Novel Antitumor L‐Arabinose Derivative of Indolocarbazole with High Affinity to DNA

Novel indolocarbazole derivative 12‐(α‐L ‐arabinopyranosyl)indolo[2,3‐α]pyrrolo[3,4‐c]carbazole‐5,7‐dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)₂₀, d(GC)₂₀ and calf thymus DNA (binding constants (1.6×10⁶) M ^?1≤K_a≤(3.3×10⁶) M ^?1). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C≤1 μM ) were identical to the concentrations that triggered p53‐dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I‐mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.     

Crosslinked-Chelating Porous Sheet with High Dynamic Binding Capacity of Metal Ions

A crosslinked chelating porous sheet was prepared by cografting ethylene glycol dimethacrylate (EGDMA) with glycidyl methacrylate onto an electron-beam-irradiated porous polyethylene sheet, followed by the introduction of an iminodiacetate group. At a molar percentage of EGDMA of 1.0 mol%, the sheet exhibited a maximum dynamic binding capacity for copper ions of 0.93 mmol/g, while the equilibrium binding capacity remained the same (1.2 mmol/g) as that of a non-crosslinked chelating porous sheet. The crosslinking of the grafted chain causes copper ions to lower their diffusion rate along the sheet thickness driven by the gradient of the amount of copper ions adsorbed.     

高性能浇注型聚氨酯弹性体的动态性能研究

通过高性能浇注型聚氨酯弹性体的合成，研究了二异氰酸酯结构、多元醇结构对浇注型聚氨酯弹性体动态性能的影响，结果表明，异氰酸酯基团含量增加，聚氨酯弹性体的硬度、撕裂强度和耐热性提高。     

Distinctive Lipid Composition of the Copepod Limnocalanus macrurus with a High Abundance of Polyunsaturated Fatty Acids

We studied the copepod Limnocalanus macrurus for seasonal variation in the composition of fatty acids, wax esters and sterols in large boreal lakes, where it occurs as a glacial‐relict. Vast wax ester reserves of Limnocalanus were accumulated in a period of only two months, and comprised mono‐ and polyunsaturated fatty acids (PUFA) and saturated fatty alcohols. In winter, the mobilization of wax esters was selective, and the proportion of long‐chain polyunsaturated wax esters declined first. PUFA accounted for >50 % of all fatty acids throughout the year reaching up to ca. 65 % during late summer and fall. Long‐chain PUFA 20:5n‐3 and 22:6n‐3 together comprised 17–40 % of all fatty acids. The rarely reported C24 and C26 very‐long‐chain PUFA (VLC‐PUFA) comprised 6.2 ± 3.4 % of all fatty acids in August and 2.1 ± 1.7 % in September. The VLC‐PUFA are presumably synthesized by Limnocalanus from shorter chain‐length precursors because they were not found in the potential food sources. We hypothesize that these VLC‐PUFA help Limnocalanus to maximize lipid reserves when food is abundant. Sterol content of Limnocalanus, consisting ca. 90 % of cholesterol, did not show great seasonal variation. As a lipid‐rich copepod with high abundance of PUFA, Limnocalanus is excellent quality food for fish. The VLC‐PUFA were also detected in planktivorous fish, suggesting that these compounds can be used as a trophic marker indicating feeding on Limnocalanus.     

Chemical and Enzymatic Synthesis of Modified Sialyl Lewis X Tetrasaccharides with High Affinity for E and P‐Selectin

A series of sialyl‐Lewis X tetrasaccharide analogs 1a — d was prepared using a combined chemical and enzymatic approach. Sialic acid analogs 5b , c were obtained from 2‐azido mannose 4c or 2‐deoxy mannose 4b and pyruvate by an aldolase reaction and converted to the protected thioglycosides 3b , c that served as sialyl donors for the Lewis X acceptor trisaccharide 2 . The resulting sialyl‐Lewis X tetrasaccharides 8a — c were deprotected by deacylation and saponification of the methyl ester. Debenzylation was achieved by careful transfer hydrogenation in the presence of formic acid or ammonium formate as a hydrogen source. Three sialyl‐Lewis X derivatives 1a — c were thus obtained and the parent compound 1a was further modified by alkaline hydrolysis of the two acetamides to give the lyso sialyl‐Lewis X derivative 1d . The four sialyl‐Lewis X tetrasaccharides 1a — d were tested for their binding affinity to E and P‐selectin with the lyso sialyl‐Lewis X derivative 1d showing the highest inhibitory potency for both lectins.     

Rational Design,Synthesis and Biological Evaluation of Modular Fluorogenic Substrates with High Affinity and Selectivity for PTP1B

Protein‐tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type‐2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self‐immolative 4‐hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme‐initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B‐catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.     

Lipid monolayers: Mechanisms of protein penetration with regard to membrane models

The influence of lipid and protein on the properties of the air-water interface is analyzed with the view to formulate a mechanism of interaction of protein with lipid monolayers. The increase in surface pressure (ΔΠ) and the quantity of protein incorporated in the lipid film after injection of protein under lipid monolayers were studied as a function of both lipid structure and protein structure. With rabbit γ-globulin, the values of ΔΠ were cholesterol > phosphatidyl choline > sphingomyelin. Similar results were obtained with ribonuclease, lysozyme and serum albumin. The quantities of protein found in films of either cholesterol or phosphatidyl choline (egg lecithin) were much larger than those calculated from a geometric model in which a protein monolayer occupies the area made available by the compressed lipid. Arguments are produced against penetration based on simple mechanisms of compressibility of the lipid film. The mechanisms operating in the incorporation of protein into lipid monolayers are grouped into three categories: (a) free penetration, typical of lecithin; (b) binding-mediated penetration, typical of cholesterol and some glycosphingolipids; and (c) binding-inhibited penetration, typical of the albumin-ganglioside system and a specific lipid hapten-antibody system. A model is described in which nonspecific protein interacts with polymeric lecithin structures (surface micelles). In the sequence of events X»Y»Z, the globular protein X is activated into the expanded or extended form Y by contact with the lipid and then restructured into a compact form Z with release of water and free energy. The resulting lipid-protein assembly has a mosaic structure in which lipid and protein polar surfaces are exposed to water. Accessibility of lecithin to phospholipase A is consistent with the model and with current views on the state of protein in biological membranes; according to such views, protein is more likely structured inside the lipid milieu and not simply denatured on the lipid-water interface.     

Redesigning the Active Site of Transaldolase TalB from Escherichia coli: New Variants with Improved Affinity towards Nonphosphorylated Substrates

Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X‐ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site‐directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D ‐fructose from DHA and d,l ‐GA by using an adjusted colour assay. The best results with respect to the synthesis of D ‐fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l ‐GA (K_M=24 mM ). We demonstrated that this double mutant can use D ‐GA, glycolaldehyde and the L ‐isomer, L ‐GA, as acceptor substrates. This resulted in preparative synthesis of D ‐fructose, D ‐xylulose and L ‐sorbose when DHA was used as donor. Hence, we engineered a DHA‐dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.     

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion

In antigen–antibody interactions, the high avidity ofantibodies depends on the affinity and number of the individualbinding sites. To develop artificial antibodies with multiplevalency, we have fused the single-chain antibody Fv fragmentsto core streptavidin. The resulting fusion protein, termed scFv::strep,was found after expression in Escherichia coli in periplasmicinclusion bodies. After purification of the recombinant productby immobilized metal affinity chromatography, refolding andsize-exclusion FPLC, tetrameric complexes resembling those ofmature streptavidin were formed. The purified tetrameric scFv::strepcomplexes demonstrated both antigen- and biotin-binding activity,were stable over a wide range of pH and did not dissociate athigh temperatures (up to 70°C). Surface plasmon resonancemeasurements in a BIAlite system showed that the pure scFv::streptetramers bound immobilized antigen very tightly and no dissociationwas measurable. The association rate constant for scFv::streptetramers was higher than those for scFv monomers and dimers.This was also reflected in the apparent constants, which wasfound to be 35 times higher for pure scFv::strep tetramers thanmonomeric singlechain antibodies. We could also show that mostof biotin binding sites were accessible and not blocked by biotinylatedE.coli proteins or free biotin from the medium. These sitesshould therefore facilitate the construction of bispecific multivalentantibodies by the addition of biotinylated ligands.     

High energetic materials: Reaction of Azides with Dioxygenyl Salts

The reactivity of sodium azide, activated sodium azide and hydrazinium azide towards various dioxygenyl salts was studied. Under no circumstances the formation of dioxygenyl azide, O₂N₃, could be observed. This is in agreement with high level quantumchemical ab initio computations at correlated level (MP2, PMP2, CISD), predicting the decomposition of hypothetical O₂⁺(g)/N₃⁻(g) to be thermodynamically highly favorable (MP2: −326; PMP2: −328; CISD; −369 [kcal/mol]). The combustion of O₂BF₄ with either N₂H₅N₃ (spontaneous reaction on contact in a coaxial solid-propellant system) or NaN₃ (safe to handle, highly exothermic reaction after ignition) is discussed in terms of high-energy-density materials (HEDM).      

Lipid composition of platelets from patients with atherosclerosis: Effect of purified eicosapentaenoic acid ethyl ester administration

Although eicosapentaenoic acid (EPA) has been shown to have beneficial effects in the prevention of atherosclerosis, the mechanism by which these effects occur is not entirely clear. We investigated the lipid composition of platelets in paired subjects with and without atherosclerotic disease, either hypercholesterolemic (low density lipoprotein [LDL] cholesterol [Chol]≥170 mg/dl) or normocholesterolemic (LDL-Chol<170 mg/dl). Platelets from patients with atherosclerotic disease had a lower phosphatidylcholine (PC)/Chol ratio, when compared with those from patients without atherosclerotic disease, irrespective of LDL-Chol levels. Eleven patients with atherosclerotic disease were treated with purified. EPA ethyl ester (1.8 g/day), and changes in lipid composition of platelets were investigated. Plasma levels of total Chol and LDL-Chol decreased significantly after EPA administration. The phospholipid (PL)/Chol ratio and the PC/Chol ratio in platelets from patients with atherosclerotic disease increased significantly after 4–10 wk EPA treatment. The EPA content in platelets increased, while the arachidonic acid (AA) content decreased. EPA-induced changes in the PL/Chol and the PC/Chol ratios of platelets, as well as fatty acyl chain shifts, may be related to the beneficial effects in preventing atherosclerosis, possibly by increase in the membrane fluidity.     

Sequential Feeding of Lipid Supplement Enriches Beef Adipose Tissues with 18:3n-3 Biohydrogenation Intermediates

The present study was designed to determine if feeding steers extruded flaxseed and hay (25 and 75%; DM basis) together as a total mixed ration (TMR), or sequentially (non-TMR) would result in different enrichments of polyunsaturated fatty acids (PUFA) and their biohydrogenation intermediates (BHI) in beef adipose tissues [subcutaneous (SC) vs perirenal (PR) fat]. Forty-eight Angus cross steers (325 ± 16 kg) were stratified by weight to six pens, and pens were randomized to either TMR or non-TMR and fed ad libitum for an average of 242 days. The concentrations of α-linolenic acid increased by 18 mol% in both SC and PR in non-TMR steers compared to TMR steers (P < 0.01). trans 18:1 isomers were more concentrated in PR than SC (14.4 vs 9.5 mol%; P < 0.01) and increased by 10 mol% in both fat depots for non-TMR (P < 0.01). Other BHI including non-methylene-interrupted 18:2 (atypical dienes), conjugated linoleic acids and conjugated linolenic acids (CLnA) were affected by diet × tissue interactions (P < 0.01). The CLnA and CLA contents were higher in both fat depots when feeding the non-TMR, but the effect of diet was more pronounced in PR than in SC (P < 0.01). Atypical dienes were highest in PR from non-TMR and lowest in TMR fed steers (4.3 and 3.6 mol%) with SC contents being intermediate. The sequential feeding of lipid supplement can thus profoundly affect the enrichment of PUFA and their BHI in beef fat and their differentially enrichment is also fat depot dependant.     

Cure of epoxy resins with aromatic amines: High heat-distortion studies

Castings having unexpectedly high heat-distortion temperatures result when certain treated resins of the EPON

1 EPON is a registered trademark of the Shell Oil Company.

828 type and about 75% of the stoichiometric amount of m-phenylenediamine, are postcured for 10–20 hr. at 175–200°C. The improvement in heat-distortion temperature is about 100°C., to values as high as 250°C. A recrystallized resin has given the highest values. Other glycidyl ethers of polyphenols have shown this phenomenon to a lesser degree, but other amine curing agents, including isomers and substitution products of m-phenylenediamine, have not. Some evidence of a new curing reaction has been developed, by NMR and pyrolysis studies of model compounds, which supports the postulate that the m-phenylenediamine is alkylated with a fifth epoxy group during the postcure, presumably at a ring carbon, resulting in greater crosslinking.     

Dynamic Compaction of Aluminum Nitride Powder: Hugoniot Measurement and Comparison with Static Behavior

A limited number of shock Hugoniot measurements were performed on two unsintered aluminum nitride powder compacts having initial densities of 1.30 and 1.53 g/cm³. Stresses achieved ranged from 0.25 to 1.8 GPa, corresponding to linear compaction rates of 0.2 to 0.6 km/s. Densification is incomplete behind the first shock wave, proceeding only to ∼70% of the solid density, regardless of the initial density. On reshock, however, significantly higher densities are achieved. Initial compaction of the powder to a relative density of 65% to 70% occurs readily at stresses below 0.25 GPa. For greater stresses, however, densification is slight. Comparisons with static compression data for the same powders suggest that this resistance to compaction is an effect of compaction rate.     

Structure‐Guided Rational Design of α/β‐Peptide Foldamers with High Affinity for BCL‐2 Family Prosurvival Proteins

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐x_L but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐x_L was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐x_L. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐x_L validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.     

Wet-Chemical Passivation of InAs: Toward Surfaces with High Stability and Low Toxicity

In a variety of applications where the electronic and optical characteristics of traditional, siliconbased materials are inadequate, recently researchers have employed semiconductors made from combinations of group III and V elements such as InAs. InAs has a narrow band gap and very high electron mobility in the near-surface region, which makes it an attractive material for high performance transistors, optical applications, and chemical sensing. However, silicon-based materials remain the top semiconductors of choice for biological applications, in part because of their relatively low toxicity. In contrast to silicon, InAs forms an unstable oxide layer under ambient conditions, which can corrode over time and leach toxic indium and arsenic components. To make InAs more attractive for biological applications, researchers have investigated passivation, chemical and electronic stabilization, of the surface by adlayer adsorption. Because of the simplicity, low cost, and flexibility in the type of passivating molecule used, many researchers are currently exploring wet-chemical methods of passivation. This Account summarizes much of the recent work on the chemical passivation of InAs with a particular focus on the chemical stability of the surface and prevention of oxide regrowth. We review the various methods of surface preparation and discuss how crystal orientation affects the chemical properties of the surface. The correct etching of InAs is critical as researchers prepare the surface for subsequent adlayer adsorption. HCl etchants combined with a postetch annealing step allow the tuning of the chemical properties in the near-surface region to either arsenic- or indium-rich environments. Bromine etchants create indium-rich surfaces and do not require annealing after etching; however, bromine etchants are harsh and potentially destructive to the surface. The simultaneous use of NH(4)OH etchants with passivating molecules prevents contact with ambient air that can occur during sample transfer between solutions. The passivation of InAs is dominated by sulfur-based molecules, which form stable In-S bonds on the InAs surface. Both sulfides and alkanethiols form well-defined monolayers on InAs and are dominated by In-S interactions. Sulfur-passivated InAs surfaces prevent regrowth of the surface oxide layer and are more stable in air than unpassivated surfaces. Although functionalization of InAs with sulfur-based molecules effectively passivates the surface, future sensing applications may require the adsorption of functional biomolecules onto the InAs surface. Current research in this area focuses on the passivation abilities of biomolecules such as collagen binding peptides and amino acids. These biomolecules can physically adsorb onto InAs, and they demonstrate some passivation ability but not to the extent of sulfur-based molecules. Because these adsorbents do not form covalent bonds with the InAs surface, they do not effectively block oxide regrowth. A mixed adlayer containing a biomolecule and a thiol on the InAs surface provides one possible solution: these hybrid surfaces enhance passivation but also maintain the presence of a biomolecule on the surface. Such surface functionalization strategies on InAs could provide long-term stability and make these surfaces suitable for biological applications.     

High performance liquid chromatography of triglycerides: Controlling selectivity with reverse phase columns

Rapid separations of triglycerides by chain length and degree of unsaturation have been made by high performance liquid chromatography (HPLC) using reverse phase columns. Several different bonded columns were evaluated for use in reverse phase HPLC of triglycerides, and differences in selectivity are discussed. Selectivity was also modified by adding silver ion to the solvent, which produces marked changes in selectivity of solute triglycerides. Presented at the AOCS meeting in San Francisco, May 1979.