2 A prebiotic RNA apparatus functions within the contemporary ribosome

Ribosomes, the universal cellular machines, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region, which was identified within all contemporary ribosomes. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA molecular apparatus. Hence, we named it the “proto-ribosome”. This prebiotic pocket-like RNA entity is suggested to be capable to accommodate substrates whose stereochemistry enables the creation of chemical bonds. It could have evolved from an earlier catalytic RNA entity that we named the “pre-proto-ribosome”, presumed to be a molecular machine capable of performing various essential tasks in the RNA world, which was snatched by the amino acid invaders for producing proteins.     

Autoradiographic Analysis of [3H]Imipramine Binding in the Human Brain Postmortem: Effects of Age and Alcohol

In vitro quantitative autoradiography of high-affinity [3H]imipramine binding sites was performed on 16 human brains postmortem. The densities of binding sites were highest in the hypothalamus. Next, in descending order, were the basal and lateral nuclei of the amygdala; substantia innominata; insular cortex; the central nucleus of the amygdala; the anterior nucleus of the thalamus; the head of the caudate nucleus; portions of the frontal, parietal, and temporal cortex; claustrum; the granular layer of the dentate gyrus; substantia nigra; the pyramidal layer of CA fields; globus pallidus; red nucleus; and white matter. Imipramine binding was found to increase with age in a region-specific manner. The presence of alcohol had a similar effect, which was most pronounced in the hippocampus. Sex and time from death to autopsy did not affect imipramine binding, in our sample.     

Effect of Chronic Desipramine Treatment on Dihydroalprenolol, Imipramine, and Desipramine Binding Sites: A Quantitative Autoradiographic Study in the Rat Brain

Desmethylimipramine (DMI) administered once daily for 10 days caused a significant decrease in beta-adrenergic receptor binding, as measured by quantitative autoradiography in discrete brain regions. The decrease was observed 72 h after the last injection throughout the cortex and in hippocampus but not in other regions, much richer in beta-receptors, such as the caudate, olfactory tubercle, superior colliculus, dorsomedial thalamus, substantia nigra, or pineal. The same paradigm did not affect imipramine (IMI) binding in the cortex or in regions with high concentrations of IMI binding sites. DMI binding was not decreased, either. Significant increases in DMI binding were observed in frontal cortex and in the ventral aspect of the bed nucleus of the stria terminalis. We conclude that a reduction in tricyclic binding is not a general phenomenon following chronic treatment with tricyclic antidepressants, and changes in binding, when they do occur, are not correlated with areas of high binding site density.     

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.     

The maintenance ofBdellovibrio at low prey density

A mathematical model for the interaction ofBdellovibrio and its prey predicted that a relatively high prey density (7×10⁵ cells ml^–1) would be required for the establishment of an equilibrium in a mixed population [8]. The present report shows thatBdellovibrio can be maintained in a continuous culture when the prey cell density is much lower (2–5×10⁴ cells ml^–1), and closer to that of naturally occurring bacterial populations in sea waters.     

A Unique Ascorbate Peroxidase Active Component in the Cyanobacterium Synechococcus PCC 7942 (R2)

Ascorbate peroxidase active component (APAC) was purified and characterized in Synechococcus PCC 9742 (R2) cells. APAC was isolated from freshly harvested cells, by ion exchange chromatography on DEAE cellulose, ultrafiltration through a 3000 dalton cut off filter and high pressure liquid chromatography through a reversed phase C-18 column. APAC was found to be extremely stable to harsh treatments of boiling water for 30 min, acidification to pH 2.0 and proteolytic digestion. A close correlation between activity and iron content of APAC was observed throughout the purification steps. E.S.R. spectrum of APAC showed a resonance line at g = 4.3 in the oxidized from. Peroxide reduction by ascorbate decreased the E.S.R. signal, which reappeared upon reoxidation by H₂O₂. The affinities of APAC to H₂O₂ and ascorbate were high (0.38 mM and 0.2 mM, respectively). Amino acid composition analysis of APAC revealed the presence of glutamic acid: glycine: cysteine residues at 2: 1: 1 ratio.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Biological and biochemical characterization of HIV‐1 Gag/dgp41 virus‐like particles expressed in Nicotiana benthamiana

The transmembrane HIV‐1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus‐like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant‐optimized HIV‐1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus‐based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV‐1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV‐1.     

Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.

Methods

Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.

Results

Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD₁₀₀ of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.

Conclusions

Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.