Single-step Transformation Research Articles

Dual-Functional ROMP-Based Betaines: Effect of Hydrophilicity and Backbone Structure on Nonfouling Properties

Foundational materials for nonfouling coatings were designed and synthesized from a series of novel dual-functional zwitterionic polymers, Poly[NRZI], which were easily obtained via ring-opening metathesis polymerization (ROMP) followed by a single step transformation of the cationic precursor, Poly[NR(+)], to the zwitterion, Poly[NRZI]. The resulting unique dual-functional structure contained the anion and the cation within the same repeat unit but on separate side chains, enabling the hydrophilicity of the system to be tuned at the repeat unit level. These dual-functional zwitterionic polymers were specifically designed to investigate the impact of structural changes, including the backbone, hydrophilicity, and charge, on the overall nonfouling properties. To evaluate the importance of backbone structure, and as a direct comparison to previously studied methacrylate-based betaines, norbornene-based carbo- and sulfobetaines (Poly[NCarboZI] and Poly[NSulfoZI]) as well as a methacrylate-based sulfobetaine (Poly[MASulfoZI]) were synthesized. These structures contain the anion-cation pairs on the same side chain. Nonfouling coatings were prepared from copolymers, composed of the zwitterionic/cationic precursor monomer and an ethoxysilane-containing monomer. The coatings were evaluated by using protein adsorption studies, which clearly indicated that the overall hydrophilicity has a major influence on the nonfouling character of the materials. The most hydrophilic coating, from the oligoethylene glycol (OEG)-containing dual-functional betaine, Poly[NOEGZI-co-NSi], showed the best resistance to nonspecific protein adsorption (Γ(FIB) = 0.039 ng/mm(2)). Both norbornene-based polymers systems, Poly[NSulfoZI] and Poly[NCarboZI], were more hydrophilic and thus more resistant to protein adsorption than the methacrylate-based Poly[MASulfoZI]. Comparing the protein resistance of the dual-functional zwitterionic coatings, Poly[NRZI-co-NSi], to that of their cationic counterparts, Poly[NR(+)-co-NSi], revealed the importance of screening electrostatic interactions. The adsorption of negatively charged proteins on zwitterionic coatings was significantly less, despite the fact that both coatings had similar wetting properties. These results demonstrate that the unique, tunable dual-functional zwitterionic polymers reported here can be used to make coatings that are highly efficient at resisting protein adsorption.

Synthesis and Structural Investigations of Functionalizable Hybrid β-Hairpin

The solution and solid state conformations of a designed β-hairpin containing functionalizable α,β-unsaturated γ-amino acids at the antiparallel β-strands and a single step transformation to its saturated γ-peptide analogue are studied.

The effects of grain size on the phase transformation properties of annealed (Ti/Ni/W) shape memory alloy multilayers

(Ti/Ni/W)n multilayer films were annealed to form a two-phase (B2-TiNi and β-W) system. Grain sizes extracted from X-ray diffraction profiles of annealed films revealed that B2-TiNi decreases with increasing W, due to the immiscible W layers obstructing its grain growth. With decreasing B2-TiNi grain size the Rs (B2–R) transformation temperature is not affected but the Ms (R–B19′) transformation temperature decreases significantly. Thus the addition of W to Ti–Ni is effective to induce the B2–R single-step transformation due to grain size effects.

Practical and reliable synthesis of dialkyl N-arylphosphoramidates with nitroarenes as substrates

A new single-step transformation of readily available nitroarenes with trialkyl phosphites, which can be performed both under thermal and microwave conditions, delivers dialkyl N-arylphosphoramidates in good yields and short reaction times.

Evaluation of MAT-vector system in white poplar (Populus alba L.) and production of ipt marker-free transgenic plants by ‘single-step transformation’

Genetic transformation of an elite white poplar genotype (Populus alba L., cv. ‘Villafranca’) was performed with MAT vectors carrying the ipt and rol genes from Agrobacterium spp. as morphological markers. The effects associated with the use of different gene promoters and distinct in vitro regeneration protocols were evaluated. Poplar plantlets showing abnormal ipt and rol phenotypes were produced only in the presence of exogenous growth regulators. The occurrence of abnormal ipt and rol phenotypes allowed the visual selection of transformants. The ipt-type MAT vector pEXM2 was used to monitor the activity of the yeast site-specific recombination R/RS system in the transformed white poplar cells. Results from these experiments demonstrated that recombinase-mediated excision events occurred during the early stages of in vitro culture, thus causing the direct production of ipt marker-free transgenic plants with normal phenotype at an estimated frequency of 36.4%. Beside this unexpected finding, transgenic ipt-shooty plants were obtained at a frequency of 63.6% and normal shoots were subsequently recovered after a prolonged period of in vitro culture. Although the transformation efficiency observed in this study, using both ipt and nptII genes as selection markers, was similar to that previously reported with standard vectors carrying only the nptII gene, the easy identification of ipt transformants, the early recombinase-mediated excision events and finally the relatively short time period required to produce ipt marker-free transgenic plants support for the choice of MAT vectors as a reliable strategy for the future production of marker-free GM poplars.

Catalytic diastereo- and positionselective oxidative mono-cyclization of 1,5,9-trienes and polyenes

Ruthenium tetroxide (1 mol%) has been used as a catalyst for the oxidative mono-cyclization of 1,5,9-trienes and polyenes. The poly-unsaturated substrates underwent mono-cyclization with a high degree of diastereo- and positionselectivity to produce mono-tetrahydrofuran diols with a varying degree of unsaturation. Up to four new stereogenic centers were created in this single step transformation. The remarkable positionselectivity appears to be a result of relative electronic properties of the double bonds within the polyolefinic substrates in conjunction with conformational constraints.

Dormant Wnt-Initiated Mammary Cancer Can Participate in Reconstituting Functional Mammary Glands

The minimal residual disease foci that beget breast cancer relapse after a period of disease dormancy remain uncharacterized despite their enormous clinical importance. To model dormant breast cancer in vivo, we employed a transgenic mouse model in which Wnt1-initiated mammary cancer is doxycycline dependent. After regression of Wnt-dependent cancers, subclinical disease lesions were propagated in vivo using classical tissue recombination techniques. Surprisingly, outgrowths derived from dormant malignant tissue reconstituted morphologically normal ductal trees in wild-type mammary fat pads. Whereas hyperplasia-derived outgrowths remained benign, outgrowths derived from dormant malignancy underwent a morphological transition suggesting single-step transformation following reactivation of Wnt signaling and rapidly yielded invasive mammary tumors. Remarkably, outgrowths derived from dormant malignancy could be serially propagated in vivo and retained the potential to undergo lobuloalveolar differentiation in response to hormones of pregnancy. Matching somatic H-Ras mutations shared by antecedent tumors and descendant mammary ductal outgrowths confirmed their clonal relatedness. Thus, propagation of epithelium that possesses a latent malignant growth program reveals impressive regenerative and developmental potential, supporting the notion that dormant mammary cancers harbor transformed mammary progenitor cells. Our results define an experimental paradigm for elucidating biological properties of dormant malignancy.

Some features of Ni–Fe–Ga shape memory alloys under compression

Three Ni–Fe–Ga ferromagnetic polycrystalline alloys having a martensitic transformation close to room temperature have been produced. Some results on their stress–strain behaviour under compression will be presented in this work. At temperatures below A f (finish of the reverse martensitic transformation), multiple steps in the stress–strain curves indicate the possibility of stress-induced martensite to martensite transformations. In this temperature domain, the critical stress to induce the variant reorientation and intermartensitic transformations is nearly temperature independent. In addition, the alloys show almost perfect strain recovery on unloading, that is the so-called rubber-like effect. As the temperature is increased ( T ⩾ A f ) the multiple step stress–strain curves evolve to a single-step transformation corresponding to stress-induced martensite from the parent phase.

Calorimetric study of multiple-step transformation in TiNi shape memory alloy with partial transformation cycle

Single-step transformation was observed for Ti–50.6 at.%Ni shape memory alloy (SMA) solution annealed at 800 °C for 20 min and quenched to 0 °C. However, multiple-step transformation (MST) occurred after further aging at 380–500 °C for 1 h. Aging at 380 °C resulted in three distinct DSC peaks upon cooling and two peaks upon heating. Aging at 450 °C resulted in three distinct DSC peaks upon cooling and one peak upon heating. There were two peaks that emerge upon cooling and one peak upon heating for the samples aged at 500 °C. The multiple-step transformation was studied by differential scanning calorimetry (DSC) with partial transformation cycle. The multiple-step transformation was explained by the heterogeneous microstructures. The matrix in the precipitate Ti 3Ni 4 region near the grain boundary transformed from parent phase (B2) to R phase and further to martensite (B19′) which contribute to the first and second peaks upon cooling, and B19′→R→B2 transformation happens upon heating. Whereas the matrix in the precipitate free grain interiors B2 transformed into martensite directly which contribute to the third peak upon cooling.

Calorimetric study of multiple-step transformation in TiNi shape memory alloy with partial transformation cycle

Single-step transformation was observed for Ti–50.6 at.%Ni shape memory alloy (SMA) solution annealed at 800 °C for 20 min and quenched to 0 °C. However, multiple-step transformation (MST) occurred after further aging at 380–500 °C for 1 h. Aging at 380 °C resulted in three distinct DSC peaks upon cooling and two peaks upon heating. Aging at 450 °C resulted in three distinct DSC peaks upon cooling and one peak upon heating. There were two peaks that emerge upon cooling and one peak upon heating for the samples aged at 500 °C. The multiple-step transformation was studied by differential scanning calorimetry (DSC) with partial transformation cycle. The multiple-step transformation was explained by the heterogeneous microstructures. The matrix in the precipitate Ti 3 Ni 4 region near the grain boundary transformed from parent phase (B2) to R phase and further to martensite (B19′) which contribute to the first and second peaks upon cooling, and B19′→R→B2 transformation happens upon heating. Whereas the matrix in the precipitate free grain interiors B2 transformed into martensite directly which contribute to the third peak upon cooling.

Single Step Conversion of Chiral Carnitine and Derivatives into (S)- and (R)-β-Substituted-γ-Butyrolactones

This paper describes an efficient single step transformation of chiral carnitine and carnitine derivatives into stereoisomerically pure (S)- and (R)-β-substituted-γ-butyrolactones, obtained by intramolecular nucleophilic displacement. (S)- Or (R)-carnitine and (R)-aminocarnitine inner salts give β-hydroxy-γ-butyrolactone and β-amino-γ-butyrolactone respectively (82% and 77%) with the same configuration as the starting material. (R)-Acetylaminocarnitine inner salt gives (R)-β-acetylamino-γ-butyrolactone (90%), while (R)-acetylcarnitine gives 2(5H)-furanone under the same reaction conditions (77%, via cyclization and subsequent elimination reaction). (R)-N-Benzyloxycarnitineamide gives a mixture of pyrrolidinone (11%) and furanoyl imidate (50%) derivatives. The direct transformation of wast (S)-carnitine into the valuable (S)-β-hydroxy-γ-butyrolactone or (after acetylation) into the precious 2(5H)-furanone is of particular interest.

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4.

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.

Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system.

The ipt-type MAT vector uses the ipt gene for regeneration of marker-free transgenic plants. However, it was pointed out that this system was not suitable for most economically important crops that regenerated through auxin-dependent embryogenesis. We report a single-step transformation system of rice using MAT vector. When we transformed scutellum tissues of 5 days pre-cultured rice seeds, marker-free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming ipt-intermediates within 4 weeks after an infection. Excision of the ipt gene caused the regeneration of marker-free transgenic rice plants through embryogenic tissues. Therefore, this system needs no selective agent and no sexual crossing for identification of transgenic plants not containing a selectable marker gene. This system is highly effective for generation of marker-free transgenic plants in economically important crops.

Systems for the removal of a selection marker and their combination with a positive marker

Many systems have been developed for the removal of a selection marker in order to generate marker-free transgenic plants. These systems consist of (1) a site-specific recombination system (Cre/lox) or a phage-attachment region (attP) to remove the selectable marker gene and (2) a transposable element system (Ac) or a co-transformation system to segregate the gene of interest from the selectable marker gene. Overall, the process is more time-consuming than conventional transformation methods because two rounds of transformation - two steps of regeneration or sexual crossings - are required to obtain the desired transgenic plants. Recently, removal systems combined with a positive marker, denoted as MAT vectors, have been developed to save time and effort by generating marker-free transgenic plants through a single-step transformation. We summarize here the transformation procedures using these systems and discuss their feasibility for practical use.

Rac is Essential in the Transformation of Endothelial Cells by Polyoma Middle T

Expression of the Polyoma Middle T (PyMT) antigen in endothelial cells results in single-step transformation to hemangioma producing malignant cells. To study the mechanism of PyMT transformation, we used the PyMT induced mouse brain endothelial cell line, bEND.3, expressing constitutively active and dominant negative mutants of the small GTPase Rac. The bEND.3 cell phenotype of tumorigenesis, loss of normal growth control and formation of cysts rather than capillary tubes in fibrin gels was reversed by expression of dominant negative Rac. The mechanism of N17 Rac action in blocking the endothelial cell transformant, PyMT, did not involve effects of Rac on the actin cytoskeleton since this component of the bEND.3 cell phenotype was not affected. Furthermore, the PyMT induced activation of the plasminogen activator (PA)/plasmin system was not affected by Rac inhibition. Inhibition of the downstream effectors of Rac, phosphatidylinositol 3-kinase (PI3-K) and p70S6k, which are known to be constitutively activated by PyMT transformation, inhibited bEND.3 cell proliferation and cyst formation in fibrin gels even in cells expressing V12 constitutively active Rac, but they did not restore capillary tube formation. These results demonstrate that middle T antigen induced endothelial cell transformation requires signal transduction by Rac. The downstream Rac effectors, P13-K and p70S6k, mediate PyMT/Rac effects on cell proliferation and cyst formation, but other unknown effectors of PyMT are required for the cytoskeletal changes and activation of the PA/plasmin system.

The genetic penetrance of the activated neu oncogene for the induction of mammary cancer in vivo.

Carcinogenesis is most often viewed as a multistage disease process. An exception to this was suggested for neu transformation of mammary cells in a transgenic model (Muller et al., 1988); however, this interpretation is controversial (Bouchard et al., 1989). In order to better define neu mammary transformation in vivo, we directly measured the genetic penetrance of the neu oncogene. Mammary cells in situ were infected with replication-defective retroviral vectors carrying the activated neu oncogene (pJRneu). A limiting dilution in vivo transplantation assay was used to measure the percentage of mammary clonogenic (stem-like) cells that stably and functionally integrated this vector. Based on this, the percentage of clonogens integrating and expressing neu that could progress to mammary carcinomas was quantified to estimate the penetrance of this gene in mammary carcinogenesis. The genetic penetrance of neu was 3.6% (95% confidence interval 2.2%-5.8%). This high degree of genetic penetrance is compatible with the observations that certain neu-transgenic mice develop a very great number of mammary carcinomas (Muller et al., 1988). However, whether these data are compatible with a single-step transformation model (100% penetrance) is uncertain and is discussed.

Control of recombination rate during transformation of Streptococcus pneumoniae: an overview.

Despite the fact that natural transformation was described long ago in Streptococcus pneumoniae, only a limited number of recombination genes have been identified. Two of them have recently been characterized at the molecular level, recA which encodes a protein essential for homologous recombination and mmsA which encodes the homologue of the Escherichia coli RecG protein. After a survey of the available information regarding the function of RecA, RecG, and other proteins such as the mismatch repair proteins HexA and HexB that can affect the outcome of recombinants, the different levels at which horizontal genetic exchange can be controlled are discussed. It is shown that the specific induction of the recA gene which occurs in competent cells is required for full recombination proficiency. Results regarding the ability of the Hex generalized mismatch repair system to prevent recombination between partially divergent sequences during transformation are also summarized. A structural analysis of homeologous recombinants which suggests that formation of mosaic recombinants can occur independently of mismatch repair in a single-step transformation is also reported. Finally, arguments in favor of an evolutionary origin of transformation as a means of genome evolution are discussed and the different types of recombination events observed which could potentially contribute to S. pneumoniae genome evolution are listed.

Oxidative stress-regulated gene expression and promotion of morphological transformation induced in C3H/10T1/2 cells by ammonium metavanadate

Promoters of C3H/10TI/2 cell morphological transformation that elevate intracellular oxidant levels can be distinguished by a spectrum of induced gene expression, which includes the oxidant-responsive murine proliferin gene family. Proliferin transcripts were induced 40- to 100-fold by 20 μM ammonium metavanadate, 20-fold by 5 μM vanadium pentoxide but only three-fold by vanadium oxide sulfate. Consistent with its response to other oxidant chemicals, induction of proliferin by ammonium metavanadate was inhibited almost completely by the antioxidant N-acetylcysteine (8 mM). Ammonium metavanadate (5 μM), added as promoter in two-stage morphological transformation assays, amplified yields of Type II and Type III foci in monolayers of 20-methylcholanthrene-initiated C3H/10T1/2 cells. Ammonium metavanadate also induced formation of Type II foci in single-step transformation assays. The results suggest that pentavalent vanadium compounds could promote morphological transformation in C3H/10T1/2 cells by creating a cellular state of oxidative stress, sufficient to induce elevated expression of the proliferin gene family.

Hormonal modulation of neu-induced mammary carcinogenesis: evidence against a single-step induction of mammary cancer by neu.

When activated by a point mutation in its transmembrane domain neu is an extremely potent transforming oncogene when expressed in rodent mammary parenchyma. This extreme potency led Muller et al. (13) to suggest the possibility that neu might transform mammary cells by a single step. A corollary to a single-step transformation hypothesis is that transformation efficiency cannot be modulated. Here we test this corollary by introducing mutated neu into in situ rat mammary cells using retroviral vectors and following infection by hormonally modulating recipient rats. Hormonal modulation included raising prolactin levels and lowering glucocorticoid levels. This modulation promotes radiation, chemical and ras initiated mammary carcinogenesis. Here we report that this hormonal promotion protocol also increased the yield of mammary carcinomas following the introduction of the mutated neu gene. We conclude that mutated neu is not a single-step transformer but is a very potent event in the multistage process of mammary carcinogenesis.

Pennsylvania railroad shops at West Philadelphia

Reaction of sodium 2-((E)-((Z)-4-hydroxypent-3-en-2-ylidene)amino)succinate and sodium (E)-2-((1-(2-hydroxyphenyl)ethylidene)amino)succinate (L1HNa2 and L2HNa2) in a 1:2 M ratio with Bz2SnCl2 in methanol gave dinuclear diorganotin(IV) complexes of composition [Bz2Sn(Lx)SnBz2Cl] (Lx = L1 (1) and L2 (2)). Reaction of L2HNa2 and Bz3SnCl (1:2) in anhydrous toluene under reflux conditions yielded the analogous chlorine-free product of composition [Bz2Sn(L2)SnBz3] (3) via cleavage of a Sn-C bond for one of the triorganotin precursor equivalents with elimination of toluene. In addition, the tributyltin(IV) compounds of composition [Bu3Sn(LxH)SnBu3] (Lx = L2 (4) and L3 (5)) were prepared in a single-step transformation by combining equimolar proportions of (Bu3Sn)2O, (S)-2-aminosuccinic acid and 2'-hydroxyacetophenone/2-hydroxybenzaldehyde in anhydrous methanol. The structures of the organotin(IV) compounds in solution were assessed using 1H, 13C and 119Sn NMR spectroscopic techniques, while the molecular structures of 1–5 were analyzed by single-crystal X-ray diffraction. In the solid state, complexes 1-3 exhibit one-dimensional coordination polymers with alternate six- and seven-coordinate tin atoms embedded in distorted monocapped trigonal-bipyramidal (1-2)/octahedral (3) and pentagonal-bipyramidal (1-2)/bicapped trigonal-bipyramidal (3) coordination environments. On the contrary, compounds 4–5 are two-dimensional coordination polymers based on 30-membered tetranuclear macrocyclic ring structures, in which the tin atoms comprise distorted trigonal-bipyramidal coordination polyhedra. However, in solution the polymeric structures of 1–5 dissociate to give dinuclear complexes, even in non-coordinating solvents such as chloroform.