Development of an immunochromatographic test system for the detection of Helicobacter pylori antigens

Applied Biochemistry and Microbiology - A test system based on immunochromatography in the sandwich format and intended for express detection of Helicobacter pylori antigens has been developed....     

Spatial remapping in the primate visual system

Summary The lateral geniculate body performs a spatial remapping operation. This remapping may help to preserve the apparent distance of objects under symmetrical eye movements, thereby stabilizing the appearance of visual space. In addition, a related, and perhaps more basic function of the geniculate remapping may be to increase the efficiency of the neural matrix which encodes depth information. For distant fixation, the majority of cells in this cortical matrix would be responsive to crossed disparities, but for near fixation, many of these same cells might be converted into uncrossed disparity detectors. Two types of models for the geniculate are presented, together with supporting evidence: (1) a quantitative psychophysical model describing its steady-state properties, and (2) a qualitative neurophysiological model describing the function of the geniculate laminae.This work was supported by the U.S. Air Force under contract No. AFORSR-F 44620-67-C0085. Supplementary funding was received from NASA and NIMH under grants Ns G 496 and MH 05673 awarded to Prof. H.-L. Teuber, Chairman, Dept. of Psychology, M. I. T., Cambridge, Mass.     

Neural network simulations of the primate oculomotor system

The performance of a neural network that simulates the vertical saccade-generating portion of the primate brain is evaluated. Consistent with presently available anatomical evidence, the model makes use of an eye displacement signal for its feedback. Its major features include a simple mechanism for resetting its integrator at the end of each saccade, the ability to generate staircases of saccades in response to stimulation of the superior colliculus, and the ability to account for the monotonic relation between motor error and the instantaneous discharge of presaccadic neurons of the superior colliculus without placing the latter within the local feedback loop. Several experimentally testable predictions about the effects of stimulation or lesion of saccaderelated areas of the primate brain are made on the basis of model output in response to “stimulation” or “lesion” of model elements.     

Development of a detection system for ferric pseudobactin using monoclonal antibodies

Certain root-colonizing fluorescent pseudomonads have been shown to promote plant growth and prevent plant disease in part through the production of siderophores. However, these favorable results have not been reproduced consistently from the laboratory to the greenhouse or from the greenhouse to the field. In some circumstances siderophores appear to play no role in disease prevention. In order to understand the dynamics of competition for iron in the rhizosphere it is essential that the localization and concentration of siderophores produced by both biocontrol agents and plant pathogens be determined. We have produced monoclonal antibodies (MAbs) to ferric pseudobactin, the siderophore of plant growth-promoting Pseudomonas B10. Three IgG1 MAbs cross-react with certain ferric pseudobactins but not with others. A competitive ELISA has been developed to detect and quantify ferric pseudobactin.     

Development of an immunochromatographic test system for the detection of human epidermal growth factor

A method was developed for the rapid detection of human epidermal growth factor based on a sandwich-format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid flow movement across the membrane components of the test strip, immunochemical reactions, and the formation of colored lines. Requirements on the configuration of the test system in order to achieve the lowest limit of detection were defined in the course of the development of the assay. It was shown that this method enables the detection of human epidermal growth factor within 5 min at concentrations as low as 10 pg/mL in aqueous solutions, urine, and the blood serum and plasma. The developed test system can be used for point-of-care diagnostics.     

Development of detection system of extraterrestrial microorganisms]

A noble method for the exploration of terrestrial and extraterrestrial soil microorganisms, especially targeted for Mars, has been developed. The method is based on the microscopic observation using fluorescence techniques. Microorganisms could be fluorescent by adsorption, enzymatic cleavage of extrinsic fluorescence chromophores such as acridine orange, ANS and SFDA, and also by intrinsic chromophores. The characteristic points of our fluorescence method are shown below. 1. The present method detected all the culturable cells tested (about 200 species from bacteria to eukaryofic cells). 2. Microorganisms in soil were much brighter than background fluorescence of soil. Cell shapes and location were clearly observed. 3. An esterase substatum SFDA, discriminated vital (reproductive) cells from dead. On the other hand, a membrane probe, ANS, detected both vital and dead cells. 3. Pre-treatment of cells with bleaching reagents improved the detection efficiency. Especially, this pretreatment was effecfive in Fungi with black chromophores. 4. Some anaerobic microorganisms such as methanogenic bacteria with intrinsic chromophores can be detected without stain. 5. Application of the technique to terrestrial soil revealed that more than 100 times larger cell density was obtained compared to the value obtained by the classic plate counting technique. Vertical distribution of microorganism of soil microorganisms from Mt. Shigayama showed that, at surface, cell density was small and maximum was shown below 15 cm from surface. 6. Some pre-biotic cell (cell like aggregates composed of amino acids) could be detected by SFDA or ANS. It can be concluded that the fluorescence technique is one of the most promising method for the exploration of extraterrestrial microorganisms.     

Development of a bead-based aptamer/antibody detection system for C-reactive protein

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10 mg/L in diluted serum with acceptable recoveries (extrapolated values of 70–130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer–target systems could increase the number of analytes measurable using xMAP-type assays.     

A computerized system for scoring primate behavior using standard keyboards

A scoring system has been developed for primate behavior which uses standard keyboards and minicomputers or microcomputers. The mnemonic, alphanumeric code used is easily learned, highly flexible, and can be recorded in longhand for later entry into a computer if a keyboard is not immediately available. The software consists of two programs, both of which can be written in BASIC. SCORE is used for data acquisition and appends the test time to each behavioral sequence. DATSUM decodes and summarizes the test data using table-driven logic. The minimum hardware required is a 16K microcomputer, an alphanumeric keyboard, a display, and cassette storage.     

IPTG-inducible episomal expression system for exogenous genes in primate cells

An isopropyl beta-D-thiogalactopyranoside (IPTG)-inducible episomal expression system has been established for the human breast carcinoma cell line MCF7. This two-component system includes: (i) a primate cell-specific episomal vector, pEpiLac, that contains an IPTG-inducible promoter and (ii) a cell line derived from MCF7, MCF7/LAP5, which expresses the IPTG-dependent transactivator LAP267. Treatment of MCF7/LAP5 cells with IPTG results in efficient inducible expression of exogenous genes from the inducible promoter in pEpiLac. Up to 300-fold induction can be observed when luciferase is used as a reporter. Inducible expression of the p27KIP1 cyclin-dependent kinase (CDK) inhibitor, the orphan nuclear receptor Nur77 and the angiogenic inducer Cyr61 has also been demonstrated. Expression of the exogenous gene is promptly halted on removal of IPTG. Moreover, the episomal vector can be stably maintained in and easily recovered from MCF7/LAP5 cells. Taken together, this inducible expression system should be applicable for the regulated expression of exogenous genes, especially growth inhibitory or cytotoxic genes, in cells of primate origin.     

Evolution of the primate visual system: Anterograde degeneration studies of the tecto-pulvinar system

A partial answer to the question of the precocious development of the temporal lobe in fossil lemurs is offered by the presentation of evidence that in the tree shrew, in prosimians, and probably in all primates the temporal lobe contains primary visual cortex. The visual pathway to the temporal lobe is achieved by relays through the superficial layers of the superior colliculus and pulvinar nucleus. This pathway parallels the geniculo-striate system. Still a third visual system can be identified with sensory-motor connections in the deeper layers of the superior colliculus.     

Development of a novel nonhuman primate model for Rift Valley fever

Rift Valley fever (RVF) virus (RVFV) can cause severe human disease characterized by either acute-onset hepatitis, delayed-onset encephalitis, retinitis and blindness, or a hemorrhagic syndrome. The existing nonhuman primate (NHP) model for RVF utilizes an intravenous (i.v.) exposure route in rhesus macaques (Macaca mulatta). Severe disease in these animals is infrequent, and large cohorts are needed to observe significant morbidity and mortality. To overcome these drawbacks, we evaluated the infectivity and pathogenicity of RVFV in the common marmoset (Callithrix jacchus) by i.v., subcutaneous (s.c.), and intranasal exposure routes to more closely mimic natural exposure. Marmosets were more susceptible to RVFV than rhesus macaques and experienced higher rates of morbidity, mortality, and viremia and marked aberrations in hematological and chemistry values. An overwhelming infection of hepatocytes was a major consequence of infection of marmosets by the i.v. and s.c. exposure routes. Additionally, these animals displayed signs of hemorrhagic manifestations and neurological impairment. Based on our results, the common marmoset model more closely resembles severe human RVF disease and is therefore an ideal model for the evaluation of potential vaccines and therapeutics.     

Character phylogeny of the primate forelimb superficial venous system.

The ontogeny and comparative anatomy of the forelimb superficial veins were investigated in humans, non-human primates and other mammals. Adult humans and the orangutan (Pongo) possess two autonomous forelimb veins, one on the lateral (preaxial) margin of the limb, the other on the medial (postaxial) margin. All other adult primates and mammals examined possess a lateral vein alone. In African apes (Pan and Gorilla) and in 24% of human forelimbs the lateral vein is short, being essentially confined to the antebrachial region, whereas in other mammals and in 76% of human limbs the lateral vein runs from the carpus to the clavicular region. In humans the medial vein develops before the lateral vein, whereas in the rabbit and the pig the medial vein is present in early embryos but is subsequently lost. We propose that in humans, and probably also in the orangutan, the possession of a medial vein is a neotenic retention of a primitive tetrapod condition. These animals, which retain their medial vein, are united by losing a late stage in their ontogeny. Other animals subsequently pass through a stage in which the medial vein is lost, but Pongo and Homo retain this vein to adulthood. The loss of an ontogenetic stage can arise independently, and the presence of a medial vein therefore affords only weak evidence for a close phylogenetic relationship between humans and the orangutan. The polymorphic lateral vein of humans may be a character state that is intermediate between the derived (short) lateral vein of the African apes and the primitive long lateral vein of other non-human primates and mammals.     

A new coordinate system using the orbital axis for morphological analysis of primate skulls

A new coordinate system for primate skulls was defined by the orbital axis and the validity of the system was examined. This system is thought to be equivalent throughout the primates. With the aid of photogrammetry the three-dimensional coordinates of 39 points on the skulls of 479 individuals comprising 54 species including man were accurately measured. The orbital structure is morphologically stable and its axis represents a comparative horizontal. The midsagittal plane and the bilateral symmetry of the cranium are also stable. The morphological stability in angular dimensions is confirmed by a standard deviation smaller than 2.0°. The major evolutionary change in the neurocranium is the inclination of the cranial base from the orbital horizon, and the inclination is related to the neurocranial size. The ear-eye plane is generally inapplicable to the primates, because it is affected by the orbital size and the descent of the auricular part due to the inclination of the cranial base. The clivus line or the vestibular coordinate system is not desirable as the horizontal, either. The evolutionary development of the facial part of the cranium is independent of that of the neurocranium and these two parts are separated by the orbital horizon.     

Development and validation of a high throughput system for discovery of antigens for autoantibody detection

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.     

Studies of primate protein variation and evolution: Microelectrophoretic detection

Genetic variation at 16 protein and enzyme loci in Cercopithecus aethiops and several other primate species has been surveyed, using cellulose acetate microelectrophoresis. Resolution of several standard variant proteins is comparable to that achieved on starch gel or polyacrylamide gel. Although both intraspecific and interspecific variation was observed for some loci, the data generally support the concept that extracellular proteins are more likely to be polymorphic within a species, while intracellular proteins generally vary between species, if at all. These methodologies are particularly appropriate for screening multiple-locus variation in large numbers of samples; their relevance to studies of molecular evolution and evaluation of theories of kin selection is discussed.This research was supported in part by California State Agricultural Experiment Station Funds to the University of California, Berkeley, and the Wenner Gren Foundation.     

A new primate social behaviour category system

A brief critical review of some primate behavioural recording techniques is presented. A new device and scoring system, from which frequency, duration, duration/frequency, and modified frequency data of a large number of behaviours can be obtained, is described using an adding machine and printout counter. Data collected using this system is illustrated. The system, factorially arranged, differentiates five behaviour types, six directions, mobility, contact, and reciprocity. Four types of reliability are estimated using films; intercorrelations and factor analyses are presented.