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SH2D1A gene defects are the cause of X-linked lymphoproliferative disorder (XLP-1), a rare condition characterized by severe immune dysregulation. We present a patient lacking the typical symptoms of XLP-1, but experiencing a severe unusual skin condition encompassing features of dermatosclerosis and vesiculobullous skin disease. A maternal cousin of the patient was diagnosed with XLP-1 and found to carry a deletion of the SH2D1A gene. SH2D1A deletion was also identified in our patient, which offered a possible explanation for his skin symptoms. Subsequent analysis showed that the deletion in both cousins was identical and involved the whole SH2D1A gene and a part of the adjacent ODZ1 gene. High phenotypic variability of XLP-1 observed in this family prompted us to analyze the genotype-phenotype correlation of 2 different-sized deletions involving SH2D1A and ODZ1 in 5 patients from 2 families, and we report the clinical and laboratory data on these individuals. Our findings illustrate the wide clinical variability of XLP-1, both inter- and intrafamilial, which may complicate the diagnosis of this condition. The comparison of phenotypes of our patients argues against a strong involvement of the ODZ1 gene in the skin disorder and other symptoms observed in our index patient. His hitherto not described severe skin condition extends the phenotypic range of XLP-1.

For the first time, the application of a commercial Shimadzu microchip electrophoresis system MCE-2010 equipped with an imaging UV detector for isoelectric focusing (IEF) of therapeutic proteins is reported. By proper adjustment of the pH gradient, samples with pI values ranging from 2.85 to 10.3 can be focused to the imaged part of the separation channel. Three therapeutic proteins (hirudin, erythropoietin, and bevacizumab) have been successfully focused on the microchip, and the results have been compared to conventional capillary IEF in terms of peak profile, pI values, and reproducibility.

An electrophoretic method for simultaneous determination of catecholamines and their O-methoxylated metabolites on the microchip as well as in the capillary is presented. A complex separation system employing sodium dodecyl sulfate (SDS) micelles, dendrimers forming a second pseudostationary phase and borate complexation is needed for the satisfactory separation of the selected compounds on the short migration length. A carbon nanotube-modified working electrode has been applied for the sensitive amperometric detection with submicromolar detection limits. The applicability of this new method for the analytics of real samples is demonstrated by analysis of mouse brain homogenate on the microchip and human urine by capillary electrophoresis.

Electrophoretic mobilities and shapes of zones of sanguinarine and chelerythrine in aqueous media around pH 7 are affected by limited solubility of their uncharged forms and by the pH-dependent chemical equilibrium between cationic and uncharged forms of these alkaloids. The sanguinarine solubility in sodium MOPS of pH 7.4 was estimated at 50 micromol x L(-1). Sanguinarine zones in this buffer have the shape of tailed peak with concentration-independent mobility if the injected sanguinarine concentration exceeds this solubility limit only slightly. The chelerythrine solubility is higher because of lower dissociation constants of its cations. Precipitation of sanguinarine and chelerythrine with the phosphate anions decelerates their electrophoretic transport in phosphate buffer. Sanguinarine solubility is 5 micromol x L(-1) at the most in 13 mmol x L(-1) sodium phosphate buffer of pH 7.4. Acidifying of the sample up to pH 3 decreases the tailing of the peaks of sanguinarine and chelerythrine and contributes to the rise of sharp maxima of their migrating zones. Any capillary coating deteriorates the peak shape.

Detection of biogenic monoamines in nanomolar concentrations is of great importance for probing the brain chemistry and for their analytics in biological fluids. The sensitivity enhancement of amperometric detection of neurotransmitters (NTs) and their metabolites after their electrophoretic separation on a microchip is presented and is based on coupled enzymatic reactions. The current response of the analyte is amplified by cyclic oxidation on a gold electrode mediated by reduced nicotinamide dinucleotide coenzyme and glucose oxidase enzyme present in the electrophoresis buffer. Using this approach, detection limits of about 10 nM for NTs and their metabolites can be reached.

Capillary zone electrophoresis was applied for the investigation of interactions of some quaternary isoquinoline alkaloids, namely sanguinarine, chelerythrine, berberine, and jatrorrhizine, with DNA constituents and with DNA. None of these alkaloids attach covalently to nucleotides or to the whole DNA under physiological conditions. The interaction with DNA constituents is a noncovalent complexation based on weak intermolecular forces. Electrostatic attraction participates in the interaction but other types of intermolecular forces are involved as well. Cations were identified as the most probable interacting forms of the alkaloids. The interaction with compounds derived from purine was always stronger than those derived from pyrimidine. All alkaloids behaved analogously and similarly to ethidium bromide, the classic DNA intercalator. Stability constants K (in l.mol(-1)) for sanguinarine and chelerythrine in phosphate buffer of pH 7.4 (I(S) = 30 mM) ranged from tens to hundreds.

Electrophoretic and photometric experiments strongly indicate that monovalent anions, which arise by deprotonation of the nitrogen atom in zwitterionic Good's buffers 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) and 3-morpholinopropanesulfonic acid (MOPS), spontaneously aggregate. Cationic migration of sanguinarine (SA) and chelerythrine (CHE) in highly alkaline 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris-propane), in which the concentration of cations of both alkaloids is negligible, may be explained by the existence of an aggregate, which contains uncharged sanguinarine or chelerythrine and one monovalent cation of Bis-Tris-propane at least. Tendency of tris(hydroxymethyl)aminomethane (Tris), bis (2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) and Bis-Tris-propane cations to ion pairing with synthetic cluster borane anions and with fused silica markedly rises up with the size and charge of these cations. The drop in mobility of cluster borane compounds sometimes exceeds 50% of their mobility found at identical pH and ionic strength in buffers with sodium cation. The electroosmosis drop approached 70% if background electrolyte contained Bis-Tris-propane cations instead of sodium cations. Nitrate, taken as a model inorganic ion, and four randomly chosen organic anions interacted markedly less with Tris, Bis-Tris and Bis-Tris-propane cations than cluster borane anions. 2-(N-morpholino)ethanesulfonic (MES) acid anions present in background electrolyte affect the ion pairing of Tris, Bis-Tris and Bis-Tris-propane cations with anionic analytes and, in this way influence also mobilites of these anionic analytes. Limited hydrophilicity at least one of interacting species appears to be the most probable cause of observed intermolecular interactions of biological buffers.

Capillary zone electrophoresis with UV detection was used for determination of dissociation constants of alkaloids sanguinarine and chelerythrine. Despite the limited solubility of the uncharged forms of the alkaloids resulting in insufficient analytical signal at higher pH the reliable dissociation constants were obtained when acidified samples containing low amount of the alkaloid were injected into the capillary. The precipitation of the alkaloid in the capillary induced by injecting sample of low pH into the background electrolyte of higher pH does not affect the mobility of the alkaloid if its concentration injected exceeds the solubility only to a small extent. Dissociation constants (pK(R+)) of sanguinarine and chelerythrine calculated to 8.3 +/- 0.1 and 9.2 +/- 0.1, respectively, are relevant to Good buffers of ionic strength of 30 mM.

Using mercaptoethanol and (L)-cysteine as representatives of mercapto compounds and capillary zone electrophoresis as experimental technique, it was evidenced that sanguinarine and chelerythrine do not react with the mercapto group of organic compounds at pH 7.4. Their interaction is fast and reversible complexation based on non-bonding intermolecular interaction in which enter uncharged forms of sanguinarine or chelerythrine. A negatively charged group, either bound to the mercapto ligand or supplied from solution, participates in the complexation. Simple 1:1 interaction scheme reported in literature holds therefore only for mercapto compounds bearing anionic group. Stoichiometric binding constants corrected for the abundance of the uncharged alkaloid form at pH 7.4 are of the order of magnitude of 10(4) l/mol and depend on both cations and anions of the background electrolyte. Interaction of sanguinarine and chelerythrine with human or bovine serum albumins does not qualitatively differ from their interaction with simple mercapto compounds. Stoichiometric binding constants for the binding of sanguinarine with human and bovine serum albumins in sodium phosphate buffer pH 7.4, corrected for the abundance of the interacting uncharged form, are 332,000+/-38,400 and 141,000+/-14,400 l/mol, respectively. The former agrees well with the value K=385,000 (or log K=5.59) from static photometric experiments. Constants for the complexation of uncharged chelerythrine with human and bovine serum albumins are 2,970,000+/-360,000 and 1,380,000+/-22,600 l/mol, respectively.