Classification of suppressor additives based on synergistic and antagonistic ensemble effects

Three fundamental types of suppressor additives for copper electroplating could be identified by means of potential Transient measurements. These suppressor additives differ in their synergistic and antagonistic interplay with anions that are chemisorbed on the metallic copper surface during electrodeposition. In addition these suppressor chemistries reveal different barrier properties with respect to cupric ions and plating additives (Cl, SPS). While the type-I suppressor selectively forms efficient barriers for copper inter-diffusion on chloride-terminated electrode surfaces we identified a type-II suppressor that interacts non-selectively with any kind of anions chemisorbed on copper (chloride, sulfate, sulfonate). Type-I suppressors are vital for the superconformal copper growth mode in Damascene processing and show an antagonistic interaction with SPS (Bis-Sodium-Sulfopropyl-Disulfide) which involves the deactivation of this suppressor chemistry. This suppressor deactivation is rationalized in terms of compositional changes in the layer of the chemisorbed anions due to the competition of chloride and MPS (Mercaptopropane Sulfonic Acid) for adsorption sites on the metallic copper surface. MPS is the product of the dissociative SPS adsorption within the preexisting chloride matrix on the copper surface. The non-selectivity in the adsorption behavior of the type-II suppressor is rationalized in terms of anion/cation pairing effects of the poly-cationic suppressor and the anion-modified copper substrate. Atomic-scale insights into the competitive Cl/MPS adsorption are gained from in situ STM (Scanning Tunneling Microscopy) using single crystalline copper surfaces as model substrates. Type-III suppressors are a third class of suppressors. In case of type-land type-II suppressor chemistries the resulting steady-state deposition conditions are completely independent on the particular succession of additive adsorption. In contrast to that a strong dependence of the suppressing capabilities on the sequence of additive adsorption ("first comes, first serves" principle) is observed for the type-IIIsuppressor. This behavior:is explained by a suppressor barrier that impedes not only the copper inter-diffusion but also the transport of other additives (e.g. SPS) to the copper surface. (C) 2011 Elsevier Ltd. All rights reserved.

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.

Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Development of Solid Oxide Fuel Cell Electrodes with High Conductivity and Enhanced Redox Stability

The intent of this study was the development of new ceramic SOFC anode materials which possess electrical conductivity as well as redox stability.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Two alpha subunits and one beta subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

GH mutant (R77C) in a pedigree presenting with the delay of growth and pubertal development: structural analysis of the mutant and evaluation of the biological activity

A heterozygous missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C), which was previously reported to have some GH antagonistic effect, was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (-2.5 SDS) at the age of 6 years. His mother and grandfather were also carrying the same mutation, but did not differ in adult height from the other unaffected family members. Hormonal examination in all affected subjects revealed increased basal GH, low IGF-I concentrations, and subnormal IGF-I response in generation test leading to the diagnosis of partial GH insensitivity. However, GH receptor gene (GHR) sequencing demonstrated no abnormalities. As other family members carrying the GH-R77C form showed similar alterations at the hormonal level, but presented with normal final height, no GH therapy was given to the boy, but he was followed through his pubertal development which was delayed. At the age of 20 years he reached his final height, which was normal within his parental target height. Functional characterization of the GH-R77C, assessed through activation of Jak2/Stat5 pathway, revealed no differences in the bioactivity between wild-type-GH (wt-GH) and GH-R77C. Detailed structural analysis indicated that the structure of GH-R77C, in terms of disulfide bond formation, is almost identical to that of the wt-GH despite the introduced mutation (Cys77). Previous studies from our group demonstrated a reduced capability of GH-R77C to induce GHR/GH-binding protein (GHBP) gene transcription rate when compared with wt-GH. Therefore, reduced GHR/GHBP expression might well be the possible cause for the partial GH insensitivity found in our patients. In addition, this group of patients deserve further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity. This might be responsible for the delay of growth and pubertal development. Finally, we clearly demonstrate that GH-R77C is not invariably associated with short stature, but that great care needs to be taken in ascribing growth failure to various heterozygous mutations affecting the GH-IGF axis and that careful functional studies are mandatory.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).

Metal-ion-dependent biological properties of a chelator-derived somatostatin analogue for tumour targeting

Somatostatin-based radioligands have been shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [(55)Co(dotatoc)] (dotatoc =4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetyl-D-Phe-(Cys-Tyr-D-Trp-Lys-Thr-Cys)-threoninol (disulfide bond)) as a new radiopharmaceutical agent for PET has been evaluated. (57)Co was used as a surrogate of the positron emitter (55)Co and the pharmacokinetics of [(57)Co(dotatoc)] were investigated by using two nude mouse models. The somatostatin receptor subtype (sst1-sst5) affinity profile of [(nat)Co(dotatoc)] on membranes transfected with human somatostatin receptor subtypes was assessed by using autoradiographic methods. These studies revealed that [(57)Co(dotatoc)] is an sst2-specific radiopeptide which presents the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing an AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst-receptor-expressing tissues, which reflects the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [(57)Co(dotatoc)] and [(67)Ga(dotatoc)] are discussed in terms of the structural parameters found for the chelate models [Co(II)(dota)](2-) and [Ga(III)(dota)](-) whose X-ray structures have been determined. Both chelates show six-fold coordination in pseudo-octahedral arrangements.

Novel sst5-selective somatostatin dicarba-analogues: synthesis and conformation-affinity relationships

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

Development of a direct evaporation bismuth Hall thruster

Hall thrusters have been under active development around the world since the 1960’s. Thrusters using traditional propellants such as xenon have been flown on a variety of satellite orbit raising and maintenance missions with an excellent record. To expand the mission envelope, it is necessary to lower the specific impulse of the thrusters but xenon and krypton are poor performers at specific impulses below 1,200 seconds. To enhance low specific impulse performance, this dissertation examines the development of a Hall-effect thruster which uses bismuth as a propellant. Bismuth, the heaviest non-radioactive element, holds many advantages over noble gas propellants from an energetics as well as a practical economic standpoint. Low ionization energy, large electron-impact crosssection and high atomic mass make bismuth ideal for low-specific impulse applications. The primary disadvantage lies in the high temperatures which are required to generate the bismuth vapors. Previous efforts carried out in the Soviet Union relied upon the complete bismuth vaporization and gas phase delivery to the anode. While this proved successful, the power required to vaporize and maintain gas phase throughout the mass flow system quickly removed many of the efficiency gains expected from using bismuth. To solve these problems, a unique method of delivering liquid bismuth to the anode has been developed. Bismuth is contained within a hollow anode reservoir that is capped by a porous metallic disc. By utilizing the inherent waste heat generated in a Hall thruster, liquid bismuth is evaporated and the vapors pass through the porous disc into the discharge chamber. Due to the high temperatures and material compatibility requirements, the anode was fabricated out of pure molybdenum. The porous vaporizer was not available commercially so a method of creating a refractory porous plate with 40-50% open porosity was developed. Molybdenum also does not respond well to most forms of welding so a diffusion bonding process was also developed to join the molybdenum porous disc to the molybdenum anode. Operation of the direct evaporation bismuth Hall thruster revealed interesting phenomenon. By utilizing constant current mode on a discharge power supply, the discharge voltage settles out to a stable operating point which is a function of discharge current, anode face area and average pore size on the vaporizer. Oscillations with a 40 second period were also observed. Preliminary performance data suggests that the direct evaporation bismuth Hall thruster performs similar to xenon and krypton Hall thrusters. Plume interrogation with a Retarding Potential Analyzer confirmed that bismuth ions were being efficiently accelerated while Faraday probe data gave a view of the ion density in the exhausted plume.

Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.