Tritosol: A new scintillation cocktail based on Triton X-100

A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for³H are about 47%, for¹⁴C about 87%, and for⁴⁵Ca about 80%.     

Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.     

Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.     

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Rooting and vitality of poinsettia cuttings was increased by arbuscular mycorrhiza in the donor plants

In this paper, we provide evidence that the rooting performance of cuttings can be improved by the arbuscular mycorrhizal (AM) symbiosis of donor plants. Poinsettia stock plants were inoculated with the Glomus intraradices isolate H510 and grown in three different cultivation systems (two organic and one conventional). AM colonization was not related to P availability in the substrate. Decay of the excised cuttings in response to unfavorable postharvest storage conditions was significantly reduced by AM colonization of the stock plants. In most cases, AM significantly promoted the formation of adventitious roots in the stored cuttings. The strongest effect of AM was found when donor plants were grown in a modified organic substrate; then AM-conditioned cuttings showed higher leaf sugar levels and a changed kinetic of carbohydrates during storage. Analyses of N, P, and K in cuttings did not indicate a nutritional effect. The results support the idea that an altered carbohydrate metabolism and plant hormones can contribute to improved rooting performance of cuttings excised from mycorrhizal donor plants.     

Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.