A Multiplex Label-Free Approach to Avian Influenza Surveillance and Serology

Influenza serology has traditionally relied on techniques such as hemagglutination inhibition, microneutralization, and ELISA. These assays are complex, challenging to implement in a format allowing detection of several types of antibody-analyte interactions at once (multiplex), and troublesome to implement in the field. As an alternative, we have developed a hemagglutinin microarray on the Arrayed Imaging Reflectometry (AIR) platform. AIR provides sensitive, rapid, and label-free multiplex detection of targets in complex analyte samples such as serum. In preliminary work, we demonstrated the application of this array to the testing of human samples from a vaccine trial. Here, we report the application of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow.     

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.     

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.     

EEG spatial organization during intense hyperventilation (cyclic breathing): II. EEG correlates of psychovisceral phenomena

EEG correlates of two main effects of long-term hyperventilation (cyclic breathing similar to rebirthing), namely, psychotic manifestations and sensations in internal organs, are considered. Standard EEGs of 44 subjects were recorded and subsequently treated by multiparametric methods. It was shown that, in the case of psychotic phenomena (hallucinations, visual and auditory images, different scenes, virtual travels, out-of-body experiences, etc.), the changes in the spatial organization of bioelectric potentials as compared with the baseline have different patterns than in the case of sensations in internal organs. The changes are observed in the frontal regions of the cortex: a decrease in spatial synchronization (linear processes) during an increase in spatial disarray (nonlinear processes) of bioelectric potentials and the intensifying of high-frequency β activity. If sensations in internal organs appear, a decrease in the above parameters was observed in the right hemisphere and in posterior cortical areas. The data are discussed from the viewpoint of qualitative specificity of changes in functional states during an altered state of consciousness with manifestations of different psychovisceral phenomena.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovora subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovora subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria produced the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovora subsp. atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the D-xylose-containing nutrient medium of 0.1–1.5% D-glucose did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovora subsp. atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.