Effect of water potential on seed germination

The response of seed germination to substrate water potential was determined for several plant species of the arctic tundra. Seeds were collected from Cape Thompson and Eagle Summit, Alaska and germinated on dialysis membranes over water solutions of polyethylene glycol with osmotic potentials of 0 to −6 bars. Germination did not occur with potentials below −3 bars, except for three fellfield species. Germination was delayed at lower osmotic potentials. Because the response of most species was similar, substrate water potential is probably not a factor affecting the establishment of most tundra plant species from seeds.     

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.     

Properties of detergent-dispersed myocardial adenylate cyclase

Adenylate cyclase from rabbit ventricle was solubilized in 30 to 50% yield by the nonionic detergent Lubrol PX. The detergent, when present in the assay at concentrations above 0.05%, rapidly inactivated the enzyme in assays conducted above 26 °C; assays were valid only when conducted below this temperature. The solubilized enzyme was eluted from diethylaminoethyl (DEAE)-Bio-Gel A (DEAE-agarose) with 100 mm NaCl in a yield of 25% and was free of detergent. Several properties of the solubilized detergent-free enzyme were similar to properties of the native membrane-bound species. The K_m for substrate was 0.1 mm, the K_a for Mg²⁺ was 2.5 mm, and ATP in excess of Mg²⁺ was inhibitory. The enzyme was activated by F^? and guanyl-5′-yl imidodiphosphate [Gpp(NH)p] in a time- and temperature-dependent manner, and activation by the latter was persistent. Activation by F^? and Gpp(NH)p reduced the K_a for Mg²⁺. Activation by Gpp(NH)p was increased by Mg²⁺; the apparent K_a for activation was 0.1 μm. Multiple binding sites for Gpp(NH)p were present: one class with a K_d value of 0.11 μm was probably associated with activation of the enzyme. The soluble enzyme was insensitive to catecholamines, in both the presence and the absence of Gpp(NH)p. Sensitivity to catecholamines was not restored by the addition of phospholipids, particularly phosphatidyl inositol, in either the presence or the absence of Gpp(NH)p, and this phospholipid did not increase the sensitivity of the membrane-bound enzyme to epinephrine. Catecholamine binding sites were present, and their association with adenylate cyclase was seemingly not affected by phospholipids.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B12.

We have proposed that the "doublet" EPR spectra observed during catalysis by a number of coenzyme B12-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B12r. By varying the magnitude of the exchange of coupling we have quite accurately simulated the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammon-ia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.     

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.     

Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob

Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K_D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.     

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.