bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amino acid transport systems in animal cells: Interrelations and energization
Authors: Christensen Halvor N. 
Published: May 2005

AbstractAfter summarizing the discrimination of the several transport systems of neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends to run forward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali‐ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate‐aspartate shuttle. After depletion of the energy reseves in the Ehrilich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restoration by pyruvate but in poor correlation with the restoration of alkali‐ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly senstitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali‐ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na+‐independent system, as well as to one entering by the principal Na+‐dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali‐ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali‐ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.
Glycosphingolipids in membrane architecture
Authors: Sharom Frances J. , Grant Chris W. M. 
Published: May 2005

AbstractAs part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin‐labeled derivatives of 2 families of carbohydrate‐bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip‐flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca2+‐induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.
Carbon‐13 as a tool for the study of carbohydrate structures, conformations and interactions
Authors: Nunez H. A. , Walker T. E. , Fuentes R. , O'Connor J. , Serianni A. , Barker R. 
Published: May 2005

AbstractThe application of 13C‐NMR spectroscopy to problems involving the structures and interactions of carbohydrates is described. Both 13C‐enriched and natural abundance compounds were used and some advantages of the use of the stable isotope are described. Carbon‐carbon and carbon‐proton coupling constants obtained from 1‐13 C enriched carbohydrates were employed in the assignment of their chemical shifts and to establish solution conformation. In all cases studied thus far, C‐3 couples to C‐1 only in the β‐anomers while C‐5 couples to C‐1 only in the α‐anomers. C‐6 and C‐2 always couple to C‐1 in both anomeric species. The alkaline degradation of glucose [1‐13 C] to saccharinic acids was followed by 13C‐NMR. The conversion of glucose [1‐13 C] to fructose‐1,6‐bisphosphate [1,6‐13 C] by enzymes of the glycolytic pathway was shown as an example of the use of 13C‐enriched carbohydrates to elucidate biochemical pathways. In a large number of glycosyl phosphates the 31P to H‐1 and 31P to C‐2 coupling constants demonstrate that in the preferred conformation the phosphate group lies between the O‐5 and the H‐1 of the pyranose ring. The influence of paramagnetic Mn2+ ions on the proton decoupled 13C‐NMR spectra of uridine diphosphate N‐acetylglucosamine indicates that the Mn2+ interacts strongly with the pyrophosphate moiety and with the carbonyl groups of the uracil and N‐acetyl groups.
On the rate limiting step in downhill transport via the LacY permease of Escherichia coli
Authors: Rotman Boris 
Published: May 2005

AbstractStrains of Escherichia coli K12 were constructed for the specific purpose of evaluating the inducibility of the influx mechanism controlled by the lacY gene. These strains are heteromerodiploids characterized by a high and relatively constant level of β‐D‐galactosidase which is not affected significantly by induction of the Lac operon. These properties were obtained by introducing episomal lacI+,Oc,Z+,Y− genes into the cells. In these merodiploids the rate of o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG) hydrolysis of extracted cells is 50‐times that of intact cells. This difference indicates that the rate limiting step in the ONPG hydrolysis by intact cells is influx.Using a set of merodiploids with and without the LacY transport system, we were able to demonstrate a specific induction of ONPG influx. However, the increase in influx due to induction was only 3.5‐fold as compared to the 40‐fold increase observed when the LacY permease was measured by intracellular accumulation of [14C] TMG.
An electron microscopic and enzymic study of rat liver peroxisomal nucleoid core and its association with urate oxidase
Authors: Lata Gene F. , Mamrak Frank , Bloch Phillip , Baker Barbara 
Published: May 2005

AbstractThe appearance of the characteristic crystalloid core of rat liver peroxisomes is emulated by the electron microscopic (EM) appearance of highly purified urate oxidase prepared from the same tissue. The purity of the enzyme preparation was established by gel electrophoresis under various conditions and the specific enzyme activity was at least as high as any previously reported. The amino acid composition of urate oxidase was determined. As additional evidence for close association of the peroxisomal core with urate oxidase, it was demonstrated that the biphasic changes in rat liver urate oxidase activity in response to prolonged starvation were paralleled by changes in the EM appearance of peroxisomes. Under comparable conditions catalase, another peroxisomal enzyme, did not show the same changes in activity as did urate oxidase. Evidence for the possible identity of urate oxidase with the peroxisomal crystalloid of rat liver has been presented, all materials having been obtained from, and experiments performed with, the rat.
Small‐angle X‐ray scattering study of human serum low‐density lipoproteins with differential reactivity for an arterial proteoglycan
Authors: Mateu L. , Kirchhausen T. , Padrón R. , Camejo G. 
Published: May 2005

AbstractThe structure and thermal behavior of human serum low‐density lipoproteins showing either a high or a low reactivity against a proteoglycan isolated from human arteries have been found to be different from each other. It is suggested that modifcations in the lipoprotein surface structure induced by the physical state of the neutral lipids could modulate the affinity of the macromolecule for the arterial component.
Ganglioside structures and distribution: Are they localized at the nerve ending?
Authors: Ledeen R. W. 
Published: May 2005

AbstractGengliosides generally provide a small portion of the complex carbohydrate content of cell surfaces. An exception is the central nervous system where they comprise up to 5–10% of the total lipid of some membranes. This tissue is unique in that the quantity of lipid‐bound sialic acid exceeds that of the protein‐bound fraction. Over 30 different molecular species have been characterized to date. These range in complexity from sialosylgalactosyl ceramide with 2 sugars to the pentasialoganglioside of fish brain with 9 carbohydrate units. Virtually all cellular and subcellular fractions of brain that have been carefully examined contain gangliosides to one degree or another, but the majority of brain ganglioside is located in the neurons. Their mode of distribution within the neuron has not been entirely clarified by subcellular studies. Calculations based on reported values for axon terminal density and synaptosomal ganglioside concentration in the rat reveal that nerve endings contribute less than 12% of total cerebral cortical ganglioside. It is concluded that the plasma membranes of neuronal processes contain most of the neuronal ganglioside. These and other considerations suggest the possibility that gangliosides may be distributed over the entire neuronal surface.
Immunochemical purification of probe‐labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface
Authors: Comoglio Paolo M. , Tarone Guido , Bertini Marilena 
Published: May 2005

AbstractThe probe 2,4,6‐trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate‐polyacrylamide gels of probe‐labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus‐transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.
Glycosaminoglycans of the fat globule membrane of cow milk
Authors: Lis Dora , Monis Benito 
Published: May 2005

AbstractMembranes of fat globules of cow milk contained 163 μg/100 mg (dry weight) of glycosaminoglycans (expressed as uronic acid); 62.5% of the uronic acids corresponded to hyaluronic acid, the remaining consisted of sulfated glycosaminoglycans (chondroitin‐4‐(‐6) sulfates, and dermatan and heparan sulfates) with different degrees of sulfation.
Distribution of glycophorin on the surface of human erythrocyte membranes and its association with intramembrane particles: An immunochemical and freeze‐fracture study of normal and En(a−) erythrocytes
Authors: Gahmberg Carl G. , Taurén Georg , Virtanen Ismo , Wartiovaara Jorma 
Published: May 2005

AbstractHuman erythrocyte membranes of the En(a–) blood group lack the major sialoglycoprotein (glycophorin). By absorption of a crude antiglycophorin antiserum with En(a–) membranes a specific antiglycophorin antiserum was obtained. By immune electron microscopy we showed that glycophorin is randomly distributed on the surface of normal erythrocytes. When polycationized ferritin, which mainly binds to glycophorin, was used as a marker a similar even labeling of normal erythrocyte membranes was seen. En(a–) membranes bound much less of this marker. In freeze‐fracturing the intramembrane particles of both membrane types had a similar distribution and appeared in equal amounts. However, partial removal of spectrin from these membranes, followed by incubation at pH 6 resulted in more extensive aggregation of the particles in En(a–) membranes than in normal membranes. The results may be interpreted as glycophorin contributing by electrostatic repulsion to the random distribution of the intramembrane particles in normal cells. This repulsion is weakened in En(a–) cells by the lack of glycophorin.
The spectrin‐actin complex and erythrocyte shape
Authors: Pinder Jennifer C. , Ungewickell E. , Bray D. , Gratzer W. B. 
Published: May 2005

AbstractThe effects of phosphorylation of spectrin on the properties of the cytoskeletal network of the human erythrocyte have been studied. A suspension of the cytoskeletal residues obtained after extraction of the ghosts with the nonionic detergent Triton X‐100 forms a gel on addition of membrane kinase and ATP. Phosphorylation has no effect on the association state of purified spectrin. No species higher than a tetramer of polypeptide chains is formed in vitro; in the absence of divalent cations, this tetramer is an entity liberated from and evidently present in the membrane. It has not so far proved possible to detect any F‐actin in the cytoskeleton before or after phosphorylation. It is suggested that the consequence of phosphorylation is formation of additional interactions between spectrin and monomeric actin molecules. This view is supported by the formation, after phosphorylation of the Triton‐extracted cytoskeleton, of an insoluble mass of protein on treatment with a cross‐linking reagent. In the absence of divalent cations, a series of oligomeric species is progressively liberated from the cytoskeleton on extraction with solutions of low ionic strength. These oligomers contain actin as well as spectrin, and are thought to result from disruption of the network by random denaturation of the mono meric actin in the absence of divalent metal ions. A schematic view of the effects of phosphorylation on the structure of the cytoskeleton is presented.
Regulation of hemopoietic cell differentiation and proliferation
Authors: Burgess Antony W. , Metcalf Donald , Watt Suzanne M. 
Published: May 2005

AbstractDifferentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and erythropoietin. The rate of proliferation of the GM‐progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM‐CSF (10−11 M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte‐macrophage differentiation appears to be related to (a) the concentration of GM‐ CSF and (b) the different sensitivity of a subpopulation of monocyte colony‐forming cells which are responsive to GM‐CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM‐CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM‐CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of 125I‐labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Elucidation of the receptor‐bound conformation of the enkephalins
Authors: Gorin Fredric A. , Balasubramanian T. M. , Barry C. David , Marshall Garland R. 
Published: May 2005

AbstractThe biologically relevant conformers of enkephalin predicted by solid state, solution state, and theoretical energy studies have been compared with the published structure‐activity data on these compounds. No conformational technique proposes a model consistent with all the pharmacological data; the shortcomings of each approach are evaluated. An alternative approach, which correlates the structure‐activity data of opiate compounds with that of the enkephalins, is described and shown to produce a model consistent with the available structure‐activity data.
Neural uptake of catecholamines and their molecular structures: A histopharmacologic study
Authors: Uno H. , Fellman J. H. 
Published: May 2005

AbstractUsing ultrastructural and histofluorescence methods, we investigated the uptake mechanism of catecholamines by the nerve terminals in the cutaneous smooth muscles of stump‐tailed macaques (Macaca arctoides). This in vivo approach ultilized the observed cytotoxic effects of 6‐hydroxydopamine on these catecholamine‐containing terminals and the protective effects of simultaneous treatment with catecholamines (dopamine, norepinephrine, and epinephrine), their 3–0‐methylated derivatives (metanephrine and normetanephrine), and catechol acids (3,4‐dihydroxymandelic acid and 2, 4, 5‐trihy‐droxymandelic acid). Both catecholamines and 3–0‐methylated derivatives protected these nerve terminals from destruction by 6‐hydroxydopamine, but catechol acids did not. However, the 3–0‐methylated derivatives were less effective than the catecholamines. The degree of protection afforded by these amines depended largely on their concentration. Only catecholamines intensified the electron density of the intravesicular mass or the fluorescence in the nerve terminals; therefore, 3–0‐methylated derivatives may inhibit 6‐hydroxydopamine uptake at axoplasmic membrane sites, but not inside the axon.These observations led to the discovery that there are two sites for the catecholamine uptake process. One site is the axoplasmic membrane. The terminals are protected by catecholamines and their 3–0‐methylated derivatives from 6‐hydroxydopamine uptake and thus destruction. The other site is the intra‐axonal compartments. Here competitive binding between the vesicular protein and both 6‐hydroxydopamine and the catecholamines plays a main role.
Effects of PTH and Ca2+ on renal adenyl cyclase
Authors: Nielsen Susan T. , Neuman William F. 
Published: May 2005

AbstractThe effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal‐lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity.Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH‐ or Gpp(NH)p‐stimulated activity was given by approximately 50 μM Ca++. Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half‐maximal stimulation of cAMP formation (1.8 × 10−8 M) was unchanged by the presence of calcium.These data suggest that calcium acts at some point other than the initial hormone‐receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex.
Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones
Authors: Baker Michael E. , Vaughn Duke A. , Fanestil Darrell D. 
Published: May 2005

AbstractBinding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl‐lysine chloromethyl ketone, and tosylamide‐phenylethyl‐chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero‐receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha‐fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.
Activation of cyclic AMP‐dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C‐1300 murine neuroblastoma
Authors: Green Richard D. , Noland Thomas A. 
Published: May 2005

AbstractDEAE‐cellulose chromatography of the 20,000g supernatant fraction of homogenates of C‐1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP‐dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP‐dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ‐32P] ATP, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP‐dependent protein kinase selectively stimulate the incorporation of 32P into several proteins. This stimulation is most prominent in the 15,000‐dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP‐dependent protein kinase system.