Anomalous carbonylation of [Pd(dppm)(O2CCF3)]2 to give an asymmetric μ-CO complex

The reactive palladium dimer, [Pd(dppm)(O₂CCF₃)]₂, is carbonylated to [Pd(dppm)(O₂CCF₃)]₂(μ-CO) in a reversible reaction with K = c. 7.2(2)x10⁴ atm⁻¹ (P_1/2 = c. 2.4 Torr). This is significantly larger than is expected based on the λ_max = 280 nm in the electronic spectrum. The product can be isolated in analytically pure form by crystallization under a CO atmosphere. It forms crystals in the monoclinic space group Cc with a = 18.584(5), b = 28.65(1), c = 11.164(3) Å and β = 95.16(2)°. The structure is significantly distorted. The bonding about the two palladium atoms is quite asymmetric. While one is close to a square planar geometry with a Pd---C(O) distance of 1.90(2) Å, the other is significantly pyramidalized and has a longer (2.00(2) Å) bond to the bridging CO. The Pd---Pd distance is only 2.896(2) Å, much shorter than that usually observed for formally non-bonded Pd atoms.     

Description of the oöcysts of three new species of coccidia (Apicomplexa: Eimeriidae) from lizards in Namibia

Three new species of coccidian were recovered from the intestinal contents and faeces of lizards in Namibia, southwest Africa. Oöcysts of Eimeria barnardi n. sp. are described from Rhoptropus barnardi (Gekkonidae) and are ellipsoidal, 24.3 × 19.9 (21–26.5 × 16–22) m; shape index (length/width) 1.22 (1.12–1.30). A micropyle and oöcyst residuum are absent but a fragmented polar granule is present. Sporocysts are subspherical, 9.2 × 8.3 (8–11 × 7.5–9) m; shape index 1.11 (1.02–1.27). Oöcysts of Eimeria pachybibroni n. sp. were found in Pachydactylus bibroni (Gekkonidae) and are ellipsoidal, 26.2 × 18.2 (21.5–28 × 16–19) m; shape index 1.44 (1.30–1.52). A micropyle and oöcyst residuum are absent but a polar granule is present. Sporocysts are subspherical, 8.9 × 8.0 (8–9.5 × 7–8.5) m; shape index 1.12 (1.03–1.20). Oöcysts of Isospora spilogaster n. sp. are reported from Mabuya spilogaster (Scincidae) and are subspherical, 27.4 × 26.0 (21.5–35 × 21–35) m; shape index 1.05 (1.00–1.13). Micropyle, oöcyst residuum and polar granules are absent. Sporocysts are ellipsoidal, 13.2 × 9.7 (10.5–15 × 9–11) m; shape index 1.36 (1.08–1.50).     

Description of the oöcysts of two new species of Eimeria (Apicomplexa: Eimeriidae) from the rough earth snake Virginia striatula (Serpentes: Colubridae) in Texas and Arkansas

Coccidian oöcysts recovered from the faeces of rough earth snakes Virginia striatula (Serpentes: Colubridae) were found to represent two previously unreported eimerians. Oöcysts of Eimeria desotoensis n. sp. were found in 5/32 (16%) of the snakes and were spherical to ellipsoidal, 18.4 × 17.2 (15–21.5 × 15–19.5) μm, with a thin, single-layered wall; their shape-index (length/width) was 1.07 (1.00–1.23). A micropyle and oöcyst residuum were absent; polar granule were present in 33% of the oöcysts. The sporocysts were ovoidal, 11.5 × 7.6 (10.5–13 × 7–8) μm, with a Stieda body; their shape-index was 1.51 (1.30–1.68). The sporocyst residuum was moderate in size and composed of a cluster of granules. Oöcysts of Eimeria hobartsmithi n. sp. were found in 2/32 (6%) of the snakes and were subspherical to ellipsoidal, 18.0 × 15.7 (16–20 × 15–17) μm, with a thin, single-layered wall; their shape-index was 1.15 (1.02–1.32). A micropyle, oöcyst residuum and polar granule were absent. The sporocysts were elongate, 13.2 × 6.3 (12–14.5 × 6–6.5) μm, with a Stieda body; their shape-index was 2.10 (1.88–2.34). A large sporocyst residuum was present in each sporocyst, often obscuring the sporozoites. In addition to the two new species, oöcysts of E. striatula Upton & McAllister, 1990 were observed in 38% of the snakes.     

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. $\text{In}\text{vitro}$ cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL₂ result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.     

An allied reprogramming,selection, expansion and differentiation platform for creating hiPSC on microcarriers

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs.     

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.     

Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge

The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residues results in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceutical properties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistry and production costs high because of complex purification procedures. Our solution to these fundamental problems in PEGylating proteins has been to exploit the latent conjugation selectivity of the two sulfur atoms that are derived from the ubiquitous disulfide bonds of proteins. This approach to PEGylation involves two steps: (1) disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which PEG was covalently attached. During this process, irreversible denaturation of the protein did not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using alpha,beta-unsaturated beta'-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiary structure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) the steric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormone somatostatin, the tetrameric protein l-asparaginase, and to the disulfides in interferon alpha-2b (IFN). SDS-PAGE, mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. The biological activity of the l-asparaginase did not change after the attachment of four PEG molecules. In the case of IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). A significantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biological activity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with all other forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previously been described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragments can be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroying their tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach also enables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticals as cost-effective medicines for global use.