The importance of Paracallisoma coecus and myctophid fishes to nesting fork-tailed and Leach's storm-petrels in the Queen Charlotte Islands, British Columbia

The diet of fork-tailed (Oceanodroma furcata) and Leach's storm-petrel(O.leucorhoa) was studied at Petrel Island, a small island northof Hippa Island in the Queen Charlotte Islands, British Columbiain 1983. The diet of the two storm-petrel species consistedmostly of the amphipod Paracallisoma coecus and fish. Most fisheswhich could be identified were myctophids, and may have beenacquired by storm-petrels at night. Fishes increased while P.coecusdeclined in importance as a food over the nestling season ofthe storm-petrels. The amount of oil also declined in the Leach'sstorm-petrel diet over the season. Since P.coecus contains muchoil, the seasonal decline of both P.coecus and oil was probablyassociated. Other less important foods were jellyfishes, copepods,isopods, euphausiids, shrimp, squid and octopus. Jellyfishes(Velella velella) only occurred in the diet of Leach's storm-petrels,which may relate to that storm-petrel feeding in open oceanwhere V. velella occurs most frequently. The most numerous copepodwas Neocalanus cristatus, which was only observed in the storm-petreldiet early on during the study period. An abyssal copepod, Bathycalanusbradyi, and the isopod Cirolana californiensis (tentativelyidentified, the latter only previously known from sea canyonsoff California) were surprising food items of storm-petrels.Most euphausiid prey consisted of Thysanoessa spinifera andEuphausia pacifica.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

Summary The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4° C some LDL-or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8° C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37° C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37° C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Vitamin K-dependent carboxylation of poly-L-glutamate.

Poly-L-glutamate preparations of varying chain length were used as substrates for bovine liver vitamin K-dependent carboxylase. The quality of these substrates (as measured by their apparent kinetic constants) was comparable to that of the more commonly used tri- and pentapeptides, but the maximal reaction rate increased at increasing chain length.     

Fucose in the surface deposits of axenic and field grown roots ofZea mays L.

Summary The location of materials containing terminal fucose residues on the surface of axenic and field grown roots of corn has been determined.Binding patterns of FITC-labelled,Lotus purpureus Moench lectin indicate the presence of the fucose residues in the cell walls and mucilage of the peripheral region of the root cap. During development, fucose residues also appear in the outer periclinal walls and overlying mucilage of columnar epidermal cells. Surface material rich in these residues persists between the mature root hairs but is not found on their surface. Fucose-rich mucilage is present on the exposed surface of aerial roots and at the point where they enter the soil. No lectin binding residues are indicated elsewhere in the roots.     

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters

Synopsis The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper.Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the whorls observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative.Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid, in a molar ratio 721. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K₄[Fe(CN)₆] containing OsO₄ in the post-fixation step we were able to demonstrate an increase in the visualization of membranous structures (liposomes).     

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.     

Nanoparticulate transport of oximes over an in vitro blood-brain barrier model

Background

Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB.

Methodology/Principal Findings

In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes.

Conclusions/Significance

With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.     

TatB functions as an oligomeric binding site for folded Tat precursor proteins

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.