Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

When are metal ion-dependent hydroxyl and alkoxyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide artifacts?

The formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/.OH adduct of the spin trap DMPO has been reported to occur through nucleophilic addition of water in the presence of aqueous ferric chloride (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080). Due to the serious implications of these findings with respect to many spin trapping studies, the suitability of DMPO as a hydroxyl radical spin trap was studied in typical Fenton systems. Using 17O-enriched water, we show conclusively that nucleophilic addition of water occurs at the nitrone carbon (or C-2 position) of DMPO in the presence of either Fe or Cu ions. Furthermore, our results demonstrate that this nucleophilic reaction is a major pathway to the DMPO/.OH adduct, even during the reaction of Fe(II) or Cu(I) with hydrogen peroxide. Primary alkoxyl adducts of DMPO also form in aqueous solution through nucleophilic addition in the presence of both Fe(III) and Cu(II). Attempts to obtain secondary and tertiary alkoxyl adducts by this mechanism were unsuccessful, possibly due to steric effects. When the reaction is carried out in various buffers, however, or in the presence of metal ion chelators, nucleophilic addition to DMPO from Fe(III) is effectively suppressed. Chelators also suppress the reaction with Cu(II). Hence, under most common experimental conditions in biochemical free radical research, nucleophilic addition to DMPO should not be of major concern.     

Development of chicken embryos in a pulsed magnetic field

Six independent experiments of common design were performed in laboratories in Canada, Spain, Sweden, and the United States of America. Fertilized eggs of domestic chickens were incubated as controls or in a pulsed magnetic field (PMF); embryos were then examined for developmental anomalies. Identical equipment in each laboratory consisted of two incubators, each containing a Helmholtz coil and electronic devices to develop, control, and monitor the pulsed field and to monitor temperature, relative humidity, and vibrations. A unipolar, pulsed, magnetic field (500-microseconds pulse duration, 100 pulses per s, 1-microT peak density, and 2-microseconds rise and fall time) was applied to experimental eggs during 48 h of incubation. In each laboratory, ten eggs were simultaneously sham exposed in a control incubator (pulse generator not activated) while the PMF was applied to ten eggs in the other incubator. The procedure was repeated ten times in each laboratory, and incubators were alternately used as a control device or as an active source of the PMF. After a 48-h exposure, the eggs were evaluated for fertility. All embryos were then assayed in the blind for development, morphology, and stage of maturity. In five of six laboratories, more exposed embryos exhibited structural anomalies than did controls, although putatively significant differences were observed in only two laboratories (two-tailed Ps of .03 and less than .001), and the significance of the difference in a third laboratory was only marginal (two-tailed P = .08). When the data from all six laboratories are pooled, the difference in incidence of abnormalities in PMF-exposed embryos (approximately 25 percent) and that of controls (approximately 19 percent), although small, is highly significant, as is the interaction between incidence of abnormalities and laboratory site (both Ps less than .001). The factor or factors responsible for the marked variability of inter-laboratory differences are unknown.     

Mitogens and hepatocyte growth control in vivo and in vitro

The online version of the original article can be found at     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the "endotoxic principle" of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg2+] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70 degrees C. These data were correlated with measurements of the beta----alpha phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges--in the absence of Mg2(+)-from 45 degrees C at high to 56 degrees C at low water content, and-at an equimolar content of Mg2(+)-from 52 degrees C at high to 59 degrees C at low water concentrations. In the gel phase-in which the lipid A acyl chains are more disordered than those from saturated phospholipids-cubic phases are adopted at high water content (greater than 60%) and at high [lipid A]:[Mg2+] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal HII state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg2+ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand.

Human immunodeficiency virus type 1 isolates of envelope genotype E are contributing substantially to the global pandemic. These strains appear to be mosaics, with the gag gene from clade A and the envelope from clade E; the parental clade E strain has not been found. Here we report the first full genomic sequence of one such mosaic virus, isolate CM240 from Thailand. Multiple breakpoints between the two parental genotypes have been found in a CM240 virus. The entire gag-pol region and most, if not all, of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41; thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be principally derived not from clade E, as previously thought, but from clade A. Another small segment not belonging to any recognized clade and presumably also contributed by the parental E strain has been found in the long terminal repeat. It may be significant that the implied virion structure resembles a pseudotype virus with the matrix and core from one clade and the outer envelope from another. In the long terminal repeat, differences were observed between CM240 and other clades in the number of NF-kappa B binding sites, the sequence of the TATA box, and the putative secondary structure of the transactivation response region stem-loop. The mosaic structure of a CM240 virion is suggestive of phenotypic differences which might have contributed to the emergence of this variant.     

Gliding motility in slide cultures of Myxococcus xanthus in stable and steep chemical gradients.

A method was devised to construct stable and steep chemical gradients in slide cultures to study the movements of gliding cells. The movement of Myxococcus xanthus individual cells and small swarms was studied in these gradients. There was no response to gradients of Casitone and yeast extract that were previously reported to stimulate a positive chemotactic response with M. xanthus.     

Central Mechanisms of Pheromone Information Processing

An advantage of using pheromones in olfactory studies is thatthey are chemical signals for which receptor neurons are evolvedand thus elicite biologically relevant odour-information tobe processed in the brain. In many vertebrate and insect species,the olfactory system is separated into a ‘main’and an ‘accessory’ division, the latter mediatingpheromone information. In moths, the pheromone information isfirst processed in the brain in a large and sexually dimorphicstructure, the macroglomerular complex (MGC) of the antennallobe (AL). Also in vertebrates the pheromone information isprocessed in specific or modified glomerular complexes. Oneprinciple question is whether individual olfactory glomeruliare functional units, processing specific information concerningboth the chemical quality and spatiotemporal features of thestimulus, like the pheromone plume. Indeed it has been shownthat the axons of different pheromone-selective receptor neuronsproject into different MGC-glomeruli. Intracellular recordingsfrom the AL projection (output) neurons also show that informationabout single components of the pheromone blend is preservedin some output pathways, whereas other output neurons respondin a unique fashion to the blend. The information about interspecificsignals, which interrupts pheromone attraction, is processedin a specific MGC-glomerulus and is to a large extent kept separatedfrom the pheromone information throughout the AL. Many of theoutput neurons accurately encode changes in the temporal characteristicsof the stimulus. Chem. Senses 21: 269–275, 1996.