Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine

Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.     

Multiplicity theorems for superlinear elliptic problems

In this paper we study second order elliptic equations driven by the Laplacian and p-Laplacian differential operators and a nonlinearity which is (p-)superlinear (it satisfies the Ambrosetti–Rabinowitz condition). For the p-Laplacian equations we prove the existence of five nontrivial smooth solutions, namely two positive, two negative and a nodal solution. Finally we indicate how in the semilinear case, Morse theory can be used to produce six nontrivial solutions. This paper was completed while the first author was visiting the University of Aveiro as an Invited Scientist. The hospitality and financial support of the host institution are gratefully acknowledged. The second and third authors acknowledge the partial financial support of the Portuguese Foundation for Science and Technology (FCT) under the research project POCI/MAT/55524/2004.     

Monotonicity of some functions involving the gamma and psi functions

Let , where is Euler's gamma function. We determine conditions for the numbers so that the function is strongly completely monotonic on . Through this result we obtain some inequalities involving the ratio of gamma functions and provide some applications in the context of trigonometric sum estimation. We also give several other examples of strongly completely monotonic functions defined in terms of and functions. Some limiting and particular cases are also considered.

     

Low regularity global well-posedness for the Zakharov and Klein-Gordon-Schrödinger systems

We prove low regularity global well-posedness for the 1d Zakharov system and the 3d Klein-Gordon-Schrödinger system, which are systems in two variables and . The Zakharov system is known to be locally well-posed in and the Klein-Gordon-Schrödinger system is known to be locally well-posed in . Here, we show that the Zakharov and Klein-Gordon-Schrödinger systems are globally well-posed in these spaces, respectively, by using an available conservation law for the norm of and controlling the growth of via the estimates in the local theory.

     

Fabrication of hollow titania microspheres with tailored shell thickness

Submicron hollow spheres are an interesting class of materials that receive significant attention nowadays. Closed and mechanically robust homogeneous hollow titania microspheres with as much shell thickness as 130 nm were fabricated by coating polystyrene beads with titania nanoparticles using sol–gel chemistry and subsequently removing the core either via heating or a chemical dissolution process. The thickness of the titania shell deposited on polystyrene core was finely tuned between 100 and 130 nm by varying the concentration of titania precursor, i.e., Ti(OEt)₄ salt from 0.5 to 2 mM during the coating process. The obtained hybrid core–shell particles and hollow microspheres were characterized by scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, X-ray diffraction, and thermo-gravimetric analysis. The approach employed is well suited to the preparation of titania-coated polystyrene hybrid particles and hollow titania spheres, which can find their applications as novel building blocks with unique optical properties for fabrication of advanced materials, catalyst, and drug delivery system.     

Platinum group metals bulk analysis in automobile catalyst recycling material by laser-induced breakdown spectroscopy

Development and application of an in-situ applicable method to provide rapid determination of platinum group metals (platinum, palladium, and rhodium) elemental concentration in automobile catalyst scrap is reported. Application is based on laser-induced breakdown spectroscopy (LIBS). Actual automobile catalyst slurry in powder form was used to develop the application. With a method requiring approximately 1.5 min of examination per sample, calibration curves are presented with linear regression coefficients close to 0.99 and stability better than 3.0%.     

Synthesis of novel tantalum oxide sub-micrometer hollow spheres with tailored shell thickness

Sub-micrometer-sized hollow tantalum oxide (Ta2O5) spheres with tunable shell thickness and void size have been fabricated exploiting beta-diketone-functionalized polystyrene (PS) beads as sacrificial templates in a sol-gel process. First, a controlled precipitation of Ta2O5 nanoparticles was carried out on the template surface by hydrolyzing tantalum ethoxide (Ta(OEt)5) at room temperature, and subsequently, the polymer core was removed either via chemical treatment with toluene or calcination at 650 degrees C. The thickness of the tantala shell precipitated on the PS core during the coating process was tuned between 100 and 142 nm by varying the concentration of tantala precursor in the reaction media. The obtained Ta2O5-coated PS particles and hollow microspheres were characterized by scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis. Due to the unique optical and dielectric properties, these nanostructured materials are envisaged to be used in applications such as novel building blocks for the fabrication of advanced materials, surface coatings, catalysts, and drug delivery systems.     

NMR study of 5-substituted pyrazolo[3,4-c]pyridine derivatives

Substituted pyrazolopyridines are potent inhibitors of phosphodiesterases and cyclin-dependent kinases. In this study, NMR was used to investigate the potential N1-H and N2-H tautomerism of 5-substituted pyrazolo[3,4-c]pyridine derivatives. Six compounds were fully characterized by using (1)H, (13)C, and (15)N chemical shifts and indirect (1)H--(13)C and (1)H--(15)N coupling constants. The (1)H NMR spectra were measured over a broad range of temperatures. All of the compounds were shown to exist predominantly in the N1-H tautomeric form. Complementary quantum-chemical calculations of the chemical shieldings and indirect spin-spin couplings support the structural conclusions drawn.     

Synthesis, characterization, and remarkable biological properties of cyclodextrins bearing guanidinoalkylamino and aminoalkylamino groups on their primary side

The introduction of aminoalkylamino and guanidinoalkylamino substituents on the primary side of beta- and gamma-cyclodextrin (CDs) resulted in a series of novel compounds that were extensively characterized by NMR spectroscopy and mass spectrometry. Bromination of the primary side of beta- and gamma-CD, and reaction with neat alkylene diamines at a pressure of 7 atm afforded aminoalkylamino derivatives that were then guanylated at the primary amino group to give the corresponding guanidinoalkylamino-CDs. These compounds are water soluble and display pK(a) values that allow them to be mostly protonated at neutral pH; for example, pK(a(1)) approximately 6.4 and pK(a(2)) approximately 9.5 for the aminoethylamino-beta-CD and pK(a(1)) approximately 7.8 and pK(a(2)) approximately 11.0 for the guanidinoethylamino-beta-CD. The title CDs are rigid, cyclic alpha-D-glucopyranose oligomers (heptamers or octamers) with branches that resemble lysine and arginine side chains that enable multiple interactions with suitable substrates. Thus, they bear similarities to known cell-penetrating peptides. Indeed, the compounds were found to cross the membranes of HeLa cells and penetrate inside the cytoplasm quickly, the guadinylated ones within 15 min, as shown by fluorescence microscopy using fluorescein-labeled derivatives. The toxicity of the compounds, measured by performing MTT tests, ranged from 50 to 300 microM. Furthermore, some of the aminated CDs could facilitate the transfection of DNA expressing the green fluorescent protein (GFP) in HEK 293T cells, with effectiveness comparable to the commercial agent Lipofectamine 2000. Circular dichroism, atomic force microscopy and electrophoresis experiments confirmed the strong interaction of the compounds with DNA. Because of their carbohydrate, non-peptide nature the title compounds are not anticipated to be enzymatically labile or immunogenic, and thus they fulfill many of the criteria for non-hazardous transport vectors in biological and pharmaceutical applications.