How initiation factors tune the rate of initiation of protein synthesis in bacteria

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.     

Common motor mechanisms support body load in serially homologous legs of cockroaches in posture and walking

We studied the mechanisms underlying support of body load in posture and walking in serially homologous legs of cockroaches. Activities of the trochanteral extensor muscle in the front or middle legs were recorded neurographically while animals were videotaped. Body load was increased via magnets attached to the thorax and varied through a coil below the substrate. In posture, tonic firing of the slow trochanteral extensor motoneuron (Ds) in each leg was strongly modulated by changing body load. Rapid load increases produced decreases in body height and sharp increments in extensor firing. The peak of extensor activity more closely approximated the maximum velocity of body displacement than the body position. In walking, extensor bursts in front and middle legs were initiated during swing and continued into the stance phase. Moderate tonic increases in body load elicited similar, specific, phase dependent changes in both legs: extensor firing was not altered in swing but was higher after foot placement in stance. These motor adjustments to load are not anticipatory but apparently depend upon sensory feedback. These data are consistent with previous findings in the hind legs and support the idea that body load is countered by common motor mechanisms in serially homologous legs.     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.     

Immunodetection of the recombinant GroEL by the Nanobody NbBruc02

Brucella has a great impact on health and economy in Syria, thus much effort is being placed on the development of diagnostics and vaccines. In this context, a wide Nanobody "immune" library was previously established, from which several Brucella-specific binders were isolated. One of these camel genetically engineered heavy-chain antibody fragments was referred to as NbBruc02. The precise antigen of NbBruc02 was presumed to be, according to proteomic approaches, the Brucella heat shock protein of 60?kDa (HSP-60). HSP-60, or alternatively named GroEL, is an interesting Brucella immunodominant antigen with important roles in the parasite life cycle, mainly adhesion and penetration during the infection of macrophages. In the present work, the capacity of NbBruc02 to filtrate the native GroEL from Brucella total extract was tested by immunochromatography approach. The interaction between NbBruc02 and its antigen was further confirmed using recombinant GroEL from Brucella. Interestingly, NbBruc02 was able to immunodetect the native as well as the denatured forms of the rGroEL in ELISA and immunoblotting, respectively. In agreement with previously reported data, NbBruc02 was able only to detect the denatured Yersinia rGroEL. Using surface plasmon resonance (SPR) biosensor, NbBruc02 showed a strong interaction, with nanomolar affinity (K (D)?=?~10(-8)?M), with the native rGroEL of Brucella and not of Yersinia. Because the casual conformational changes in the GroEL 3D structure make the base of its function, NbBruc02 by its ability to recognize a "conformational epitope," could open wide perspectives to study the role of GroEL in Brucella physiology.     

The large carpenter bees of central Saudi Arabia, with notes on the biology of Xylocopa sulcatipes Maa (Hymenoptera, Apidae, Xylocopinae)

The large carpenter bees (Xylocopinae, Xylocopa Latreille) occurring in central Saudi Arabia are reviewed. Two species are recognized in the fauna, Xylocopa (Koptortosoma) aestuans (Linnaeus) and Xylocopa (Ctenoxylocopa) sulcatipes Maa. Diagnoses for and keys to the species of these prominent components of the central Saudi Arabian bee fauna are provided to aid their identification by pollination researchers active in the region. Females and males of both species are figured and biological notes provided for Xylocopa sulcatipes. Notes on the nesting biology and ecology of Xylocopa sulcatipes are appended. As in studies for this species from elsewhere, nests were found in dried stems of Calotropis procera (Aiton) (Asclepiadaceae) and Phoenix dactylifera L. (Arecaceae).     

Origin of low mammalian cell toxicity in a class of highly active antimicrobial amphipathic helical peptides

We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316-12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.     

A versatile synthetic route to chiral quinoxaline derivatives from amino acids precursors

Summary The synthesis ofN-protected L-amino acid (3-benzylquinoxalin-2-yl) hydrazide derivatives is reported here. 3-Benzyl-2-hydrazinoquinoxaline was prepared and then coupled withN-Boc-L-amino acids including; Alanine, Valine, Leucine, Phenylalanine, Tyrosine, Serine and Proline in the presence of HBTU as a coupling reagent to provide the expected product with high yield and purity. The products were deprotected by p-toluenesulphonic acid in acetonitrile and then the tosylate salts were evaluated for antibacterial and antifungal activity. Abbreviations: HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide Boc,t-butyloxycarbonyl, DIEA, diisopropylethylamine; DMF, N,N-dimethylformamide; PTSA, p-toluenesulphonic acid, TEA, triethyl amine. Amino acids are abbreviated and designated following the rules of the IUPAC-IUB Commission of BiochemicalNomenclature (J. Biol. Chem., 247 (1972) 977).     

A Nuclear Marker for Mammalian Cells and Its Use with Intracerebral Transplants

The Hoechst dye staining method has been successfully applied to the central nervous system in mammals and its use has been demonstrated in intracerebral transplantation. The technique is rapid, simple and based on intrinsic nuclear properties. It was found to be permanent and valid whatever the animal strains or ages, allowing the distinction of rat cells from those of mouse, studied either separately or in a cross-transplantation model. It permitted the detection of grafted cells in the area of transplantation and the observation of early dispersion around the implantation site. Moreover, it can be combined with immunohistochemistry as demonstrated by a myelin marker in a relevant model. Immunodetection can thus help to directly observe grafted cells, at distance from the locus of transplantation, confirming their presence in the graft-type myelin patches.

Because of its rapid performance, this technique can be used systematically after transplantation to check for the presence of grafted cells in the host.