Molecular Mechanisms of Translation Initiation in Mammals Studied by in vitroReconstruction of Initiation Complexes

Papers on the mechanisms of translation initiation in mammals studied by reconstruction of initiation complexes from individual components are reviewed. The author points to the constraints of this approach and to the pitfalls ignoring which one might come to erroneous conclusions and even artifacts. In addition, some methods employed in the field as well as some technical problems are discussed in the paper, together with the means of obviating them. The review could be a guidebook for newcomers into this quite labor-consuming field.     

Reactivities of the coordinated organonitriles in molybdenum(0) and tungsten(0) phosphine complexes: protonation of the nitrile carbon and cleavage of the CN triple bond

The molybdenum and tungsten dinitrogen-organonitrile complexes trans-[M(N₂)(NCR)(dppe)₂] (2, M=Mo; 4, M=W; R=Ph, C₆H₄Me-p, C₆H₄OMe-p, Me; dppe=Ph₂PCH₂CH₂PPh₂) underwent double protonation at the nitrile carbon atom with loss of N₂ and a change in oxidation state to +4 on treatment with hydrochloric acid to afford the cationic imido complexes trans-[MCl(NCH₂R)(dppe)₂]⁺. The solid-state structure of trans-[WCl(NCH₂CH₃)(dppe)₂][PF₆]·CH₂Cl₂ was determined by single-crystal X-ray analysis. Protonation of complexes 2 by fluoroboric acid or hydrobromic acid also formed the similar imido complexes trans-[MoX(NCH₂R)(dppe)₂]⁺ (X=F, Br). In contrast, the dinitrogen complex trans-[Mo(N₂)₂(dppe)₂] reacted with two equiv. of benzoylacetonitrile, a nitrile with acidic CH hydrogen atoms, to give the nitrido complex trans-[Mo(N)(NKCCHCOPh)(dppe)₂] (12), which was accompanied by evolution of dinitrogen and the formation of 1-phenyl-2-propen-1-one in high yields. For complex 12, the zwitterionic structure, where the anionic enolate ligand PhC(O⁺)=CHCN coordinates to the cationic Mo(IV) center through its nitrogen atom, was confirmed by spectroscopic measurements and single-crystal X-ray analysis. A unique intermolecular aromatic C---HO hydrogen bonding was observed in that crystal structure. Complex 12 is considered to be formed via the cleavage of the CN triple bond of benzoylacetonitrile on the metal. A reaction mechanism is proposed, which includes the double protonation of the nitrile carbon atom of the ligating benzoylacetonitrile on a low-valent molybdenum center.     

Diversity of Anopheles species and trophic behavior of putative malaria vectors in two malaria endemic areas of northwestern Thailand

We determined the species diversity, blood‐feeding behavior, and host preference of Anopheles mosquitoes in two malaria endemic areas of Tak (Mae Sot District) and Mae Hong Son (Sop Moei District) Provinces, located along the Thai border with Myanmar, during a consecutive two‐year period. Anopheline mosquitoes were collected using indoor and outdoor human‐landing captures and outdoor cow‐baited collections. Mosquitoes were initially identified using morphological characters, followed by the appropriate multiplex AS‐PCR assay for the identification of sibling species within Anopheles (Cellia) complexes and groups present. Real‐time PCR was performed for parasite‐specific detection in mosquitoes (Plasmodium spp. and Wuchereria bancrofti). A total of 7,129 Anopheles females were captured, 3,939 from Mae Sot and 3,190 from Sop Moei, with 58.6% and 37% of all anophelines identified as An. minimus, respectively. All three malaria vector complexes were detected in both areas. One species within the Minimus Complex (An. minimus) was present along with two related species in the Funestus Group, (An. aconitus, An. varuna), two species within the Dirus Complex (An. dirus, An. baimaii), and four species within the Maculatus Group (An. maculatus, An. sawadwongporni, An. pseudowillmori, and An. dravidicus). The trophic behavior of An. minimus, An. dirus, An. baimaii, An. maculatus, and An. sawadwongporni are described herein. The highest An. minimus densities were detected from February through April of both years. One specimen of An. minimus from Mae Sot was found positive for Plasmodium vivax.     

Rare earth thiocyanate complexes with tripiperidinophosphine oxide: synthesis and characterization. Crystal structure of Pr(SCN)3(tpppO)3

The synthesis and characterization of rare-earth (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y) thiocyanate adducts with tripiperidinophosphine oxide (tpppO) with general formula (RE)(SCN)₃(tpppO)₃ are reported. Conductance measurements in acetonitrile indicate the non-electrolytic nature of the complexes. Infrared absorption spectra evidence that the SCN⁻ ion coordinates through the nitrogen atom (isothiocyanate form) and that tpppO coordinates through the phosphoryl oxygen. X-ray powder patterns suggest the existence of three different crystal forms: (1) La; (2) an isomorphous series including Ce, Nd and Pr; and (3) another isomorphous series, including Sm, Gd, Eu, Ho, Er, Tb, Lu and Y. The visible spectra of the Nd adduct and the calculated parameters β = 0.98, b^1/2 = 0.072 and δ = 1.06 indicate that the metal-ligand bonds are essentially electrostactic. The emission spectra of the Eu compound showed ⁵D₀ → ⁷F_J bands (J = 0, 1, 2), suggesting a C_3v symmetry for the coordination polyhedron. The lifetime of the ⁵D₀ state is 1.28 ms. The emission spectra of the Tb complex presented ⁵D₄ → ⁷F_J bands (J = 4, 5, 6) and the Dy complex showed the ⁴F_9/2 → ⁶H_13/2 band. The structure of the Pr complex showed that the coordination polyhedron is a trigonal antiprism, with the isothiocyanate anions in one base and three tpppO ligands in the other. Thermal analyses (TG-DTG) were carried out for the Ce, Nd and Gd adducts. Mass losses start between 250 and 334 °C. The final residues at 1300 °C are the corresponding phosphates.     

Protein processing as a regulatory mechanism in the synthesis of the photosynthetic antenna in Chloroflexus

Chloroflexus aurantiacus can be induced to shift from respiratory to photosynthetic energy production by introducing light and/or lowering the oxygen concentration of a culture. After induction, cells synthesize bacteriochlorophyll and proteins for the formation of a functional photosynthetic apparatus. Bacteriochlorophyll is detectable within 2 h after induction. Chlorosome polypeptides are detected after 8–12 h. Two proteins, M_r 60,000 and M_r 47,000, are present in both induced and noninduced cells and react specifically with antibodies against chlorosome polypeptides. Immunological data suggest that these proteins (M_r 60,000 and 47,000) are polyproteins which are transcribed and translated in the dark. When cells are exposed to light or low oxygen tension these proteins are processed into functional polypeptides required in the assembly of the chlorosome. The reaction center polypeptide (M_r 26,000) appears to be part of a separate genetic control system.Dedicated to Prof. G. Drews on occasion of his 60th birthday     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.     

Ruthenium ammine complexes as electron acceptors for growth stimulation by plasma membrane electron transport

Ammineruthenium(III) complexes have been found to act as electron acceptors for the transplasmalemma electron transport system of animal cells. The active complexes hexaammineruthenium(III), pyridine pentaammineruthenium(III), and chloropentaammineruthenium(III) range in redox potential (E ₀) from 305 to –42 mV. These compounds also act as electron acceptors for the NADH dehydrogenase of isolated plasma membranes. Stimulation of HeLa cell growth, in the absence of calf serum, by these compounds provides evidence that growth stimulation by the transplasma membrane electron transport system is not entirely based on reduction and uptake of iron.     

Further evidence from sectioned material in support of the existence of a linear terminal complex in cellulose synthesis

Summary Transmembrane linear terminal complexes considered to be involved in the synthesis of cellulose microfibrils have been described in the plasma membrane ofBoergesenia forbesii. Evidence for the existence of these structures has been obtained almost exlusively using the freeze etching technique. In the present study an attempt has been made to complete these studies using conventional fixation, staining, and sectioning procedures. In developing cells ofBoergesenia forbesii, strongly stained structures traversing the plasma membrane and averaging 598.9 nm ± 171.3 nm in length, 28.7 nm ± 4.2 nm in width, and 35.2 nm ± 6.6 nm in depth have been demonstrated. These structures are considered to be linear terminal complexes. At their distal (cell wall) surface, they appear to be closely associated with cellulose microfibrils. At the proximal (cytoplasmic) surface, they are associated with microtubules and polysomes. A model of the possible interrelation of the terminal complexes and microtubules leading to the generation of cell wall microfibrils is proposed.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Farnesylacetone, a sesquiterpenic hormone of Crustacea, inhibits electron transport in isolated rat liver mitochondria

Farnesylacetone (C18 H30 0) is a male hormone extracted from the androgenic gland of crab, Carcinus maenas. Appropriate enzymatic assays, as well as spectrophotometric studies, indicate that micromolar concentrations of farnesylacetone interact with the electron transport pathway of rat liver mitochondria. By the use of artificial electron donors and electron acceptors, it is shown that farnesylacetone immediately inhibits the electron transfer within complex I (NADH ubiquinone reductase activity) and complex II (succinate ubiquinone reductase activity). It is proposed that farneylacetone could interact with these two complexes of the respiratory chain at the level of the iron-sulfur centers implicated in the dehydrogenase activities. These observations are compared with the results obtained with terpenic molecules which interact with mitochondrial respiration.