Deuteration of human carbonic anhydrase for neutron crystallography: Cell culture media, protein thermostability, and crystallization behavior.

Deuterated proteins and other bio-derived molecules are important for NMR spectroscopy, neutron reflectometry, small angle neutron scattering, and neutron protein crystallography. In the current study we optimized expression media and cell culture conditions to produce high levels of 3 different deuterated human carbonic anhydrases (hCAs). The labeled hCAs were then characterized and tested for deuterium incorporation by mass spectrometry, temperature stability, and propensity to crystallize. The results show that is possible to get very good yields (>10 mg of pure protein per liter of cell culture under deuterated conditions) and that protein solubility is unaffected at the crystallization concentrations tested. Using unlabeled carbon source and recycled heavy water, we were able to get 65-77% deuterium incorporation, sufficient for most neutron-based techniques, and in a very cost-effective way. For most deuterated proteins characterized in the literature, the solubility and thermal stability is reduced. The data reported here is consistent with these observations and it was clear that there are measurable differences between hydrogenous and deuterated versions of the same protein in Tm and how they crystallize.

Localization of sunitinib, its metabolites and its target receptors in tumour-bearing mice: a MALDI-MS imaging study.

BACKGROUND AND PURPOSE: The clinical effects of anti-angiogenic agents remain controversial. Therefore, elucidating the pharmacological properties of these compounds is a pivotal issue. EXPERIMENTAL APPROACH: The effects of treatment with sunitinib on tumour and normal tissues of mice bearing C-26 adenocarcinoma cells were analysed by matrix-assisted laser desorption ionization MS imaging (MALDI-MSI). Expression of the key targets of sunitinib--angiogenic receptors--was studied by immunofluorescent labelling. KEY RESULTS: MALDI-MS assays showed that sunitinib and its fragment ions were present throughout tumour and normal tissues. Major metabolites were identified in blood and solid tissues, while minor drug metabolites were detectable only in blood. Tumour growth and intratumour VEGF receptor-2 expressions were significantly reduced in sunitinib-treated mice, while the expression of the other targeted receptors, PDGF receptor -α or -ß and fibroblast growth factor receptor-1, remained unaffected. Within tumour tissue, the close proximity of sunitinib metabolites to the precursor ion suggested in situ metabolism of the administered drug. There were intratumour areas where the signal intensity of sunitinib correlated with expression of VEGF receptor-2. CONCLUSIONS AND IMPLICATIONS: This is the first study that demonstrates MALDI-MSI is a versatile platform to study the intratumour localization of an unlabelled anti-angiogenic drug. The combination of MALDI-MSI and immunofluorescence analysis can provide further insights into the molecular interaction of drug compounds and their targets within tumour tissue.

Congenic strains displaying similar clinical phenotype of arthritis represent different immunologic models of inflammation.

Proteoglycan (PG)-induced arthritis (PGIA) is an autoimmune inflammatory disease controlled by multiple genes in the murine genome. BALB/c x DBA/2 congenic strains carrying four major PGIA chromosome loci were immunized, and positions of loci on chromosomes 3, 7, 8 and 19 (loci Pgia26, Pgia21, Pgia4 and Pgia12, respectively) were confirmed. Each congenic strain exhibited a different pattern of regulation of clinical and immunologic features of PGIA, and these features were significantly influenced by gender. Locus Pgia26 delayed PGIA onset in males and females, and the effect was associated with a lower rate of antigen-induced lymphocyte proliferation and lower production of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4). Pgia12 similarly delayed onset in males, but the effect was achieved by elevated proliferation of PG-specific lymphocytes and enhanced production of IFN-gamma and IL-4. The effect of the Pgia21 locus was arthritis-suppressive in females but PGIA-permissive in congenic males. These opposite effects are attributed to two-fold higher serum autoantibody and IL-6 levels in males than in females. Our study supports the idea that each congenic strain represents a different immunologic subtype of PGIA, providing an explanation for the complex etiology and various clinical phenotypes of rheumatoid arthritis.

Systemic sclerosis-rheumatoid arthritis overlap syndrome: a unique combination of features suggests a distinct genetic, serological and clinical entity.

OBJECTIVE: To determine the genetic, clinical and serological characteristics of systemic sclerosis (SSc)-rheumatoid arthritis (RA) overlap syndrome. METHODS: Clinical manifestations and immunolaboratory features of 22 SSc-RA patients were assessed. The HLA-DR genotype of the 22 SSc-RA patients determined by SSP-PCR was compared with that of 38 SSc patients, 100 RA patients and 50 healthy controls. RESULTS: All overlap patients fulfilled the American College of Rheumatology (ACR) criteria for SSc and RA. Five of the 22 patients (23%) had diffuse cutaneous SSc (dcSSc) and 17 patients (77%) had limited cutaneous SSc (lcSSc). Antinuclear antibody, anti-Scl70, IgM rheumatoid factor and anti-CCP antibody positivity were detected in 22 (100%), 5 (23%), 16 (73%) and 18 patients (82%), respectively. Seventeen patients (77%) had pulmonary fibrosis, 12 (55%) had oesophageal dismotility, 11 (50%) had cardiac and five (23%) had renal involvement. Hand joint destruction was observed in 18 patients (82%). Significantly increased frequencies of HLA-DR3 (36% vs 5%), HLA-DR7 (9% vs 4%), HLA-DR11 (36% vs 7%) and HLA-DRw53 (23% vs 5%) were observed in SSc-RA compared with RA patients (P < 0.05). Allele frequencies of the 'shared epitope' (HLA-DR1 and -DR4) were significantly increased in SSc-RA (32% and 27%, respectively) and RA patients (46% and 31%, respectively) in comparison with SSc patients (10.5% and 16%, respectively) or healthy controls (16% and 14%, respectively) (P < 0.05). CONCLUSIONS: To date this is the largest SSc-RA overlap cohort. Genetics, clinical and immunolaboratory features suggest a mixed phenotype. Our data suggest that SSc-RA overlap syndrome may be a distinct genetic, immunological and clinical entity.

Enantioseparation of hydroxy acids on easy-to-prepare continuous beds for capillary electrochromatography.

This paper deals with the enantioseparation of hydroxy acids by ligand-exchange capillary electrochromatography. A chiral continuous bed was easily prepared by in situ polymerization of monomers, including an L-4-hydroxyproline derivative. This phase showed chiral recognition for several hydroxy acids, in addition to amino acids.

A new easy-to-prepare homogeneous continuous electrochromatographic bed for enantiomer recognition.

Completely homogeneous polyacrylamide-based gels were used for capillary electrochromatography (CEC) of drug enantiomers. Like continuous beds (also called continuous polymer rods, silica rods, monoliths) they do not require frits to support the bed because it is covalently linked to the capillary wall. A long lifetime is an important feature of the beds. The gel matrices can be prepared in any laboratory and for specific interactions they can be derivatized with appropriate ligands. The application range is, therefore, broad. For chiral electrochromatography, negatively and positively charged polyacrylamide gels copolymerized with 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) were prepared. The latter monomer was synthesized from beta-CD and allylglycidyl ether by a very simple one-step procedure. Eight acidic, neutral and basic drug compounds were resolved into their enantiomers, most of them with baseline separation. Interestingly, the resolution is independent of the electroendosmotic velocity, i.e., rapid analyses will not give low resolution. Upon increasing this velocity, the plate height for the fast enantiomer did not change (or decreased slightly), whereas that for the slow enantiomer increased. Only the last term in the van Deemter equation contributed significantly to the total plate height. The composition of the gel was chosen such that the "pores" became large enough to guarantee a satisfactory electroendosmotic flow (EOF). This open gel structure explains why acetone diffused as in free solution, i.e., independently of the presence of the gel matrix. This finding also indicates that the separation of small molecules in polyacrylamide gels cannot be explained by "molecular-sieving", but rather by some type of adsorption ("aromatic adsorption"?).

Chiral separation of amino acids by ligand-exchange capillary electrochromatography using continuous beds.

A chiral ligand-exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one-step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica-based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.

Chiral separation of alpha-amino acids by ligand-exchange capillary electrophoresis using N-(2-hydroxy-octyl)-L-4-hydroxyproline as a selector.

The direct chiral resolution of underivatized alpha-amino acids by capillary zone electrophoresis (CZE) based on the principle of ligand exchange is described. An N-(2-hydroxyoctyl)-L-4-hydroxyproline/Cu(II) complex was used as a chiral selector. Besides amino acids containing aromatic residues, the basic amino acid histidine was resolved. Baseline separations were obtained for all amino acids investigated. The influence of selector concentration, electrolyte composition and pH on the resolution was investigated. It was found that there is a correlation between pI of the amino acids and the optimal pH.

New set-up for capillary isoelectric focusing in uncoated capillaries.

Substituted aminomethylphenol dyes, low-molecular-mass isoelectric point (pI) markers and hemoglobin samples from normal individuals and diabetic patients were used to test a new set-up of capillary isoelectric focusing (cIEF) in uncoated capillaries. In previous cIEF methods, a mixture of sample components and carrier ampholytes was applied in the capillary and analyzed. In the new set-up a fractionated injection protocol is used to apply a 'sandwich' ampholyte-sample-ampholyte plug in the capillary for analysis. This new set-up allows the separation of amphoteric compounds having pI values outside the pH region of the ampholytes applied in the capillary with high precision. The high resolution power of this technique was proven with the analysis of hemoglobin variants.

An approach to ideal separation media for (electro)chromatography.

In packed chromatographic beds, both Eddy diffusion and the relatively long time the analytes stay in the mobile phase until they collide and interact with the ligands attached to the beads (the residence time in the mobile phase) contribute considerably to zone spreading. One should not expect Eddy diffusion to occur in macroscopically homogeneous separation media, such as gels or polymer solutions, and residence time should be shorter, since the pore size of these media is much smaller than the average distance between the beads in a packed bed. Accordingly, these separation media (which can be regarded as homogeneous continuous beds [monoliths]) should theoretically give very high resolution, which has been verified experimentally: Frontal analysis of a neutral marker, acetone, showed that electroendosmosis in a homogeneous gel displaced the boundary without any distortion except that caused by diffusion (the marker was selected not to interact with the gel). All other common disturbing phenomena in chromatography (for instance, Eddy diffusion and nonspecific adsorption) were, accordingly, negligible, indicating that electrochromatography in homogeneous gels may be the ideal chromatographic method. However, to fully utilize these desirable chromatographic properties of homogeneous continuous beds, one has to choose analyte/bed interactions with sufficiently high association-dissociation rate constants (i.e., the residence time of the analytes in the stationary phase must be kept very short), which will be the subject of forthcoming studies. The electrophoretic counterpart of capillary electrochromatography (CEC), electrophoresis of noncharged analytes in a solution of charged polymers, seems to give a somewhat larger zone broadening, probably due to their high viscosity and conductivity, with attendant longer analysis (= diffusional) times compared to gels. Since the mobile phase is propelled through the gels by electroendosmosis, the theoretical and experimental requirements for high electroendosmotic flow in gels, i.e., short analysis times, are discussed. The electroendosmotic velocity v(x) can be estimated by the simple equation v(x) = Vmax (1-e-kappa x) (1/kappa = the thickness of the double layer; Vmax = the plug flow velocity) when the distance from the channel wall, x, << the radius R of the channel (pore). For kappa R > or = 5, the equation obtains with good approximation for all R values. An initially straight zone in a gel pore should be heavily distorted by the electroendosmotic flow, according to this equation (see Figure 1). However, due to rapid diffusion and other leveling effects, the zone is transported as in perfect plug flow, as is shown experimentally. A plot of electroendosmotic mobility obtained by frontal analysis against 1/(1 + square root of mu) can be used to estimate roughly the pore size in a gel and permits quantitative examination of the current theory of electroendosmosis. It is not a trivial problem to synthesize ligand-containing gels with pores large enough to allow a high electroendosmotic flow. Therefore, we have described a universal method: a polymer containing phenylboronate and acrylic acid groups was synthesized and entrapped in a standard agarose gel (for automated runs, replaceable methoxylated agarose should be used). Both of these charged groups serve to generate the electroendosmotic flow required in electrochromatography. This gel was designed to have the dual property of separating compounds that contain vicinal OH groups in the cis-configuration (exemplified by ribonucleosides) by reaction with the boronate groups, and aromatic substances by virtue of the acrylic acid residues (and perhaps also the phenyl groups) in the polymer and the agarose chains. The latter interaction, the so-called aromatic adsorption, has the advantage that it does not require a time-consuming attachment of ligands. (ABSTRACT TRUNCATED)