Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles.

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

Indirect influence of modality on direct memory for words and their modality: closed-head-injured and control participants.

Twenty closed-head-injured (CHI) patients and 28 control participants were tested on recall and recognition of words. In addition, memory for modality (i.e., visual vs. auditory) of word presentation was measured directly (i.e., recognition) and indirectly (i.e, by its influence on word and modality recognition). As predicted, the CHI patients were impaired relative to controls on all of the direct memory tasks; that is, word recall, word recognition, and modality judgment. However, the CHI and control groups did not differ significantly on the magnitude of the modality effect (i.e., facilitation due to correspondence of modality in learning and test). The findings are interpreted in the theoretical framework that distinguishes between item (i.e., words) and source (i.e., modality) memory and between direct and indirect measures of memory.

Tandem mass spectrometry study of p38α kinase inhibitors and related substances.

The p38 mitogen-activated protein kinase α (p38α) is an important drug target widely investigated for therapy of chronic inflammatory diseases. Its inhibitors are rather lipophilic and as such not very favourable lead compounds in drug discovery. Therefore, we explored various approaches to access new chemical space, create diversity, and generate lead libraries with improved solubility and reduced lipophilicity, based on known p38α inhibitors, e.g., BIRB796 and TAK-715. Compound modification strategies include incubation with human liver microsomes and bacterial cytochrome P450 mutants from Bacillus megaterium and treatment by electrochemical oxidation, H2O2, and intense light irradiation. The MS/MS fragmentation pathways of p38α inhibitors and their conversion products have been studied in an ion-trap-time-of-flight MS(n) instrument. Interpretation of accurate mass MS(n) data for four sets of related compounds revealed unexpected and peculiar fragmentation pathways that are discussed in detail. Emphasis is put on the usefulness of HRMS(n)-based structure elucidation in a screening setting and on peculiarities of the fragmentation with regard to the analytes and the MS instrument. In one example, an intramolecular rearrangement reaction accompanied by the loss of a bulky group is observed. For BIRB796, the double-charge precursor ion is used in MS(2), providing a wider range of fragment ions in our instrument. For TAK-715, a number of related compounds could be produced in a large-scale incubation with a Bacillus megaterium mutant, thus enabling comparison of the structure elucidation by (1)H NMR and MS(n). A surprisingly large number of homolytic cleavages are observed. Competition between two fragmentation pathways involving either the loss of CH3(•) or OH(•) radicals was observed for SB203580 and its conversion products.

High-resolution metabolic profiling towards G protein-coupled receptors: rapid and comprehensive screening of histamine H4 receptor ligands.

In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H(4) receptor (hH(4)R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH(4)R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic 'hotspots' for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

Artificial neural network processing of stripping analysis responses for identifying and quantifying heavy metals in the presence of intermetallic compound formation.

Feed-forward neural networks have been trained to identify and quantify heavy metals in mixtures under conditions where there were significant complications due to intermetallic compound formation. The networks were shown to be capable of (i) correlating voltammetric responses with individual heavy metals in complex mixtures, (ii) determining the relationship between responses and concentrations (including nonlinear relationships due to overlapping peaks and intermetallic compound formation), and (iii) rapidly determining concentrations of individual components from mixtures once trained. Using simulated data, modeled after complex interactions experimentally observed in samples containing Cu and Zn, it has been demonstrated that networks containing two layers of neurons (a nonlinear hidden layer and a linear output layer) can be trained to calculate concentrations under a variety of complicated situations. These include, but are not limited to, cases where the response of the intermetallic compound formed is observed as a shoulder of one of the pure metals and cases where the response of the intermetallic compound formed is not observed in the potential window. In addition, the network described above was trained to simultaneously determine concentrations of four metals (Cu, Pb, Cd, and Zn) in a concentration range where all responses were complicated by intermetallic compound formation (1-500 ppb).

The intravenous use of coconut water.

Medical resources routinely used for intravenous hydration and resuscitation of critically ill patients may be limited in remote regions of the world. When faced with these shortages, physicians have had to improvise with the available resources, or simply do without. We report the successful use of coconut water as a short-term intravenous hydration fluid for a Solomon Island patient, a laboratory analysis of the local coconuts, and a review of previously documented intravenous coconut use.