Attempts to observe through-chain energy transfer and electron transfer on α-helical polypeptide chain

Singlet singlet energy transfer between the two terminal chromophores attached to an α-helical polypeptide chain has been studied. The transfer efficiency was satisfactorily explained by Förster's theory when the interchromophore distance was calculated from the α-helical structure. Therefore, it was concluded that no particular effect from the possible energy band structure of the α-helical conformation was detected in the end-to-end energy transfer. Similarly, end-to-end electron transfer was attempted between the electron donor acceptor pair attached to the ends of α-helcial polypeptide chain. However, no intramolecular interaction was found between the donor acceptor pair, indicating that the exciton structure of the α-helical polypeptides is not effective enough to realize through-chain electron transfer.     

Codon-induced transfer RNA association. A property of transfer RNA involved in its adaptor function?

It is shown by equilibrium sedimentation that the binding of cognate codons to tRNAPhe (yeast), tRNAPhe (Escherichia coli), tRNALys, tRNAfMet and of the wobble codon UUU to tRNAPhe (yeast) induces dimerization of codon transfer RNA complexes. Analysis of the sedimentation profiles with a quantitative evaluation of the coupling between sedimentation and association equilibrium provides dimerization constants in the range from 1 X 10(4) to 6 X 10(4) M-1. These results on various tRNAs from different organisms suggest that the codon-induced tRNA association is a general phenomenon. Probably the codon-induced tRNA association facilitates the aminoacyl transfer reaction.     

Function of the phosphatidylinositol transfer protein gene family: is phosphatidylinositol transfer the mechanism of action?

Phosphatidylinositol transfer proteins (PITPs) bind and facilitate the transport of phosphatidylinositol (PI) and phosphatidylcholine between membrane compartments. They are highly conserved proteins, are found in both unicellular and multicellular organisms, and can be present as a single domain or as part of a larger, multi-domain protein. The hallmark of PITP proteins is their ability to sequester PI in their hydrophobic pocket. Ablation or knockdown of specific isoforms in vivo has wide ranging effects such as defects in signal transduction via phospholipase C and phosphoinositide 3-kinase, membrane trafficking, stem cell viability, Drosophila phototransduction, neurite outgrowth, and cytokinesis. In this review, we identify the common mechanism underlying each of these phenotypes as the cooperation between PITP proteins and lipid kinases through the provision of PI for phosphorylation. We propose that recruitment and concentration of PITP proteins at specific membrane sites are required for PITP proteins to execute their function rather than lipid transfer.     

Two-staged nuclear transfer can enhance the developmental ability of goat�Csheep interspecies nuclear transfer embryos in vitro

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.     

Is ubiquinone diffusion rate-limiting for electron transfer?

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 10⁹ M^–1 sec^–1 corresponding to a diffusion coefficient in the range of 10^–6 cm²/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA 81, 2606–2610, 1984) is 3×10^–9 cm²/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theK _m of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.     

Förster excitation energy transfer in peridinin-chlorophyll-a-protein

Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the F?rster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the F?rster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.     

Plastocyanin–ferredoxin oxidoreduction and endosymbiotic gene transfer

Sequence similarities of proteins associated with plastocyanin-ferredoxin oxidoreduction (PcFdOR) activity of Photosystem I (PSI) were grouped and compared. PsaA, psaB, psaC, and petG represent genes that have been retained in the chloroplasts of both green- and red-lineage species. PsaD, psaE, psaF, and petF represent genes that have been retained in the chloroplast of red-lineage species, but have been transferred to the nuclear genome of green-lineage species. Translated sequences from red- and green-lineage proteins were compared to that of contemporary cyanobacteria, Synechocystis PCC 6803, and Gloeobacter violaceus PCC 7421. Within the green lineage, a lower level of sequence conservation coincided with gene transfer to the nuclear genome. Surprisingly, a similar pattern of sequence conservation existed for the same set of genes found in the red lineage even though all those genes were retained in their chloroplast genomes. This discrepancy between green and red lineage is discussed in terms of endosymbiotic gene transfer.     

Liposome‐mediated DNA transfer to chicken sperm cells

Abstract

The efficacy of using liposomes to transfer DNA to chicken sperm cells was investigated. Liposomes were prepared from dilauroyl (12:0) phosphatidylcholine (DLPC), dimyristoyl (14:0) phosphatidyl choline (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EYPC) or lipids extracted from sperm cell membranes. The efficiency of trapping of DNA into the liposomes, transfer of the DNA from the liposomes to the sperm cells and the effect of the liposomes on the fertilizing ability of the sperm cells were determined. Increasing the concentration of lipid in the liposome preparations increased the trapping efficiency of DNA into liposomes but lowered the transfer of DNA to sperm. Including stearylamine (SA) in the liposomes increased the incorporation of DNA into the liposomes and the DNA transfer to sperm cells, while including lauroyllysophosphatidylcholine (LPC) along with SA resulted in the highest transfer efficiency from liposomes to sperm. The transfer of DNA from liposomes to sperm cells was lowered by increasing the number of sperm cells, while decreasing the number of sperm cells lowered the fertility. The sperm cells remained fertile after exposure to low levels of DPPC or lipofectin reagent or to high levels of SA and LPC. The best conditions for liposome‐mediated gene transfer to chicken sperm cells are thus using either lipofectin reagent at .006 to .06 μmol/ml and 5 × 10⁷ sperm or with DPPC liposomes comprised of 10 μmol/ml total lipid including 5 mol% SA and 20 mol% LPC with 2.5 × 10⁸ sperm cells. The use of liposomes to enhance the transfer of DNA to sperm cells may make the use of sperm cells as gene transfer vectors possible.     

Lateral gene transfer: when will adolescence end?

The scope and impact of horizontal gene transfer (HGT) in Bacteria and Archaea has grown from a topic largely ignored by the microbiological community to a hot-button issue gaining staunch supporters (on particular points of view) at a seemingly ever-increasing rate. Opinions range from HGT being a phenomenon with minor impact on overall microbial evolution and diversification to HGT being so rampant as to obfuscate any opportunities for elucidating microbial evolution - especially organismal phylogeny - from sequence comparisons. This contentious issue has been fuelled by the influx of complete genome sequences, which has allowed for a more detailed examination of this question than previously afforded. We propose that the lack of common ground upon which to formulate consensus viewpoints probably stems from the absence of answers to four critical questions. If addressed, they could clarify concepts, reject tenuous speculation and solidify a robust foundation for the integration of HGT into a framework for long-term microbial evolution, regardless of the intellectual camp in which you reside. Here, we examine these issues, why their answers shape the outcome of this debate and the progress being made to address them.     

Gene transfer in bacteria: speciation without species?

Although Bacteria and Archaea reproduce by binary fission, exchange of genes among lineages has shaped the diversity of their populations and the diversification of their lineages. Gene exchange can occur by two distinct routes, each differentially impacting the recipient genome. First, homologous recombination mediates the exchange of DNA between closely related individuals (those whose sequences are sufficient similarly to allow efficient integration). As a result, homologous recombination mediates the dispersal of advantageous alleles that may rise to high frequency among genetically related individuals via periodic selection events. Second, lateral gene transfer can introduce novel DNA into a genome from completely unrelated lineages via illegitimate recombination. Gene exchange by this route serves to distribute genes throughout distantly related clades and therefore may confer complex abilities--not otherwise found among closely related lineages--onto the recipient organisms. These two mechanisms of gene exchange play complementary roles in the diversification of microbial populations into independent, ecologically distinct lineages. Although the delineation of microbial "species" then becomes difficult--if not impossible--to achieve, a cogent process of speciation can be predicted.     

Asymmetric nuclear reprogramming in somatic cell nuclear transfer?

Despite the progress achieved over the last decade after the birth of the first cloned mammal, the efficiency of reproductive cloning remains invariably low. However, research aiming at the use of nuclear transfer for the production of patient-tailored stem cells for cell/tissue therapy is progressing rapidly. Yet, reproductive cloning has many potential implications for animal breeding, transgenic research and the conservation of endangered species. In this article we suggest that the changes in the epi-/genotype observed in cloned embryos arise from unbalanced nuclear reprogramming between parental chromosomes. It is probable that the oocyte reprogramming machinery, devised for resident chromosomes, cannot target the paternal alleles of somatic cells. We, therefore, suggest that a reasonable approach to balance this asymmetry in nuclear reprogramming might involve the transient expression in donor cells of chromatin remodelling proteins, which are physiologically expressed during spermatogenesis, in order to induce a male-specific chromatin organisation in the somatic cells before nuclear transfer.     

The contribution of surface residues to membrane binding and ligand transfer by the α-tocopherol transfer protein (α-TTP)

Previous work has shown that the α-tocopherol transfer protein (α-TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which α-TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of α-TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of α-TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired α-TTP-assisted secretion of α-tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.     

Trace metals in barnacles:the significance of trophic transfer

Barnacles have very high accumulated trace metal body concentrations that vary with local trace metal bioavailabilities and represent integrated measures of the supply of bioavailable metals. Pioneering work in Chinese waters in Hong Kong highlighted the potential value of barnacles (particularly Balanus amphitrite) as trace metal biomonitors in coastal waters, identifying differences in local trace metal bioavailabilities over space and time. Work in Hong Kong has also shown that although barnacles have very high rates of trace metal uptake from solution, they also have very high trace metal assimilation efficiencies from the diet. High assimilation efficiencies coupled with high ingestion rates ensure that trophic uptake is by far the dominant trace metal uptake route in barnacles, as verified for cadmium and zinc. Kinetic modelling has shown that low efflux rate constants and high uptake rates from the diet combine to bring about accumulated trace metal concentrations in barnacles that are amongst the