bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the F1-ATPase in mitochondria of Vigna unguiculata (L.)Walp. cv. Pitiuba by K+
Authors: Pinheiro Moema Rodrigues , Lima Maria Silva , de Melo Dirce Fernandes , Peter Horst W.
Published: December 2009

Mitochondria from the bean Vigna unguiculata contain an ATPase system that is strongly activated by K+ and Na+ in the presence of low concentrations of substrate. Optimum activation by physiological concentrations of K+ is achieved in the presence of 0.1 mM MgATP. At the substrate optimum of the total ATPase, which is 2.3 mM, the enzyme is only poorly activated by K+. The K+-activated component of the total F1-ATPase behaves like an individual enzyme with kinetic properties different to the total F1-ATPase. On the basis of enzyme kinetic measurements, we determined the mechanism of activation by K+ as follows:[Formula: see text]K+ is not required for the binding of the substrate MgATP to the enzyme but for the splitting of the enzyme–substrate complex into the reaction products.
The effect of methylation of phosphatidylethanolamine on the behaviour of lipid monolayers at the air–water interface
Authors: Keough K. M. W. , Hawco M. W. , Parsons C. S.
Published: December 2009

Monolayers of DPPE and its N-methylated derivatives including DPPC have been investigated at 23 and 37 °C using a modified Langmuir–Wilhelmy surface balance. The monolayers have been subjected to dynamic compression and expansion, and some characteristics of the surfaces have been determined. The minimum surface tension attained by surfaces containing the lipids (maximum surface pressures sustained by the films) depended on the extent of methylation of the head group. Monolayers of DPPE or N-MeDPPE collapsed at surface tensions of 12–16 mN∙m−1, whereas those containing N,N-diMeDPPE and DPPC could be compressed to near zero surface tension. The areas per molecule occupied by these lipids under high compression varied slightly and not systematically with head-group methylation. Monolayers containing mixtures of DPPC and DPPE were also studied under the same conditions. The monolayers showed some deviation from the behaviour expected if they were to have characteristics of ideally mixed systems. The minimum surface tensions attained suggested that monolayers containing 50 mol% or more DPPC might be further enriched during compression by some selective exclusion of the DPPE. At high surface pressures, some positive deviations in nominal areas per molecule from that expected for ideal mixing were observed in the monolayers made with 50 mol% or more DPPC. These deviations might be caused by packing disruptions associated with the explosion of lipid from the films.
Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung
Authors: Mok Amy , Wong Tanya , Filgueiras Octavio , Casola Paul G. , Nicholson Don W. , McMurray William C. , Harding Paul G. , Possmayer Fred
Published: December 2009

CDPdiacylglycerol pyrophosphatase (E. C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6–8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 μM and Vmax values of 311 and 197 pmol∙min−1∙mg protein−1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.
Purification et quelques propriétés de l'AMPc phosphodiestérase d'Arthrobacter crystallopoietes
Authors: Schneider François , Lefebvre Gérard , Ribolzi-Chery Michèle , Bertin Jean-Michel , Gay Robert
Published: December 2009

cAMP phosphodiesterase was purified 8250-fold from extracts of Arthrobacter crystallopoietes, primarily by hydrophobic chromatography. The molecular weight of this enzyme was estimated as 51 000 by gel filtration and density-gradient centrifugation. The results suggest that the enzyme consists of two subunits with a molecular weight of 25 600. Properties of this enzyme are reported, including its negative cooperativity. This phosphodiesterase specifically catalyzes the hydrolysis of 3′,5′-cyclic nucleotides. Divalent ions either have no effect on activity or are weak inhibitors. Photooxidation of the enzyme with methylene blue and treatment with mercuribenzoates suggest that this enzyme may possess an imidazole group within its active site. The effects of thiols and Fe2+ on activity suggests that this enzyme may be a metalloenzyme.
Bacterial and mammalian thioredoxin systems activate iodothyronine 5′-deiodination
Authors: Das Arun K. , Hummel Brian C. W. , Gleason Florence K. , Holmgren Arne , Walfish Paul G.
Published: December 2009

The identity of a dithiol (designated DFB) of relative mass (Mr) = 13 000, reported previously to be present infraction B of rat liver cytosol and to participate as a cofactor in the 5′-deiodination of iodothyronines, has been investigated. Substitution of highly purified thioredoxin from Escherichia coli for fraction B or of highly purified thioredoxin reductase from either E. coli or rat liver for cytosolic fraction A (containing DFB reductase) permits deiodination of 3,3′,5′-[l25I]triiodothyronine by rat liver microsomes to proceed. Addition of antibodies to highly purified rat-liver thioredoxin or thioredoxin reductase inhibits deiodination. Thus, the thioredoxin system largely accounts for the activity of the cytosolic cofactor system supporting 5′-deiodination of 3,3′,5′-triiodothyronine in rat liver.
Control models of cell cycle transit, exit, and arrest
Authors: Skehan Philip
Published: December 2009

Several distinct cycles mediate the events which occur between one cell division and the next. In micro-organisms there are generally two cycles. One governs biomass growth, the other DNA synthesis and cell division. In higher eukaryotes there can be as many as four distinct cycles, with growth, DNA synthesis, cell division, and nuclear division each possessing its own functional sequence of events. These cycles are controlled and coordinated by several different regulatory mechanisms. Restriction points are specific steps in the cycle whose completion is governed by external regulatory agents. One set of restriction points requires nutrients and growth hormones for step completion. Another set serves as receptors for differentiating factors which cause cycle arrest and initiate cellular differentiation. There is currently a debate as to whether restriction point inhibition involves permanent arrest or temporary arrest with a stochastic arrested-state residence time controlled by a transition probability mechanism. Tissue sizing is a process of negative feedback inhibition mediated by intercellular communication via cell surface contact and the extracellular matrix. Sizers commonly operate throughout broad portions of the cycle and appear to cause a slowing of cycle transit velocity rather than arrest. Sizers are probably the major regulatory mechanism for cell growth under conditions of nutrient and growth factor excess. They also generate compensatory proliferation following wounding or cell death. A growing body of evidence suggests that both the transit velocity, with which cells move through their several cycles, and the coordination of the cycles are controlled by intracellular regulatory mechanisms which behave as biological oscillators. These oscillators trigger complex sequences of events such as DNA synthesis and cell division.
Freezing tolerance in plants: a biochemical overview
Authors: Singh J. , Laroche A.
Published: December 2009

Tolerance to extracellular freezing is an inducible and hereditable trait in many plants. Cellular membranes have been accepted as the sites of extracellular freezing injury, although not all cellular membranes exhibit similar degrees of sensitivity to freezing. Plasma membrane function and photosynthetic activity are among the first cellular activities to be affected after a freeze–thaw cycle. The structural nature of irreversible membrane alterations leading to cell injury during lethal freezing are well understood. However, although numerous biochemical changes have been observed during cold hardening, the exact changes that enable membranes of hardened plant cells to withstand the dehydrating and mechanical stresses of extracellular freezing have not been unequivocally elucidated. Similarly, the biochemical events subsequent to initial exposure of the plant cell to environmental or chemical triggers required for the induction of freezing tolerance are not known. The role of protein synthesis and altered gene expression during the induction of freeing tolerance is discussed.
Characterization and partial purification of rat submandibular gland protein kinase C
Authors: Pon D. J. , Lintlop S. P. , Sen A. K.
Published: December 2009

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 μM and 5 μg, respectively. Addition of 10−6 M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 μM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.
Luminescence-specific synthesis of myristic acid in the bioluminescent bacterium Vibrio harveyi
Authors: Byers David M.
Published: December 2009

The effect of bioluminescence development on the metabolism of fatty acids, in particular myristic acid (14:0), which is required to form the aldehyde substrate of luciferase, has been investigated in the luminescent marine bacterium Vibrio harveyi. The induction of luminescence in late exponential growth was not accompanied by substantial changes in the fatty acid composition. However, pulse-label studies revealed a small but significant increase in the ratio of labeled 14:0 to 16:0 (palmitic acid) when induced cells were incubated with [1-14C]acetate in growth medium. Evidence consistent with a decrease in the specific radioactivity of the acetate precursor in actively luminescing cells was also obtained. No free 14C-labeled fatty acid or aldehyde was detected under normal growth conditions. In contrast, unesterified [14C]14:0 (up to 15% of the total lipid label) transiently accumulated when wild type cells were labeled with [1-14C]acetate in a nutrient-free medium. No preferential synthesis of 14:0 or free fatty acid was detected under any conditions in preinduced cells or in a dark mutant of V. harveyi (mutant M17), which is defective in a luminescence-related acyltransferase and is thus incapable of providing fatty acid for light emission. Combined with previous evidence indicating that this acyltransferase can catalyze the deacylation of fatty acyl-acyl carrier protein (acyl-ACP) in vitro, the present results support a model in which 14:0 is diverted from phospholipid synthesis to the luminescence system at the level of 14:0-ACP. Under normal growth conditions, the requirement of 14:0-ACP via this pathway does not appear to substantially alter the overall fatty acid composition during the development of bioluminescence.
Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells
Authors: Hernanz A. , Fuente M. de la
Published: December 2009

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.
Transcriptional and translational events during reovirus infection
Authors: Lemay Guy
Published: December 2009

This short review focuses on the mechanisms involved in transcription and translation in mouse L cells infected with reoviruses. The viral genomic RNA (double-stranded), retained in the inner capsid following removal of the outer capsid of the infecting virion, is transcribed by a viral polymerase. The synthesized viral mRNA is blocked at the 5′ end by a cap structure similar to the cap structure of cellular mRNA but synthesized by the viral enzymes of the inner capsid. This viral mRNA is also used as the first strand and template for the synthesis of the second strand of viral genomic RNA; the newly replicated genome is retained in an inner capsid structure to generate the progeny subviral particles. These particles are active at the transcriptional level but do not synthesize the cap, owing to the absence of the guanylyltransferase activity involved in the formation of this structure. The uncapped mRNA, or late viral mRNA, constitutes the bulk part of viral mRNA. The transcription of the viral genome is finally arrested upon addition of outer capsid proteins to obtain a mature virion. During viral multiplication, there is a gradual inhibition of host-cell protein synthesis, concomitant with stimulation of late viral mRNA translation. The two phenomena are apparently distinct. Furthermore, the inhibition of host-cell protein synthesis has been shown to be dispensable for normal virus multiplication; however, it might accelerate it. The mechanisms responsible for inhibition are still unclear but might involve modifications in the activity of cellular cap-binding proteins. This last point suggests an analogy with poliovirus infection; the two systems are thus briefly compared. Possible significance of the absence of a poly(A) tract at the 3′ end of reovirus mRNA, in contrast to the occurrence of such a sequence at the end of cellular mRNA, is also examined. Different models involving cap discrimination, competition between mRNAs, or alteration of cell membrane permeability have been proposed to explain the events observed at the translational level in reovirus-infected cells. These different models are compared. Finally, recent data implicating the viral sigma 3 capsid protein in efficient translation of late viral mRNA are discussed.
Purification of benzylamine oxidase from cultured porcine aortic smooth muscle cells
Authors: Hysmith Robert M. , Boor Paul J.
Published: December 2009

Soluble benzylamine oxidase (BzAO) from cell homogenates and the conditioned culture medium of porcine aortic smooth muscle cells was purified by anionic HPLC methods and characterized with regard to enzyme kinetics and inhibition by semicarbazide, phenelzine, cuprizone, diethyldithiocarbamate (DDC), and p-chloromercuriphenylsuphonate (PCMPS). BzAO from both the cell homogenates and the conditioned culture medium had an Mr of 130 000, as determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Using [methylene-14C]benzylamine hydrochloride as substrate, BzAO from cell homogenates and the conditioned culture medium had Km values of 5.1 and 6.1 μM, respectfully, and Vmax values of 89 and 53 nmol∙mg protein−1∙h−1. Both enzymes were sensitive to inhibition by semicarbazide and phenelzine, but insensitive to inhibition by the copper chelating agent DDC. BzAO isolated from the conditioned culture medium was more sensitive to inhibition by lower concentrations of cuprizone and PCMPS than the enzyme isolated from cell homogenates. Antisera raised against BzAO from cell homogenates reacted with BzAO from the conditioned culture medium and from porcine plasma.
Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments
Authors: Darasch S. , Mosser D. D. , Bols N. C. , Heikkila J. J.
Published: December 2009

Continuous exposure of a Xenopus laevis kidney epithelial cell line, A6, to either heat shock (33 °C) or sodium arsenite (50 μM) resulted in transient but markedly different temporal patterns of heat-shock protein (HSP) synthesis and HSP 70 and 30 mRNA accumulation. Heat-shock-induced synthesis of HSPs was detectable within 1 h and reached maximum levels by 2–3 h. While sodium arsenite induced the synthesis of some HSPs within 1 h, maximal HSP synthesis did not occur until 12 h. The pattern of HSP 70 and 30 mRNA accumulation was similar to the response observed at the protein level. During recovery from heat shock, a coordinate decline in HSPs and HSP 70 and 30 mRNA was observed. During recovery from sodium arsenite, a similar phenomenon occurred during the initial stages. However, after 6 h of recovery, HSP 70 mRNA levels persisted in contrast to the declining HSP 30 mRNA levels. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of 5 HSPs in the HSP 70 family, of which two were constitutive, and 16 different stress-inducible proteins in the HSP 30 family. In conclusion, heat shock and sodium arsenite induce a similar set of HSPs but maximum synthesis of the HSP is temporally separated by 12–24 h.
Active-site-directed inactivation of Schizophyllum commune cellulase by 4′, 5′-epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside
Authors: Clarke Anthony John
Published: December 2009

4′,5′-Epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside (4) was synthesized by a Koenigs–Knorr reaction of 4-penten-1-ol and acetobromcellobiose, promoted by silver trifluoromethanesulfonate and N,N′-tetramethylurea, and tested as a potential active-site-directed irreversible inhibitor of the Schizophyllum commune cellulase. Incubation of the S. commune cellulase with 4 resulted in a time-dependent irreversible inactivation of the enzyme. The inactivation process obeyed pseudo-first-order kinetics and the hyperbolic plot of kobs as a function of inhibitor concentration provided values for Kd and k2 of 150 mM and 2.0 × 10−4 s−1, respectively, at pH 5.5 and 25 °C. The binding of a competitive inhibitor, cellobiose, to the cellulase prior to incubation with 4 protected the enzyme from rapid inactivation, suggesting that the inactivation is due to attack at the active site. The dependence of the inactivation on pH is consistent with the participation of carboxyl groups. Treatment of the affinity-labeled enzyme with [14C]methoxyamine resulted in the near stoichiometric formation of a stable radiolabelled adduct, suggesting that one inhibitor molecule binds per enzyme active site of the enzyme.
Developmentally regulated slow troponin C messenger RNA in chicken skeletal and cardiac muscles
Authors: Berezowsky C. , Bag J.
Published: December 2009

Regulation of slow troponin C gene expression was examined in both skeletal and cardiac muscle at various stages of development in chicken. The steady-state levels of troponin C mRNA were initially measured by Northern blot analysis. It was observed that the level of troponin C mRNA reached its maximum in both skeletal and cardiac muscle of 16- to 18-day-old embryos. A drop in troponin C mRNA level was observed just prior to hatching. The level of actin mRNA, myosin heavy chain mRNA, and mRNA for a nonmuscle protein, vimentin, was also similarly regulated during development of chicken muscles. Further studies were carried out to determine the level of slow troponin C mRNA using nuclease S1 protection analysis. A significant amount of slow troponin C mRNA was found in the skeletal muscle of adult chicken, which predominantly consists of the fast isoform of troponin C. This observation suggests the possibility of post-transcriptional control of slow troponin C synthesis in skeletal muscle. Primary cultures of cardiac myocytes were also used to determine how the troponin C mRNA level is regulated in a culture of cardiac muscle cells. Measurements of the steady-state levels of slow troponin C mRNA by nuclease S1 protection analysis show that it was maximal in 60-h-old cultures. A drop in the level of this mRNA was observed after these cells were maintained in culture for 4 days.
Appearance of prostaglandins, thromboxane B2, and hepoxilin A3 in the circulation of the normal and diabetic (BB) rat after arachidonic acid administration — correlation with plasma insulin
Authors: Pace-Asciak C. R. , Martin J. M. , Lee S.-P.
Published: December 2009

The temporal in vivo expression of the eicosanoids (products of the cyclooxygenase pathway and one product of the 12-lipoxygenase pathway, hepoxilin A3) was investigated after bolus intravenous injection of arachidonic acid in the normal rat and in the genetic rat model of type I insulin-dependent diabetes, the diabetic BB rat. The temporal relationship between the expression of these products and plasma insulin concentrations was also investigated to determine whether any correlation existed between the rise in plasma insulin levels and any of the newly formed eicosanoids. Measurements of the eicosanoids present in whole blood were carried out using the deuterium isotope dilution technique involving separation of pentafluorobenzyl esters, O-methyl oximes, and trimethylsilyl ether derivatives by high-resolution gas chromatography and specific detection by negative ion chemical ionisation mass spectrometry in the selected ion mode. Injection of arachidonic acid resulted in large and statistically significant increases in the blood concentrations of all products within 1 min, with thromboxane B2 (the stable product of thromboxane A2) and trioxilin A3 (the stable product of hepoxilin A3) being the highest (4.5–12 ng/mL). The mean concentrations of thromboxane B2 and trioxilin A3 in blood appeared greater in the diabetic BB rat than in the normal rat, while the opposite was found for 6-keto PGF1α (the stable product of prostacyclin). The apparent greater ratio of thromboxane B2 to 6-keto PGF1α in the diabetic BB rat than in the normal rat supports a prothrombotic nature of platelets associated with diabetes.
Membrane D2 T antigen: characterization and comparison with its nuclear counterpart
Authors: Ismail Alnashir A. , Hand Roger
Published: December 2009

Up to 40% of membrane T antigen (D2 T antigen) produced by the adenovirus 2–simian virus 40 hybrid, Ad2+D2, remained tightly associated with membranes under alkaline conditions up to pH 11.5. The antigen could not be totally solubilized by treatment with ionic detergents, nonionic detergents, or both. These properties are characteristic of integral membrane proteins. Sephacryl S-300 chromatography in high salt in the presence of Brij-99 showed that the nuclear form of D2 T antigen was dissociated to low molecular weight species, while the membrane form eluted as a complex of high molecular weight. The membrane form, therefore, is able to bind more detergent than the nuclear form, indicating a difference in supramolecular structure.
Unusual nucleoprotein structures in the vicinity of the replication origins of the extrachromosomal rDNA of the slime mould Physarum polycephalum
Authors: Pauli Urs , Kuenzler Peter , Braun Richard
Published: December 2009

The nucleoprotein structure in the vicinity of the four replication origins of the extrachromosomal rDNA of the slime mould Physarum polycephalum was examined. At limited extents of digestion with micrococcal nuclease, a series of cleavage sites were found in the proximity of the orgins of replication. The distance between the sites reflected an altered nucleosomal organization of rDNA chromatin. It could be demonstrated that the cleavage sites were not the result of sequence-specific cutting of micrococcal nuclease. Such a different organization of rDNA chromatin was supported by recent findings that regions close to the origins of replication were inaccessible to cross-linking with psoralen. In addition, the same area contained sites that were protected from restriction endonuclease digestion (i.e., possible anchoring sites for the rDNA to the nucleolar matrix). Therefore, our results suggest that the nucleoprotein complexes presented in this paper are involved in anchoring and (or) in the replication of the rDNA.
Synthesis of heparan sulfate proteoglycans by the isolated glomerulus
Authors: Templeton Douglas M. , Khatchatourian Marina
Published: December 2009

Incorporation of [35S]sulfate into newly synthesized macromolecules was studied in the isolated rat glomerulus and found to be linear between 6 and 24 h. When whole glomeruli were treated under conditions that dissociate proteoglycan aggregates, >90% of incorporated label was extracted. Of this, 80–90% was found to be the heparan sulfate proteoglycan. Similarly, a linear incorporation of [35S]sulfate into a glomerular basement membrane-enriched fraction was due almost entirely to proteoheparan sulfate. This predominance of heparan sulfate among the newly sulfated glycosaminoglycans has previously been observed in vivo and in the perfused kidney, but different patterns have hitherto been described in vitro. The present results suggest that under certain conditions, the isolated glomerulus is a suitable in vitro model for the study of proteoglycan synthesis. The pattern of incorporation of proteoglycans into the glomerular basement membrane reflects the time course and distribution of their synthesis by the whole glomerulus.
Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum
Authors: Siu Chi-Hung , Wong Lu Min , Lam Tak Yee , Kamboj Rajender K. , Choi Anthony , Cho Aesim
Published: December 2009

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in respose to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell–cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr 80 000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell–cell adhesion. One of them has a Mr of 150 000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell–cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.
Nuclear magnetic resonance in the investigation of cerebral tumors and cerebral edema: a clue to the cellular alterations that may affect the distribution of water
Authors: Megyesi Joseph F. , Maestro Rolando F. Del
Published: December 2009

The understanding of tumor-associated cerebral edema involves an elucidation of the mechanisms involved in the altered distribution of water in the vicinity of cells. Changes in cellular macromolecules such as intracellular proteins, extracellular matrix components, and cell-membrane proteins may alter the water interactions in and around cells. The technique of pulsed nuclear magnetic resonance (NMR) gives a measure of the relaxation properties of protons in water molecules in such systems. The T1 and T2 relaxation times are increased in cerebral tumors and peritumoral tissue compared with normal brain. The in vitro study of cerebral tumors requires a tumor model that possesses the properties of the actual tumor under study. The C6 astrocytoma cell line has many of the properties of glioblastoma multiforme. An NMR study of C6 astrocytoma cells grown in monolayer, as spheroids of varying sizes and when implanted into rat hosts, has been undertaken. Results show that T1 and T2 relaxation times are not a static feature of the tumor cells but may reflect changing microenvironments that result from the contribution of a number of interacting factors present in the growing tumor.
Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies
Authors: Burdett Elena , Klip Amira
Published: December 2009

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55 000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze–thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters. It is concluded that the antibody may be reacting with exofacial regions of the glucose transporter in intact cells. This suggests that the antibodies may be used to detect relative changes in glucose transporter number on the cell surface.
A comparison of acetylation in vitro of microsomal, homogenate, and Golgi fractions of rat liver
Authors: Sambasivam Hari , Murray Robert K.
Published: December 2009

The activity of acetyltransferase was detected in the microsomal fraction of rat liver by incubation with [3H]acetyl-CoA and by analyses using sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Endogenous membrane proteins of relatively high molecular weight were found to serve as substrates. Optimal conditions for assay of the enzyme were defined. A deacetylase activity was also detected, which was inhibited by 2 mM ethylenediaminetetraacetic acid. Further subfractionation disclosed that the acetyltransferase activity was most enriched in the Golgi fraction, in which its specific activity was some ninefold greater than in the total homogenate. The radioactive labelling of Golgi-associated proteins observed was relatively intense, exceeding that of histone and ribosomal proteins in the homogenate. Analysis of the acetylated Golgi fraction by two-dimensional electrophoresis revealed approximately 90 radioactive polypeptides. Various treatments demonstrated that a minimum of 80% of the incorporated radioactivity was present as derivatives of N-acetylneuraminic acid, principally N-acetyl-9-mono-O-acetylneuraminic acid (Neu5,9Ac2). The sialic acid O-acetyltransferase activity detected is thus probably identical to that reported by Varki and Diaz; the intense labelling of proteins reflects the ability of Golgi apparatus fractions to take up and concentrate acetyl-CoA. Protein-bound radioactive Neu5,9Ac2 was also detected in the medium of hepatocytes incubated with N-[3H]acetylmannosamine, demonstrating that these cells synthesize certain proteins containing acetylated sialic acids, some of which may be secreted. The data confirm that the Golgi apparatus is a major site of acetylation of protein-bound sialic acids in rat liver in vitro and provide new information showing that many glycoproteins undergo this particular type of modification.
Composition of phospholipid molecular species from mammary tumors
Authors: Ishinaga Masataka , Carroll Kenneth K.
Published: December 2009

The molecular phospholipid species of mammary tumors induced by 7,12-dimethylbenz[a]anthracene in rats that were fed diets containing 20 or 3% sunflower-seed oil and different levels of calcium were analyzed by high-pressure liquid chromatography. Twenty-seven molecular species of phospholipids were identified. Phosphatidylcholine was predominantly composed of palmitoyl-arachidonoyl (16:0-20:4) (17–21%), palmitoyl-oleoyl (16:0-18:1) (19–21%), stearoyl-arachidonoyl (18:0-20:4) (12–13%), and 1,2-dipalmitoyl (16:0-16:0) (10–14%) species. The major molecular species of phosphatidylethanolamine were 18:0-20:4 (37–39%) and 16:0-20:4 (10–11%). The composition of diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species from rat mammary tumors was not greatly affected by the different diets.
Effect of microenvironment and cell-line type on carbohydrate-binding proteins of macrophage-like cells
Authors: Gabius Hans-Joachim , Vehmeyer Katalin
Published: December 2009

The pattern of sugar inhibition of rosette formation, a model for intercellular interaction between cultured cells and glutaraldehyde-fixed, trypsinated rabbit erythrocytes, served to infer the presence of carbohydrate-binding proteins. This profile from cell extracts for the two murine macrophage-like cell lines, P388D1 and J774A.1, was comparatively analyzed by affinity chromatography on supports with immobilized carbohydrates (lactose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and maltose) or with the immobilized mannose-rich yeast glycoprotein mannan or fetuin-derived glycopeptides containing sialic acid residues. After elution with specific sugar in the absence of Ca2+ ions, the proteins were separated by sodium dodecyl sulfate – polyacrylamide slab gel electrophoresis. The composition of carbohydrate-binding proteins of the two lines clearly exhibited quantitative and qualitative differences. Moreover, the pattern of P388D1 cells was also demonstrated to change significantly in response to alterations in the conditions of the physiological environment. These alterations were imposed by in vitro growth, by subsequent in vivo growth in nude mice, and by re-adaptation of cells to culture after in vivo passage. Collectively, our observations and other physiological and biochemical reports on macrophage lectins indicate that the presence of sugar receptors with different specificities may be an indicator of macrophage differentiation, being reversibly modulated to a considerable extent by external factors, e.g., microenvironment. Extensive but selective alterations in this respect could play an important role in the control of recognition and effector mechanisms within diverse functions of macrophage subpopulations.
Effects of subunit V antibodies on the topology of the subunit and the activity of beef heart cytochrome-c oxidase
Authors: Freedman Jo A. , Leece Bryan , Cooper Christopher E. , Nicholls Peter , Chan Samuel H. P.
Published: December 2009

Redox-sensitive epitopes on subunit V of beef heart cytochrome-c oxidase were demonstrated previously using polyclonal subunit-specific antibodies raised in rabbits. The antibodies only slightly inhibited electron transfer, and the accessibility of their epitopes depended on the presence of a membrane and on the redox state of the oxidase. The present paper describes additional preparations of antibodies raised against subunit V. These antibodies have an even higher subunit specificity, they are more than three times as inhibitory against electron transfer, and their binding does not require a membrane. Moreover, the redox-sensitive nature of their binding to detergent-dispersed oxidase is sensitive to the method of its isolation. We discuss inferences that can be drawn from a detailed quantitative comparison of the interactions of the two antibody preparations with the antigen in different environments. The techniques used in the comparison can be used to examine other perturbants of the oxidase as to their effects on specific segments of the enzyme.
Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei
Authors: Carruthers Vern B. , Clarke Michael W.
Published: December 2009

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.
A new bilirubin-degrading enzyme from orange peels
Authors: Wu Tai-Wing , Li Grace S.
Published: December 2009

A novel enzyme activity that catalyzes the degradation of unconjugated bilirubin (Bu) has been demonstrated in extracts of the peels of edible oranges. Unlike the few known bilirubin-oxidizing enzymes, the orange enzyme does not produce biliverdin as a product, does not seem to require oxygen, and has a unique absorption spectrum of its products. Even at the crude stage, the enzyme has a specific activity that is 10 and 20 times higher, respectively, than those reported for the crude or partially purified Bu-degrading enzymes from mushrooms and rat liver. The enzyme has apH optimum near 7.5 and a Km value of 50–100 μM for Bu. The enzyme is remarkably stable, retaining over 50% activity after prolonged digestion with proteinase K or heating at 100 °C. Similar treatments inactivated the bilirubin oxidase from Myrothecium verrucaria MT-1. The enzyme is poorly soluble in water but can be partially solubilized with cholic acid, with a doubling in specific activity.
Minor lamin polypeptides from rat liver nuclei can be cross-linked into heteropolymers by disulfide bridges
Authors: Raymond Yves , Chauvette Michel
Published: December 2009

The peripheral lamina of rat liver nuclei is characterized by the presence of three major polypeptides called lamins A, B, and C. Recent studies have identified in rat liver lamina two quantitatively minor polypeptides that have some of the biochemical and immunological properties of the lamins and were tentatively called minor lamin species. We have further characterized these minor lamin polypeptides. Both minor lamin species copurified quantitatively with the major lamins in dissociation–reassociation experiments and shared epitopes with all three major lamins as well as with intermediate filament proteins, including an epitope involved in coiled-coil interactions in lamina and filaments. Minor lamins generated partial peptide maps very similar to each other but completely different from those of lamins A, B, and C. The two minor lamin species could be cross-linked into heteropolymers containing a constant ratio of both polypeptides by exposure to O-phenanthroline – cupric ion complexes, although they did not appear to be cross-linked by disulfide bonds in the native envelope. Preliminary results suggest that the cross-linked minor lamins could be preferentially associated with lamin B. It therefore appears that in addition to the network of lamins A, B, and C, the peripheral lamina is characterized by the presence of two closely juxtaposed minor lamin polypeptides. The molecular interactions between these various polypeptides and their respective roles remain to be identified.
Metabolism of nitrogen fixed by root nodules of alfalfa (Medicago sativa L.): I. Utilization of [14C, 15N]glutamate and [14C, 15N]glutamine in the synthesis of γ-aminobutyrate
Authors: Ta T. C. , Macdowall F. D. H. , Faris M. A. , Joy K. W.
Published: December 2009

The fate of nitrogen and carbon from [14C, 15N]glutamate and glutamine, two primary assimilation products of ammonia from fixed nitrogen, was studied by vacuum infiltration of the compounds into alfalfa nodule slices. The amide group of glutamine is an important precursor in the synthesis of asparagine, a major transport compound in alfalfa; this reaction, catalyzed by asparagine synthetase, also produces glutamate. Glutamate is also synthesized by the action of glutamate synthase. Transamination plays an important role in the redistribution of the nitrogen groups to yield a range of amino acids. The rapid transfer of 15N from glutamate to aspartate provides the substrate for asparagine synthesis. Some glutamate was used in glutamine synthesis, indicating the operation of glutamine synthetase. Glutamate is also metabolized by decarboxylation to γ-aminobutyric acid (Gaba), a nonprotein amino acid abundant in alfalfa nodules; Gaba is further metabolized by transamination. Considerable amounts of carbon from both glutamine and glutamate enter the pool of organic acids and are utilized in the synthesis of amino acids. There is relatively little metabolism of glutamate by isolated bacteroids.
Studies on the structure of the oligosaccharide chains of α1 -acid glycoprotein associated with rough membranes from rat liver
Authors: Silvanovich A. , Jamieson J. C.
Published: December 2009

The high mannose form of rat α1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of α1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.Key words: α1-acid glycoprotein, mannosidase, oligosaccharide, nuclear magnetic resonance.
Dimethylmaleic anhydride, a specific reagent for protein amino groups
Authors: Escalera Santiago de la , Palacián Enrique
Published: December 2009

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.Key words: sulfhydryl modification, cysteine residues, maleic anhydride, monomethylmaleic anhydride, dimethylmaleic anhydride.
Purification and characterization of plasma T-kininogen from Wistar and brown Norway rats
Authors: Adam Albert , Damas Jacques , Renard Claire , Calay Germain , Bourdon Victor
Published: December 2009

A rapid and convenient three-step purification scheme has been developed for the purification of T-kininogen (alpha1-cysteine proteinase inhibitor) from rat plasma. The purification process includes chromatography on hydroxyapatite, immunoaffinity chromatography and gel filtration. This procedure is applied to plasma from the brown Norway rat which is known to be deficient in high and low molecular weight kininogens. The method furnished large amounts of T-kininogen from turpentine-treated Wistar rats as well as from untreated and turpentine-treated deficient brown Norway rats. The amino acid and hexose content of the three T-kininogens has been determined. While the composition of the molecules isolated from both injured rats was similar, the neutral sugar content of T-kininogen purified from untreated brown Norway rats was lower and its amino acid composition showed slight differences. The three molecules have identical behaviour and similar physicochemical and immunological properties when analysed by SDS electrophoresis, isoelectrofocusing and two-dimensional immunoelectrophoresis.Key words: T-kininogen, T-kinin, kallikrein–kininogen system.