Bifunctional DNA-gold nanoparticle conjugates as building blocks for the self-assembly of cross-linked particle layers

The DNA-directed self-assembly of surface-bound layers of gold nanoparticles offers a broad range of applications in biomedical analyses as well as in materials science. We here describe a new concept for the assembly of substrate-bound nanoparticle monolayers which employs bifunctional nanoparticles as building blocks, containing two independently addressable DNA oligomer sequences. One of the sequences was utilized for attaching the particle at the solid support, while the other sequence was used to establish cross-links between adjacently immobilized particles. AFM analyses proved the functionality of inter-particle cross-links leading to enhanced surface coverages and the formation of monolayered supramolecular aggregates attached to the substrate. We anticipate that further refinement of this approach will enable applications, for instance, the assembly of ordered layers useful as transducers in biosensing.     

ATP stimulates signal recognition particle (SRP)/FtsY-supported protein integration in chloroplasts

The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.     

Temporal variation of RAPD-PCR phenotype composition of the grain aphid Sitobion avenae (Hemiptera: Aphididae) on wheat: the role of hydroxamic acids

Hydroxamic acids (Hx) contained in wheat are active mutagens which play an important role in the defence of the plant against aphids. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) dominant markers were used to assess genetic variability in the aphid Sitobion avenae (Fabricius) in relation to hydroxamic acid levels in their host-plants. Colonies of aphids belonging to a single RAPD-PCR profile were grown on different host-plants differing in their Hx content under greenhouse conditions. The RAPD-PCR phenotypic pattern showed the appearance of two new RAPD-PCR variants after four to five generations of exposure to wheat cv. Chagual (high Hx levels), one after exposure to wheat cv. Huayún (low Hx levels), and none after exposure to oat (lacking Hx). Differential appearance of new RAPD-PCR aphid phenotypes also occurred on field-grown wheat. While the overall phenotypic 'richness' diminished during the season, the number of RAPD-PCR phenotypes decreased on cv. Huayún and increased on cv. Chagual. The preferential appearance in the field and in the greenhouse of new RAPD-PCR phenotypes of S. avenae on cv. Chagual is discussed on the basis of mutagenesis induced by hydroxamic acids and by the products of their transformation within the aphid. Aphid abundance is interpreted in terms of antixenosis and antibiosis by hydroxamic acids. The appearance on cv. Chagual of phenotypes first detected on cv. Huayún was accounted for by intercrop migrations.     

Synthesis of renin substrate by rat liver.

The present study attempts to determine if the isolated rat liver is capable of synthesizing renin substrate from 14C-labelled amino acids added in the perfusate. The renin substrate is characterized via reaction with renin, forming a substance that is subsequently identified as proangiotensin. Extensive evaluation of the reaction product is carried out by using molecular-sieve chromatography, countercurrent distribution, reactivity with converting enzyme, radioimmunological technique and bioassay. The results demonstrate that isolated rat liver perfused with artificial salt solution is capable of synthesizing a protein that reacts with renin to form a radioactive substance indistinguishable from proangiotensin.     

Molecular markers to differentiate two morphologically-close species of the genus Sitobion

The effect of allelochemicals from its host, the larva of the cabbage root fly, Delia radicum (L.) (Diptera: Anthomyiidae), and the host's food plant on the ovipositor probing response of the parasitoid Trybliographa rapae (Westw.) (Hymenoptera: Cynipidae) were investigated. Trybliographa rapae probed both cabbage root fly infested and uninfested swede (Brassica napus var. napobrassica), although significantly more wasps responded to infested swede. Antennal sensilla are likely to be the mediators of this response. The synomones and kairomones involved are extractable in water, diethyl ether and methanol. No response was observed to washed, starved cabbage root fly larvae. Wasps spent significantly longer searching infested swede than uninfested, although probing frequency remained constant. It is suggested that the initiation of probing in T. rapae is dependent on a threshold concentration of general synomones or host related synomones and kairomones, whereas time spent searching a particular area is dependent on the environment perceived by sensilla on the ovipositor.     

TRP channels as potential drug targets

Calcium (Ca2+) is an ubiquitous intracellular signal that is responsible for a plethora of cellular processes including fertilization, secretion, contraction, neuronal signaling and learning. In addition, changes in intracellular Ca2+ have been known to influence cell proliferation and differentiation for more than three decades. Recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels which respond to many different modes of stimulation both from within and outside the cell may be a primary mode of cation and Ca2+ entry into cells and may have roles in growth control. Moreover, changes in the expression of these channels may contribute to certain cancers. In the following, recent results concerning the expression and function of members of this family of ion channels are summarized.     

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.     

Splice variants of a ClC-2 chloride channel with differing functional characteristics

We identified two ClC-2 clones in a guinea pigintestinal epithelial cDNA library, one of which carries a 30-bpdeletion in the NH₂ terminus. PCR using primersencompassing the deletion gave two products that furthermore wereamplified with specific primers confirming their authenticity. Thecorresponding genomic DNA sequence gave a structure of three exons andtwo introns. An internal donor site occurring within one of the exonsaccounts for the deletion, consistent with alternative splicing.Expression of the variants gpClC-2 and gpClC-277-86 in HEK-293cells generated inwardly rectifying chloride currents with similaractivation characteristics. Deactivation, however, occurred with fasterkinetics in gpClC-277-86. Site-directed mutagenesis suggeststhat a protein kinase C-mediated phosphorylation consensus site lost ingpClC-277-86 is not responsible for the observed change. Thedeletion-carrying variant is found in most tissues examined, and itappears more abundant in proximal colon, kidney, and testis. Thepresence of a splice variant of ClC-2 modified in itsNH₂-terminal domain could have functional consequences intissues where their relative expression levels are different.

     

Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification

Nucleic acid amplification techniques are used for signal generation in antibody-based immunoassays, thereby dramatically enhancing the sensitivity of conventional immunoassays. Methodological aspects, as well as applications of this novel approach, are summarized in this review, with an emphasis on immuno-polymerase chain reaction (IPCR). IPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100-10,000-fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. The evolution of IPCR included the development of efficient reagents, the design of assay formats and the maintenance of functionality, even within complex biological matrices. Eventually, IPCR crossed the border from being a research method to a routine laboratory technique, enabling a broad range of applications in immunological research and clinical diagnostics.