Asymmetrical,water-soluble phthalocyanine dyes for covalent labeling of oligonucleotides

Two new water-soluble, porphyrazine (Pz) dyes containing an isothiocyanate function for covalent linking have each been prepared by cross condensation of two different aromatic dinitriles, one containing carboxylates for solubilizing purposes and the other containing a nitro group for conversion into the labeling function. The initial mononitrotricarboxylato Pzs have been purified to homogeneity from the mixture of Pz congeners formed in the condensation reaction by anion exchange chromatography. The phthalocyanine dye 1 has an absorption maxima at 683 nm while the trinaphthoporphyrazine dye 2 has an absorption maxima at 755 nm, due to the increased size of the aromatic system. Both dyes were successfully conjugated to oligonucleotide primers, showing their potential for use in near-infrared-based DNA diagnostic applications.     

Impact of urea on water structure: a clue to its properties as a denaturant?

A new investigation of the structure of urea-water solutions at a mole ratio of 1 urea to 4 water molecules is described. Neutron diffraction is used in conjunction with isotope labelling on the water and urea hydrogen atoms and on the nitrogen atom of urea. The diffraction data are analysed using the empirical potential structure refinement procedure to yield a set of site-site radial distribution functions and spatial density functions that are consistent with the diffraction data. The results are discussed in relation to recent and past X-ray and neutron diffraction experiments and theoretical studies of this system. It is found that urea incorporates readily into water, forming pronounced hydrogen bonds with water at both the amine and carbonyl headgroups. In addition the urea also hydrogen bonds to itself, forming chains or clusters consisting of up to approximately 60 urea molecules in a cluster. There, is however, little or no evidence of urea segregating itself from water, in marked contrast to a recent study of the methanol-water system. This behaviour is discussed in the context of the great propensity of urea to effect protein denaturation.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis

Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.     

Discovery of novel conformationally restricted diazocan peptidomimetics as inhibitors of interleukin-1beta synthesis

A novel diazocan containing dipeptide mimetic was synthesized via reductive N-N bond cleavage of a pyrazolidino-pyrazolidine using Raney-Ni and evaluated as an ICE inhibitor. This versatile 8-membered ring containing scaffold possesses an N-5 ring nitrogen that was used to explore structure-activity relationships in a cell-based assay measuring inhibition of interleukin-1beta.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ~7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ~25 min as follows: (i) DNA immobilization ~6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ~6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light.     

The hydration of the neurotransmitter acetylcholine in aqueous solution

Neutron diffraction augmented with hydrogen isotope substitution has been used to examine the water structure around the acetylcholine molecular ion in aqueous solution. It is shown that the nearest-neighbor water molecules in the region around the trimethylammonium headgroup are located either in a ring around the central nitrogen atom or between the carbon atoms, forming a sheath around the onium group. Moreover the water molecules in this cavity do not bond to the onium group but rather form hydrogen bonds with water molecules in the surrounding aqueous environment. Given that in the bound state the onium headgroup must be completely desolvated, the absence of bonding between the onium headgroup and the surrounding water solvent may be selectively favorable to acetylcholine-binding in the receptor site. Away from the headgroup, pronounced hydrogen-bonding of water to the carbonyl oxygen is observed, but not to the ether oxygen in the acetylcholine chain.     

Relationship between myeloperoxidase activity and the ontogenic response of rat gastric mucosa to ethanol.

We have examined the gastric luminal content of Na+, K+, and protein and mucosal levels of myeloperoxidase in rats between the ages of 10 and 60 days in response to luminal instillation of ethanol (20 and 50% w/v). In control animals the appearances of ions and protein and myeloperoxidase activities were low and similar in all age groups. Luminal content of cations and protein increased in response to both 20 and 50% ethanol and were greater in animals older than 20 days when compared with younger rats. However, ethanol treatment resulted in a significant degree of mucosal cellular disruption and erosions in both young and mature rats. Myeloperoxidase activities in response to ethanol were not greater than control until animals were older than 20 days. Treatment of rats aged 10-60 days with intraperitoneal glycogen (1%) resulted in peritoneal granulocyte infiltration. The concentration of peritoneal cells increased as animals aged. With the exception of day 15, the myeloperoxidase content of the peritoneal leukocytes did not vary significantly at other ages examined. These data suggest that (1) mucosal efflux of Na+, K+, and protein in response to luminal ethanol increase as rats age from 10 to 60 days; (2) the ontogenic development of ethanol-induced cation and protein appearance parallel the increase in myeloperoxidase activity in the gastric mucosa; and (3) the increase in mucosal myeloperoxidase activity in response to ethanol likely reflects increased granulocyte infiltration as rats age.