–sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.
Alterations in membrane permeability with trypsin treatment
Authors: Johnson P. A. , Johnstone R. M.
Published: December 2009

Trypsin treatment of Ehrlich cells reduced sodium-coupled amino acid transport, lowered ATP levels, and abolished cation gradients. The data suggest that the decrease in active amino acid transport results from an alteration in membrane permeability caused by DNA released from a fraction of the cells upon trypsin treatment. The reduced amino acid transport appears to be the result of an abolition of ion gradients required for transport since vesicles prepared from these cells show near normal transport activity. The altered permeability of the membrane can be restored by incubation of the cells with serum. Protein synthesis does not appear to play a role in the restoration of Na+-dependent amino acid transport since restoration is unaffected by the presence of cycloheximide. The recovery of activity does depend partly on the presence of Ca2+ in the incubation medium since reversal of the trypsin inhibition of glycine transport can be obtained on incubation with Ca2+ and glucose.
Activation and inactivation of acetylcholinesterase by metal ions
Authors: Tomlinson George , Mutus Bulent , McLennan Ian
Published: December 2009

The kinetic consequences of acetylcholinesterase peripheral site occupation by metal ions were examined using three substrates; acetylthiocholine, p-nitrophenylacetate, and 7-(dimethylcarbamoyloxy)-N-methylquinolinium iodide. Two classes of metal ion effects were noted: activation by a group including Mg2+, Ca2+, Mn2+, and Na+, and inactivation by a second group which to date includes Zn2+, Cd2+, Hg2+, Ni2+, Cu2+, and Pb2+. Activation is demonstrable only in solutions of low ionic strength whereas inactivation can be readily observed in solutions of both low and high ionic strength. Activation appears to be due to a combination of metal ion binding and ionic strength effects and involves binding to peripheral sites which are distinct from those which bind organic cationic activators such as gallamine, propidium, and 7-(dimethylcarbamoyloxy)-N-methylquinolinium. The principal activating effect is on the deacylation phase of the enzyme–substrate reaction. Inactivators effect a slow conversion of the enzyme to an unreactive form. The kinetics of inactivation are biphasic at low ionic strength but become essentially monophasic at high ionic strength. More than 80% of the enzyme activity can be recovered upon addition of EDTA provided the chelating agent is added immediately following completion of the inactivation process. Prolonged exposure to inactivators results in a progressive decrease in the amount of recoverable activity. Although peripheral ligand interactions may result in a variety of catalytic site conformations, the macroscopic properties can be accounted for in terms of three ligand-dependent states of the enzyme in which catalytic ability (actual or potential) is retained, and a fourth denatured state.
Modulation of acylglycerol synthetase activities of rat intestinal mucosa by homogenization and sonication
Authors: Manganaro F. , Kuksis A.
Published: December 2009

Diacylglycerol and triacylglycerol synthesis was compared in intact villus cells and in their homogenates and sonicates using 2-monoacylglycerols and free fatty acids as substrates along with appropriate cofactors. It was found that simple pestle homogenization of cells led to an apparent loss of 80–90% in the triacylgiycerol and 40-fold increase in the diacylglycerol synthetase activity. Sonication of the homogenate resulted in reestablishing the normal activity of cellular triacylglycerol synthesis (12 ± 1 nmol∙mg protein−1∙h−1) but the diacylglycerol biosynthesis further increased (to a maximum of 70 ± 5 nmol∙mg protein−1∙h−1). Preincubation with trypsin and postincubation treatment with pancreatic lipase indicated that different enzymes are involved in diacylglycerol and triacylglycerol biosynthesis from 2-monoacylglycerols and that these enzymes become segregated during homogenization. It is concluded that a triacylglycerol synthetase system utilizing 2-monoacylglycerols can be reassembled upon sonication.
A new graphical rule for rate laws of enzyme reactions with branched pathways
Authors: Chou Kuo-Chen
Published: December 2009

A new graphical rule is presented for deriving the rate equations of enzyme reactions. The new rule has the following advantages: (i) it is very effective in simplifying the calculations for complex mechanisms, especially for those with branched pathways; (ii) a significant and intuitive correlation between the rule and the mechanism is established through the product-releasing cycle and product-combining cycle.
Analysis of chromosome movement in crane fly spermatocytes by ultraviolet microbeam irradiation of individual chromosomal spindle fibres. II. Action spectra for stopping chromosome movement and for blocking ciliary beating and myofibril contractions
Authors: Sillers Peggy J. , Forer Arthur
Published: December 2009

Chromosome-to-pole movement in crane fly spermatocytes was temporarily blocked by ultraviolet light focussed to a 4-μm-diameter spot on single chromosomal spindle fibres. Since similar irradiation of the interzonal region did not alter chromosome-to-pole movement, this effect was specific to spindle fibres. The action spectrum for blocking chromosome movement in this specific way had two peaks, one at 270 nm and one at 290 nm. To block movement, irradiations with 280-nm-wavelength light required two to four times more energy than irradiations with 270- or 290-nm-wavelength light.Action spectra were obtained for blocking ciliary beating and for blocking myofibril contraction. The action spectrum for blocking ciliary beating had a broad peak, between 260 nm and 280 nm, whilst that for blocking myofibril contraction had two peaks, at 270 and 290 nm, just like that for blocking chromosome movement. We discuss the similarities and differences in the various action spectra, and we compare the action spectra to absorption spectra of spindle components and to other action spectra (e.g., that for depolymerizing actin-containing filaments).Absorption spectra were obtained for ultraviolet light passing through spindle fibres as well as for ultraviolet light passing through the interzone.
Differential response of adenylate cyclase to β-adrenergic agonists in white adipocyte membranes from lean and obese (ob/ob) mice
Authors: Bégin-Heick Nicole
Published: December 2009

The quality of the β-adrenergic response, as measured by the activation of adenylate cyclase, was found to differ in adipocyte membranes from lean and obese mice. In the tissue from lean mice, the response was similar to that in rat adipose tissue and could, by analogy, be classified as β1-receptor response. In the tissue preparations from the obese mice, the rank order of potency of the three classical agonists (isoproterenol, epinephrine, and norepinephrine) was more typical of a β2-receptor response.
Quantitative analysis of purine and pyrimidine metabolism in Chinese hamster ovary cells
Authors: Hunting Darel , Hordern Joyce , Henderson J. Frank
Published: December 2009

A detailed quantitative study of purine and pyrimidine metabolism in logarithmically growing Chinese hamster ovary cells has been performed. Direct measurements of the ribonucleoside and deoxyribonucleoside triphosphate pool sizes have been made, and the pathways and rates of metabolism of adenine, hypoxanthine, guanine, guanosirae, deoxyadenosine, and deoxyguanosine and of uridine, cytidine, deoxyeytidine, deoxyuridine, and thymidine have been determined.
An investigation of the destruction of the β-lactam ring of penems by the albumin drug-binding site
Authors: Bruderlein H. , Daniel R. , Perras D. , DiPaola A. , Fidelia A. , Belleau B.
Published: December 2009

The plasma proteins have been reported to exert irreversible effects on some β-lactams. The penems represent a class of antibiotics of great interest due to their wide spectrum of activity. However, many compounds of this class were found to be unstable in rat serum. A prototype compound, (±)-2-methylpenem-3-carboxylic acid (BCL-98), was selected for detailed investigations. The capacity of penems to inhibit the destruction of a chromogenic β-lactam (BCL-604) by serum was the technique selected to study the interaction of BCL-98 with serum proteins at the specific destructive site. The serum of various species was investigated and the magnitude of BCL-604 destruction was in the following order: rat = human > dog > mice. The human plasma proteins were fractionated on a Sephadex column and the fractions tested individually. The albumin fraction was found to be entirely responsible for the destruction, the globulin fractions being completely inactive. The reaction of Ellman's reagent with serum was not diminished in the presence of the BCL-98, thus demonstrating that the sulfhydryl group of albumin is not involved in this binding. Displacement of a fluorescent probe (5-dimethylaminonaphthalene-1-sulfonamide) by BCL-98 showed that the binding site for the latter is the same as the one occupied by L-tryptophan and thus involves the ε-lysine amino group of the albumin binding site. The exact nature of this binding is not yet established, but it can be inferred that it may involve acylation of the ε-lysine group at the binding site. Some analogs of BCL-98 were also investigated. The basic functionality seems to protect the molecule from the destructive site of the albumin, probably because this substituent induces binding at another nondestructive site resulting in a prolonged half-life in the blood.
"Cap" sequences is poly(A)-rich RNA from dry wheat embryos
Authors: Lane Byron G.
Published: December 2009

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5′-terminai "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5′)ppp(5′)N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C), and uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5′)ppp(5′)A, with roughly equivalent amounts of m7G(5′)ppp(5′)G and m7G(5′)ppp(5′)C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; these differences have not been previously noted in the literature and are the subject of brief comment in this paper.
Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium
Authors: Al-Jobore Ameera , Roufogalis Basil D.
Published: December 2009

The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 μM) and high (65 μM) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidyiethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1–2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1–2 μmol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 from 0.5 to 6.5 mM. Ca2+ inhibition of the enzyme was also antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenyiphosphatase partial reaction.
Hepatic microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity in the lactating rat
Authors: Walker Brian L. , Hahn Peter
Published: December 2009

Liver microsomal 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase activity increases after parturition in the female rat. The elevated activity observed within 24- to 36-h postpartum was sustained throughout lactation but declined to control levels by day 30, the natural weaning age of the pups. Premature weaning, induced by removal of the offspring, returned HMGCoA reductase activity to normal within 2–4 days. The elevation in HMGCoA reductase was not due to elimination or inversion of the natural diurnal rhythm of this enzyme, which was similar in phase, but not in amplitude, in lactating and control animals. Elevated feed intake in the lactating animal may have contributed to the increase in HMGCoA reductase since pair feeding with a nonpregnant, nonlactating control rat abolished the elevation in enzyme activity observed during lactation. Elevated food intake did not induce the elevation in HMGCoA reductase in lactating rats, however, since the major change in food intake occurred after the initial increase in enzyme activity.
The use of diafiltration for the measurement of dialysable ligand binding to macromolecules: an examination of the theoretical parameters using concanavalin A in an Amicon cell
Authors: Marks J. E. , Isenman D. E. , Painter R. H.
Published: December 2009

The technique of diafiltration as a means of determining the characteristics of small ligand binding by proteins has been evaluated by measuring the binding of 45Ca to concanavalin A. Deposition of protein on the diafiltration membrane was found to be an important source of error but which could be eliminated by presaturation of the membrane with polylysine. The association constant for concanavalin A and calcium was 1.8 × 104 M−1, with a value of n = 2.2 atoms of calcium being bound per dimer of concanavalin A.The concentration of protein relative to the dissociation constant is also an important variable, and it was found that the method of diafiltration is only useful when protein concentrations greater than the value of the dissociation constant are used.
Changes in nucleotide pools during conidial germination in Neurospora crassa
Authors: Sabina Richard L. , Dalke Paulette , Hanks Alan R. , Magill Jane M. , Magill Clint W.
Published: December 2009

The acid-soluble nucleotide pools of wild type and several adenine auxotrophs of Neurospora crassa were studied immediately prior to and during conidial germination in the presence of adenine. A two- to four-fold increase in most nucleotide pools was observed after 6 h of germination at 33 °C indicating a general increase in nucleotide pools during this developmental period. The largest components of the acid-soluble nucleotide pools were uracil-containing nucleotide–sugars, which are precursors of chitin and glucan, the major constituents of cell walls. On removal of adenine, the UDP–sugar pools decreased significantly, in adenine auxotrophs, while the pools of UTP increased significantly. ATP levels increased approximately twofold in the first 6 h of germination. After 1 h without exogenous adenine, ATP dropped twofold or more in adenine auxotrophs but not in wild type. There was a net decrease in all adenine nucleotide pools during adenine starvation and a much greater decrease was seen in adenine auxotrophs than in wild type. The adenylate energy charge remained stable despite major changes in the adenylate pools.Accumulation of intermediates was observed in germinated conidia from purine auxotrophs blocked at various steps in the purine pathway. IMP accumulated in ungerminated and in starved conidia of the adenine-8 (ad-8) strain. Ungerminated conidia of the ad-5 strain contained a large pool of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) while the ad-1 strain had smaller amounts of AICAR, but significantly more than any other strain tested. The AICAR pools disappeared from ad-5 and ad-1 in the presence of 50 mg histidine/100 mL. Similarly the IMP pools in ad-8 decreased markedly in the presence of histidine, indicating that the contribution from the histidine biosynthetic pathway to purine nucleotide formation is significant.
Effects of cyclic nucleotides on hemoglobin synthesis in fetal calf liver cells in culture
Authors: Canas P. E. , Congote L. F.
Published: December 2009

Fetal calf liver cells were cultured in a serum-free medium in the presence of dibutyryl cAMP (dbcAMP), dibutyryl cGMP (dbcGMP), or cGMP. After a 20-h incubation with the nucleotides the synthesis of the fetal hemoglobins F0 and F1 was measured by the incorporation of [3H]leucine. The three cyclic nucleotides stimulated hemoglobin synthesis at a concentration of 1 μM. dbcGMP (10 nM) inhibited the synthesis of F0 and F1 but at a concentration of 1 μM significantly stimulated the synthesis of both hemoglobins. dbcAMP (10 μM) preferentially stimulated the synthesis of the transient hemoglobin F1 whereas cCMP (1 μM)stimulated specifically the synthesis of the definitive hemoglobin F0. Prostaglandin E1 (100 nM) stimulated the synthesis of F1 in a manner similar to that observed for dbcAMP. AMP, GMP, CMP, and sodium butyrate did not stimulate hemoglobin synthesis at the concentrations indicated above. The analysis of the α- and γ-globin chains by high pressure liquid chromatography indicated that the synthesis of both chains are stimulated by the three cyclic nucleotides. These changes in hemoglobin synthesis taking place at different concentrations of cyclic nucleotides may be of importance in the maturation of erythroid cells, because in these cells differentiation is normally coupled with cell proliferation. Furthermore, some of these cyclic nucleotides may be second messengers of erythropoietic hormones. We have found that the intracellular levels of cGMP were increased 15 min after addition of step III sheep plasma erythropoietin to the cultures, whereas the amounts of cAMP remained unchanged. It is then possible that erythropoietin itself or a factor present in the erythropoietin preparation may act by a cGMP-dependent mechanism.
The submicrosomal distribution of dolichol and dolichol phosphokinase activity in rat liver
Authors: Rip Jack W. , Carroll Kenneth K.
Published: December 2009

Microsomes were isolated from rat liver and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER) components, and the purity of these fractions was determined. The dolichol content of each of the three fractions was estimated, using high-pressure liquid chromatography. Although highest concentrations (1940 ng/mg protein) were found in Golgi, the RER contained the largest absolute amounts. The presence of large quantities of dolichol in RER is consistent with the role of dolichol as an intermediate in asparagine-linked glycoprotein synthesis. RER and SER fractions contained high specific activities for dolichol phosphokinase, while the activity in Golgi was quite low. High concentrations of dolichol in Golgi and high dolichol phosphokinase activity in SER suggest that dolichol (and dolichyl phosphate) may be utilized in Golgi for glycoprotein processing and in the transmembrane movement of sugars such as galactose.
Microcalorimetric study of the interaction of human and bovine serum albumins with tryptophan and tryptophan analogs
Authors: Phuong-Nguyen Hong , Bruderlein Hélène , Delmas Geneviève , Pépin Yvon
Published: December 2009

The thermodynamic parameters for the protein–ligand binding have been obtained by microcalorimetry on three albumin samples (fatty acid free human serum albumin (I), fraction V human serum albumin (II), and fraction V bovine serum albumin (III)) bound with L-tryptophan (A) and three-ring tryptophan analogs (B and B*). The percentage, u, of binding molecules (equivalent to the number of sites) is found to be 1.0 for I and 0.65 for II (in good agreement with dialysis results on the same systems) and 1.0 for III.The large negative ΔH(bind) (−27.2 to −33.2 kJ∙mol−1) constitutes the main contribution to ΔG(bind) (−23.0 to −31.2 kJ·mol−1). The better binding of I–III towards B and B* compared with A is due to ΔS(bind) being less negative. This is interpreted as a lesser loss of entropy for the three-ring ligands than for the normal tryptophan when they are bound.Data obtained on the proteins (heat of dilution; Huggins' constant, k′) correlate well with u or Qmax. This indicates that these physicochemical data could be used to characterize and compare rapidly some albumins of different sources. The unexpected finding that the parameters of I and III are nearer to each other than they are from II is discussed.
S-Adenosylhomocysteine hydrolase from rat liver
Authors: Briske-Anderson Mary , Duerre John A.
Published: December 2009

The kinetic constants for the reversible adenosylhomocysteine hydrolase from rat liver have been determined. The Km values of the enzyme for S-adenosylhomocysteine, adenosine, and L-homocysteine were 12.3 μM, 0.94 μM, and 164 μM respectively. Under the specified conditions the Vmax for the synthetic reaction was 6.2 μmol/min per milligram, while the Vmax for the hydrolytic reaction was 0.72 μmol/min per milligram. L-Homocysteine acted as a mixed-type inhibitor of the hydrolytic reaction. Adenine, a competitive inhibitor of both the synthetic and hydrolytic reactions (Ki = 1.2 ± 0.2 μM), was found to be a product of the reaction. In the absence of L-homocysteine about 0.8 nmol of adenosine was hydrolyzed to adenine and ribose per minute per milligram enzyme.
A Col E1 hybrid plasmid containing Escherichia coli genes complementing D-xylose negative mutants of Escherichia coli and Salmonella typhimurium
Authors: Maleszka Ryszard , Wang Patrick Y. , Schneider Henry
Published: December 2009

The Clarke and Carbon bank of Col E1 – Escherichia coli DNA hybrid plasmids was screened for complementation of D-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kb. By transformation or F′-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater man mat for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F′-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a D-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.
Mitochondrial genome diversity and the evolution of mitochondrial DNA
Authors: Gray Michael W.
Published: December 2009

Mitochondrial DNA (mtDNA) is functionally conservative, encoding basically the same genes in all eukaryotes, yet mitochondrial genome organization and expression show an amazing diversity, and mtDNAs are highly variable in size and in potential information content. This review focuses on certain novel features of mitochondrial genome diversity which have emerged from recent comparative studies of mtDNA and which indicate that, in the course of evolution, mtDNAs have undergone massive sequence rearrangements, have lost (and (or) acquired) large stretches of sequence between and within genes, and have sustained, in at least some cases, an unusually high degree of primary sequence divergence. It is suggested that much of the mitochondrial genome diversity that presently exists can be accounted for by a variable (and in some cases, quite rapid) rate of evolution of mtDNA. An endosymbiotic origin of mitochondria can be accommodated in this scenario if it is assumed that the "protomitochondrion," although eubacterial in essential elements of its translation system (e.g., ribosomal RNA), was unlike modern eubacteria in containing introns and extensive intergenic "spacer" sequences in its genome. Differing evolutionary pressures on the "free-living" and "intracellular" progenitors of eubacteria and mitochondria, respectively, could have resulted in the subsequent elimination of these "extra" sequences at different rates and to different extents, leading in the case of humans and other mammals to a mitochondrial genome having little or no noncoding sequences. Viewed from this perspective, most of the introns and intergenic sequences in some larger mtDNAs are considered to be retained primitive traits, vestiges of genome organization in the universal "progenote" from which a very early divergence of the eukaryotic nuclear, eubacterial, and archaebacterial lineages has been postulated. In all but the more conservative mitochondrial genomes, rapid primary sequence divergence will have tended to obscure the phylogenetic ancestry of most mtDNA-encoded macromolecules. This could explain why animal and fungal mitochondrial tRNAs, and plant mitochondrial 5S rRNA, are not typically eubacterial. A variable rate of mtDNA evolution is indicated by the fact that plant mitochondrial ribosomes are less "atypical" than the same components in other mitochondria, with plant (wheat) mitochondrial small-subunit rRNA showing much clearer indications of a eubacterial origin than its counterparts in animal (human, mouse) and fungal (yeast) mitochondrial ribosomes. Thus, the plant mitochondrial genome may be evolving considerably less rapidly than its counterparts in fungi and (especially) vertebrate animals. Our present state of knowledge about the structure, function, and evolution of mtDNA demonstrates both the power of, and essential need for, broadly based comparative studies in this field.
Age- and ploidy-related changes in rat liver nuclear proteins as assessed by one- and two-dimensional gel electrophoresis
Authors: Brasch Klaus
Published: December 2009

Hepatocyte nuclei from young (3–5 week), mature (8–12 week), and aged (over 32 weeks) rats were isolated and characterized by flow cytometry. Nuclei were bulk separated into diploid (2C), tetraploid (4C), and octoploid (8C) enriched fractions on sucrose gradients. Total, 0.35 M NaCl soluble, and residual proteins were prepared from all nuclear stages and examined by one- and two-dimensional gel electrophoresis. Within limits of sensitivity of these techniques, the following general features emerged. (a) A majority of proteins visualized were common to and present in similar relative quantities in nuclei from all age and ploidy groups. (b) A relatively higher proportion of nonhistone proteins (NHP) were saline-soluble in 2C nuclei from young rats than at any subsequent stage of development. (c) Several age-related and to a lesser extent ploidy-related fluctuations in pattern among the NHP were evident. These reflected primarily differences in solubility rather than major quantitative changes among individual proteins. (d) Exceptions to the foregoing included a group of high molecular weight components (> 100 000), a major and a minor component between 45 000 and 50 000, and a heterogeneous group of proteins in 2C nuclei from very young animals. There were no obvious differences among the histones, although these proteins were not examined in detail. The complex pattern of changes observed are discussed in terms of known aspects of hepatocyte differentiation and are related to possible changes in nucleoplasmic, nuclear matrix and Hn-RNP associated proteins.
The anaerobic sn-glycerol-3-phosphate dehydrogenase: cloning and expression of the glpA gene of Escherichia coli and identification of the glpA products
Authors: Schryvers Anthony , Weiner Joel H.
Published: December 2009

The expression of recombinant plasmids carrying the glpA gene (anaerobic glycerol-3-phosphate dehydrogenase) and the closely linked glpT gene (glycerol 3-phosphate transport) were studied under aerobic and anaerobic conditions. When the pattern of expression of enzymatic activity in different strains was compared with sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE) analysis from the same cells the glpA products were identified. Two polypeptides of 62 000 and 43 000 relative mass correlated with enzymatic activity.Five recombinant plasmids that contained one or both of the glpT or glpA genes were isolated and subjected to restriction endonuclease cleavage analysis. The regions of overlap from the inserts in these plasmids allowed definition of the regions of DNA containing the glpT and glpA genes. SDS–PAGE analysis revealed a partial product of the glpA locus from one plasmid, pLC42-17, which allowed more precise definition of the glpA locus on the physical DNA map and prediction of the direction of transcription.
Organization and expression of leghaemoglobin genes
Authors: Brisson N. , Pombo-Gentile A. , Verma D. P. S.
Published: December 2009

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin–cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all the species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.
Chromosomal protein poly(ADP-ribosyl)ation in pancreatic nucleosomes
Authors: Aubin R. J. , Dam V. T. , Miclette J. , Brousseau Y. , Poirier G. G.
Published: December 2009

When pancreatic chromatin fragments were prepared and resolved in the presence of 80 mM NaCl, endogenous poly(ADP-ribose) polymerase activity was found to be maximal in nucleosome periodicities of four to five units and did not respond to any further increases in nucleosomal architecture. Furthermore, in nucleosome complexities spanning 1 through 14 and over unit lengths, polyacrylamide gel electrophoresis on acid–urea and acid–urea–Triton gels has shown pancreatic histone H1 to be the only actively ADP-ribosylated histone species. The extent of ADP-ribosylation of histone H1 was also demonstrated to retard the protein's mobility in acid–urea, acid–urea–Triton, and lithium dodecyl sulfate polyacrylamide gels and to consist of at least 12 distinct ADP-ribosylated species extractable in all nucleosome complexities studied. Finally, extraction and subsequent electrophoresis of total chromosomal proteins in the presence of lithium dodecyl sulfate also evidenced heavy ADP-ribosylation at the level of nonhistone chromosomal proteins of the high mobility group comigrating in the core histone region, as well as in the topmost region of the gels where poly(ADP-ribose) polymerase was found to form a poly(ADP-ribosyl)ated aggregate.
Intracellular translocation of cellular and heat shock induced proteins upon heat shock in Drosophila Kc cells
Authors: Tanguay R. M. , Vincent M.
Published: December 2009

The intracellular distribution of cellular and heat shock induced proteins (hsp) was studied during a heat shock to Drosophila Kc cells. The different hsp's are unequally distributed between the intracellular organelles; while hsp 84 shows an exclusive cytoplasmic location, hsp 70 is equally distributed between the nucleus and the cytoplasm. The low molecular weight (MW) hsp's 22–26 are mainly concentrated in the nuclear fraction. Hsp 34 also shows a preferential nuclear localization but differs from the 22–26 hsp group with regard to its solubility in salt.During this heat shock (HS) treatment, a major preexisting microsomal polypeptide of relative mass (Mr) 45 000 with some properties similar to cytoskeletal proteins is translocated to the nuclear pellet. This translocation does not occur during arsenite treatment which also induces the synthesis of some hsp's. Furthermore, the hsp's synthesized in response to this arsenite treatment do not become associated with the nucleus as during the HS. Consequently the nuclear translocation of the 45 000 Mr protein does not seem to be involved in the induction of the high MW group of hsp's. Furthermore, it suggests that the hsp's which are found in the nuclear pellet upon HS do not have a nuclear localization per se. It rather reflects a temperature-dependent translocation of certain cellular proteins and hsp to the nucleus during a HS.
Structural studies on yeast nucleosomes
Authors: Lee Kay P. , Baxter Heather J. , Guillemette Joseph G. , Lawford Hugh G. , Lewis Peter N.
Published: December 2009

Mononucleosomes isolated from micrococcal nuclease digests of stationary phase chromatin of the yeast Saccharomyces cerevisiae were compared both compositionally and physiochemically with those from chicken and bovine calf. It was found that while yeast mononucleosomes are similar in composition, their thermal denaturation profiles and circular dichroism spectra indicate a less constrained structure. Furthermore, yeast nucleosomes were discovered to be labile in solutions of low ionic strength and could not be reconstituted by methods applicable to calf and chicken nucleosomes. On the basis of the reconstitution of a hybrid nucleosome containing calf histones H2A, H2B, and H3 and yeast histone H4, it was concluded that variations in the yeast H4 sequence are unlikely to be responsible for the apparent decrease in the stability of yeast nucleosomes. Examination of histone–histone interactions in free solution revealed a change in the H3–H4 interaction and together with the previously published results of other researchers it was inferred that changes in the H3 sequence might be responsible for this structural variation.
Synthesis and turnover of proteins and mRNA in germinating wheat embryos
Authors: Grzelczak Zbyszko F. , Sattolo Mark H. , Hanley-Bowdoin Linda K. , Kennedy Theresa D. , Lane Byron G.
Published: December 2009

The most prominent methionine-labeled protein made when cell-free systems are programmed with bulk mRNA from dry wheat embryos has been identified with what may be the most abundant protein in dry wheat embryos. The protein has been brought to purity and has a distinctive amino acid composition, Gly and Glx accounting for almost 40% of the total amino acids. Designated E because of its conspicuous association with early imbibition of dry wheat embryos, the protein and its mRNA are abundant during the "early" phase (0–1 h) of postimbibition development, and easily detected during "lag" phase (1–5 h), but they are almost totally degraded soon after entry into the "growth" phase of development, by about 10 h postimbibition.The most prominent methionine-labeled protein peculiar to the cell-free translational capacity of bulk mRNA from "growth" phase embryos is not detected as a product of in vivo synthesis. Its electrophoretic properties and its time course of emergence, after 5 h postimbibition development, suggest that this major product of cell-free synthesis may be an in vitro counterpart to a prominent methionine-labeled protein made only in vivo, by "growth" phase embryos. Designated G because of its conspicuous association with "growth" phase development, the cell-free product does not comigrate with any prominent dye-stained band in electrophoretic distributions of wheat proteins. The suspected cellular counterpart to G, also, does not comigrate with a prominent dye-stained wheat protein during electrophoresis, and although found in particulate as well as soluble fractions of wheat embryo homogenates it is not concentrated in either nuclei or mitochondria, as isolated.
Effects of membrane fluidity and identification of the rate-limiting step in the protein-mediated phosphatidylcholine exchange reaction
Authors: Bozzato Richard P. , Tinker David O.
Published: December 2009

The properties of the membrane interface play a very significant role in the regulation of the exchange reaction as catalyzed by the phosphatidylcholine (PC) exchange protein from beef liver. The reaction was studied using unilamellar vesicles of egg PC as donor membranes and multilamellar vesicles (MLV's) of dipalmitoyl PC (DPPC) or egg PC containing phosphatidylglycerol as acceptor membranes. MLV's of pure DPPC were found to be poor substrates in the exchange reaction, but could be improved significantly by the addition of DPPG. Maximum exchange occurred when 5 mol% DPPG was incorporated into the liposomes, the reaction being inhibited thereafter. Progress curves for the reaction were also shown to exhibit a biphasic characteristic, where an initial rapid period is followed by a much slower phase. It is postulated that this biphasic characteristic can be accounted for by the changing properties of the donor vesicles as they become coated with DPPC. When annealed and nonannealed MLV's of DPPC were used as acceptors, marked differences in the biphasic characteristic of the progress curves were observed. The half time of the initial rapid period of exchange using nonannealed vesicles was an order of magnitude lower than that for the annealed vesicles, indicating that the exchange protein selectively recognizes structural features present in the nonannealed vesicles. The effect of membrane fluidity was also investigated by the measurement of initial reaction velocities at 37 and 45 °C, below and above the phase transition temperature of DPPC. It was found that the desorption rate constant (k−A) for the DPPC acceptors, which represents the rate-limiting step in the exchange reaction, increases fivefold above the phase transition temperature of this lipid. It seems then that a major factor in determining the rate of the exchange reaction is the facility of extraction of a lipid from the membrane interface.