Reproducibility of global and local reconstruction of three-dimensional micro-computed tomography of iliac crest biopsies.

Variation in computed tomography (CT) image gray-scale and spatial geometry due to specimen orientation, magnification, voxel size, differences in X-ray photon energy and limited field-of-view during the scan, were evaluated in repeated micro-CT scans of iliac crest biopsies and test phantoms. Using the micro-CT scanner on beamline X2B at the Brookhaven National Laboratory's National Synchrotron Light Source, 3-D micro-CT images were generated. They consisted of up to 1024 x 2400(2), 4-microm cubic voxels, each with 16-bit gray-scale. We also reconstructed the images at 16-, 32-, and 48-microm voxel resolution. Scan data were reconstructed from the complete profiles using filtered back-projection and from truncated profiles using profile-extension and with a Local reconstruction algorithm. Three biopsies and one bone-like test phantom were each rescanned at three different times at annual intervals. For the full-data-set reconstructions, the reproducibility of the estimates of mineral content of bone at mean bone opacity value, was +/-28.8, i.e., 2.56%, in a 4-microm cubic voxel at the 95% confidence level. The reproducibility decreased with increased voxel size. The interscan difference in imaged bone volume ranged from 0.86 4-microm 0.64% at 4-microm voxel resolution, and 2.64 4-microm 2.48% at 48 microm.

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter.

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.

The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2.

The three-dimensional structures of the class II anticoagulant phospholipase A2 (PLA2) toxin RVV-VD from the venom of Russell's viper, Vipera russelli russelli, and the class I neurotoxic PLA2 Notechis II-5 from the, Australian tiger snake, Notechis scutatus scutatus, were determined to 2.2 A and 3.0 A resolution, respectively. Both enzymes are monomeric and consist of 121 and 119 residues, respectively. A comparison of ten class I/II PLA2 structures showed, among other differences, that the beta-sheet of these enzymes (residues 76-83) is about 90 degrees less twisted in class I than in class II PLA2s. This, along with the insertion of some residues in the region 57-59 in class I enzymes (the elapid loop), could be the main reason for the significant difference in the anticoagulant and (presynaptic) neurotoxic properties between the two classes of PLA2. It seems apparent from sequence and structural comparisons that the toxic site of PLA2 responsible for the strong anticoagulancy of these toxins consists of a negatively charged part, Glu53, together with a positively charged ridge of lysine residues free for intermolecular interactions. These lysines differ between the two classes of PLA2.

An approach to ideal separation media for (electro)chromatography.

In packed chromatographic beds, both Eddy diffusion and the relatively long time the analytes stay in the mobile phase until they collide and interact with the ligands attached to the beads (the residence time in the mobile phase) contribute considerably to zone spreading. One should not expect Eddy diffusion to occur in macroscopically homogeneous separation media, such as gels or polymer solutions, and residence time should be shorter, since the pore size of these media is much smaller than the average distance between the beads in a packed bed. Accordingly, these separation media (which can be regarded as homogeneous continuous beds [monoliths]) should theoretically give very high resolution, which has been verified experimentally: Frontal analysis of a neutral marker, acetone, showed that electroendosmosis in a homogeneous gel displaced the boundary without any distortion except that caused by diffusion (the marker was selected not to interact with the gel). All other common disturbing phenomena in chromatography (for instance, Eddy diffusion and nonspecific adsorption) were, accordingly, negligible, indicating that electrochromatography in homogeneous gels may be the ideal chromatographic method. However, to fully utilize these desirable chromatographic properties of homogeneous continuous beds, one has to choose analyte/bed interactions with sufficiently high association-dissociation rate constants (i.e., the residence time of the analytes in the stationary phase must be kept very short), which will be the subject of forthcoming studies. The electrophoretic counterpart of capillary electrochromatography (CEC), electrophoresis of noncharged analytes in a solution of charged polymers, seems to give a somewhat larger zone broadening, probably due to their high viscosity and conductivity, with attendant longer analysis (= diffusional) times compared to gels. Since the mobile phase is propelled through the gels by electroendosmosis, the theoretical and experimental requirements for high electroendosmotic flow in gels, i.e., short analysis times, are discussed. The electroendosmotic velocity v(x) can be estimated by the simple equation v(x) = Vmax (1-e-kappa x) (1/kappa = the thickness of the double layer; Vmax = the plug flow velocity) when the distance from the channel wall, x, << the radius R of the channel (pore). For kappa R > or = 5, the equation obtains with good approximation for all R values. An initially straight zone in a gel pore should be heavily distorted by the electroendosmotic flow, according to this equation (see Figure 1). However, due to rapid diffusion and other leveling effects, the zone is transported as in perfect plug flow, as is shown experimentally. A plot of electroendosmotic mobility obtained by frontal analysis against 1/(1 + square root of mu) can be used to estimate roughly the pore size in a gel and permits quantitative examination of the current theory of electroendosmosis. It is not a trivial problem to synthesize ligand-containing gels with pores large enough to allow a high electroendosmotic flow. Therefore, we have described a universal method: a polymer containing phenylboronate and acrylic acid groups was synthesized and entrapped in a standard agarose gel (for automated runs, replaceable methoxylated agarose should be used). Both of these charged groups serve to generate the electroendosmotic flow required in electrochromatography. This gel was designed to have the dual property of separating compounds that contain vicinal OH groups in the cis-configuration (exemplified by ribonucleosides) by reaction with the boronate groups, and aromatic substances by virtue of the acrylic acid residues (and perhaps also the phenyl groups) in the polymer and the agarose chains. The latter interaction, the so-called aromatic adsorption, has the advantage that it does not require a time-consuming attachment of ligands. (ABSTRACT TRUNCATED)

The effect of beta-cyclodextrins as buffer additives on the (capillary) electrophoretic separation of peptides and proteins.

Electrophoretic and chromatographic experiments performed under straightforward conditions do not always provide satisfactory resolution. An obvious approach, then, is to manipulate the magnitude of relevant separation parameters, such as charge (zeta potential), size, and hydrophobicity, all of which can be accomplished by complex formation. This alternative has been studied with both a neutral, an anionic, and a cationic derivative of beta-cyclodextrin in an attempt to increase the resolution of peptides and proteins in free-zone electrophoresis utilizing the capillary format. The investigation showed that charged beta-cyclodextrins are suitable for this purpose. As expected, the effect seems to be most significant for substances with a small net surface charge, i.e., low mobility. Consequently, it may be advantageous to choose a pH of the buffer that is not far from the isoelectric point of the solutes. It should be emphasized that changes in the electropherograms observed upon addition of any complexing agent to the buffer may involve improvement or worsening of the resolution. Only by experimentation can one determine whether complexation with cyclodextrins favors resolution, since our knowledge about the interactions taking place is limited. However, if a positively (negatively) charged beta-cyclodextrin decreases the resolution of an acidic (basic) protein, one can expect theoretically, a negatively (positively) charged beta-cyclodextrin to increase the resolution, as was verified experimentally. The difference in mobility between two peaks caused by the complexation with cyclodextrins need not be larger than 2-3% for satisfactory resolution because the peaks are sharp. We have introduced a new definition for the resolution of two very adjacent peaks--the most common and interesting case in real-world analyses--that does not require measurement of peak widths.

Crystallographic investigation of the dependence of calcium and phosphate ions for notexin.

The crystal structure of the neurotoxic phospholipase A2, notexin, revealed three binding sites for sulphate ions which were suggested to be phosphate binding sites of importance for the activity of the toxin. The present investigation shows that the sulphate ion bound to the major binding site alters the structure of residues 60-75. In the absence of sulphate and phosphate, the structure of this loop has a conformation which partly resembles the non-neurotoxic PLA2s. The affinity of notexin for phosphate is 17 microM, as measured by the increase in fluorescence at 345 nm. Since the concentrations of phosphate and sulphate ions in blood plasma are 3 and 1 mM, respectively, the binding site must be occupied under physiological conditions. This major sulphate/phosphate binding site explains the specific affinity labelling by pyridoxal phosphate. Pyridoxal phosphate binds to this anion binding site which allows the reaction with Lys-88 or Lys-89. The structure of notexin in the presence and absence of Ca2+ shows only small local structural differences.

Bulimia: an empirical analysis of psychodynamic theory.

This study examined two aspects of psychodynamic theory concerning bulimia nervosa, that bulimic patients ascribe to a traditionally feminine sex-role and that bulimic women have difficulty differentiating emotional from visceral sensations. 18 bulimic and 18 control women were administered the Bem Sex-role Inventory and the Eating Disorder Inventory. Contrary to dynamic theory, bulimic women were not more likely classified as traditionally feminine than control subjects; however, significantly more controls than bulimic women were classified as androgynous. Analysis of scores on the Eating Disorder Inventory's Interoceptive Awareness scale indicated a significant difference between bulimic persons and controls, providing strong support for the hypothesis that bulimic women have a difficult time differentiating emotional from visceral sensations.

The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution.

The three-dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 A resolution to a crystallographic R-value of 16.5% with good stereo-chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2 s) but several parts of the molecule are distinctly different. The most significant differences from PLA2 s from bovine pancreas and rattlesnake occur in the stretches 56-80 and 85-89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The C alpha positions for residues 86-88 differ by about 6 A from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.

An optimized procedure for the separation of amino acid phenylthiohydantoins by reversed-phase HPLC.

An optimization procedure for the separation of 24 PTH-amino acids by high-performance liquid chromatography on an inexpensive Merck Superspher Si 60 RP-8, (4.0 x 250 mm) column with PTH-Nle as an internal standard is described. The effects of pH, ionic strength, temperature and gradient were investigated. Using conventional HPLC equipment, the practical detection limit is about 5 pmol.

Some biochemical characteristics and cell membrane actions of a toxic phospholipase A2 isolated from the venom of the pit viper Agkistrodon halys (Pallas).

A toxic component (AgTx) from the venom of Agkistrodon halys (Pallas) was isolated using DEAE-cellulose DE11 and CM-Sephadex C50 column chromatography and finally purified to homogeneity by FPLC on a MonoQ column. The toxin is a neutral (pI 6.9) single chain polypeptide with a mol. wt of 14,000 and an amino acid composition (123 residues) roughly similar to that of notexin. AgTx was found to have phospholipase A2 activity which was dependent on calcium and stimulated by sodium deoxycholate. The toxin caused efflux of 2-deoxy-(1-3H)-glucose-6-phosphate (a cell membrane integrity probe) as well as of [3H]acetylcholine from rat brain synaptosomes. No cell membrane damage was induced by AgTx on cultured N1E 115 neuroblastoma cells and chick myotube cultures. The LD50 ws 150 micrograms/kg (i.p.) in mice. The main symptom observed was respiratory paralysis. The results obtained show that AgTx can be classified as a toxic phospholipase A2 with a presynaptic site of action.

Conformational properties of phospholipases A2. Secondary-structure prediction, circular dichroism and relative interface hydrophobicity.

The sequences of 32 phospholipases A2 (EC 3.1.1.4) were analysed by secondary-structure prediction and the results were compared with the available crystallographic data. Good agreement is evident between prediction and experiment, especially for helical structure. Circular dichroic spectra were also determined for six enzymes from Elapid snake venom and these, in association with previously published spectra, confirm the main implication of the predictions, namely that all the homologues have qualitatively similar tertiary structures. Consideration was then given to possible structure/activity relationships in the light of the above findings. The relative hydrophobicity/hydrophilicity of the area of the enzyme thought to interact with lipid/water interfaces was predicted and certain correlations were noted with relative penetrating power, species of origin and the presence of beta-neurotoxic properties.

Identification of the thiol groups in human ceruloplasmin.

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor-bound neurotoxin.

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor bound neurotoxin. Toxicon 21, 219-229, 1983--Free Naja naja siamensis neurotoxin was acetylated with non-radioactive and acetylcholine receptor-bound neurotoxin with radioactive acetic anhydride. The toxins from the two experiments were combined and the monoacetyl derivatives isolated by chromatography on Bio-Rex 70. The yields were determined by spectrophotometry and scintillation counting. To localize the acetyl group, a radioactive monoacetyl toxin was oxidized with performic acid, digested with trypsin and a peptide with the radioactive acetyl group was isolated by gel filtration on Sephadex G-25 and high voltage paper electrophoresis. Amino acid analysis indicated from which part of the molecule the peptide was derived. In free toxin, Ac-Lys 23 and 49 account for 56% and 12%, respectively, of the monoacetyl derivatives, and in bound toxin for only 25% and 8%. Lys 49 is as reactive as Ile 1 in free toxin and 50-150% more reactive than Lys 69, 35 and 12, but it has the lowest reactivity in bound toxin, being only about half as reactive as any of these three residues. The large decrease in reactivity of Lys 23 and 49 indicates that they interact with the receptor. The proximity of the receptor makes them less accessible to acetic anhydride. The reactivities are compared to that of Lys 12, which in free toxin has the least reactive amino group. The yield of Ac-Lys 23 relative to that of Ac-Lys 12 drops from 12.4 to 1.5, or by 88%, Lys 49, 2.6 and 0.5 (81%); Ac-Ile 1, 2.6 and 1.1 (58%); Ac-Lys 69, 1.9 and 0.9 (53%); Ac-Lys 35, 1.8 and 1.0 (44%). The drop in reactivity relative to that of Lys 12 indicates a real decrease, provided that Lys 12 does not become more reactive in bound toxin. This is unlikely, since sequence homology shows that Lys 12 corresponds to Lys 15 of the neurotoxin oxiana II of Naja naja oxiana, a residue known to interact with the receptor. Sequence homology also supports the conclusion that the drop in the reactivity of Ile 1 has the same cause. The receptor-binding region of the siamensis toxin is rather large, containing the residue Lys 23 and 49, Ile 1 and probably also Lys 69 and 35.

Amino-acid sequence of the alpha-subunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus).

The amino acid sequence of the alpha-subunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan has been determined. The very basic protein, by itself a moderately neurotoxic phospholipase, consists of a single polypeptide chain of 119 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease and trypsin. Chymotryptic peptides and cyanogen bromide fragments were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin alpha-subunit is closely homologous to the other taipoxin subunits and to other elapid snake venom phospholipases A2.

complete amino-acid sequence of a non-neurotoxic, non-enzymatic phospholipase A2 homolog from the venom of the Australian tiger snake Notechis scutatus scutatus.

The complete amino acid sequence of Notechis II-1, a non-neurotoxic, non-enzymatic phospholipase A2 homolog from the venom of the Australian tiger snake Notechis s. scutatus has been determined. The protein consists of a single chain of 119 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined mainly by automated Edman degradation. Notechis II-1 contains all of the residues that appear to be invariant in elapid and pancreatic phospholipases A2 except at position 30 in the sesquence, where an otherwise invariant glycine is replaced by serine.

Chemical modification of taipoxin and the consequences for phospholipase activity, pathophysiology, and inhibition of high-affinity choline uptake.

Treatment of taipoxin with p-bromophenacyl bromide resulted in modification of single histidine residues in the alpha and beta subunits. The modification decreased the neurotoxicity (lethality) 350-fold, but the inhibitory action on high-affinity choline transport was reduced only threefold. The phospholipase activity and Ca2+-association constants for taipoxin and its subunits were determined. A model for the neurotoxicity of taipoxin indicates the alpha subunit as the ultimate cause of the disruption of synaptic transmission.

Amino acid sequence of a postsynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus.

Although 60 percent of the protein in tiger snake (Notechis scutatus scutatus) venom consists of the basic per-synaptically neurotoxic and myotoxic phospholipases notexin and Notechis II-5 and other phospholipase homologs such as Notechis II-1, several post-synaptic "curaremimetic" neurotoxins are present in small amounts. The major one of these is a typical "long" neurotoxin containing 73 amino acids in a single peptide chain cross-linked by five disulfide bridges. The formula weight calculated from the amino acid sequence is 8,051. The LD50 for intravenous injection into mice is 125 micrograms/kg.

Some biochemical responses of rat skeletal muscle to a single subcutaneous injection of a toxin (notexin) isolated from the venom of the Australian tiger snake Notechis scutatus scutatus.

1. Some biochemical responses of mammalian skeletal muscle to a single subcutaneous injection of a purified toxin from the venom of the Australian tiger snake, Notechis scutatus scutatus, have been investigated to determine the role of changes in peptide hydrolase enzymes in the muscle wasting caused by notexin administration. 2. Within 6 h of injection, serum creatine kinase activity was increased by five- to ten-fold, and remained elevated for at least 24 h. 3. There was an initial inflammatory response in the muscle adjacent to the site of injection; by 12 h after injection, muscle wet weight increased by 60%. 4. After the initial increase, wet weight fell to about 50% of normal at 7 days. Normal wet weight was achieved by 20 days after the injection. Over the period 1-20 days after the injection of the toxin, the changes in wet weight were mirrored by changes in non-collagen protein content. 5. The activities of cathepsin B and acid proteinase were increased following the injection of the toxin. By 2 days after injection, there was a ten-fold increase in the activity of cathepsin B, and a seven-fold increase in the activity of acid proteinase. The activity of both enzymes become normal by 20 days. 6. Experiments utilizing a variety of cytotoxic drugs suggested that the acid proteinase and cathespin B are primarily located within invading phagocytic cells. 7. The results are discussed with reference to the previously described pathology of toxin-damaged skeletal muscle.