Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Nerve Growth Factor: Biological Significance, Measurement and Distribution

NGF proteins probably act as informational molecules transferred from end organs to the neurons of the sympathetic nervous system. The direct demonstration of the NGF content of most end organs requires assays more sensitive than those currently available. The high levels of NGF produced by some organs are probably of some other physiological significance.     

Nickel requirement for chemolithotrophic growth in hydrogen-oxidizing bacteria

In a comparative study the requirement of several strains of autotrophic hydrogen-oxidizing bacteria for nickel was examined. Autotrophic growth was studied both in liquid media, previously freed from trace metals; and on solidified media, using a plate diffusion assay. The latter assay was based on the observation that EDTA causes complete inhibition of autotrophic growth on agar medium as a result of nickel deficiency. Nickel was shown to be required as a trace element in five strains of Alcaligenes eutrophus, in two strains of Xanthobacter autotrophicus, in Pseudomonas flava, in Arthrobacter spec. 11X and in strain 12X. In these bacteria nickel was not replaceable by cobalt, copper, manganese or zinc ions. No significant nickel requirement was detected by these methods, however, for Paracoccus denitrificans and Nocardia opaca 1b.     

Vitamin analysis with use of a yeast electrode

A vitamin B₁ (thiamin)-sensitive electrode has been devised by combining an oxygen electrode with a yeast-containing membrane. The assembly was used for assaying thiamin at concentrations down to 10^?11 gl^?1. The analytical procedure developed should allow the measurement of 10–20 samples per hour. The performance of the yeast electrode was improved when alginate membranes reinforced with a nylon network were used. An apparatus for preparing such membranes is described together with a magnetic membrane holder facilitating handling of membranes in combination with electrodes.     

Nitrate reductase activity and growth in Paul's Scarlet rose suspension cultures in relation to nitrogen source and molybdenum

Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO₃ ^- medium, activity in NO₃ ^--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO₃ ^- alone was added to NO₃ ^- or urea+Mo cultures. In NO₃ ^--Mo cultures, Mo alone or with NO₃ ^- caused a similar increase in activity, whereas urea-Mo cultures required both NO₃ ^- and Mo for enzyme induction.Abbreviations FAD flavin adenine dinucleotide - Mo molybdenum - NADH reduced nicotinamide adenine dinucleotide - NO₃ ^-+Mo standard MX₁ culture medium - NO₃ ^--Mo MX₁ medium purified of Mo and used for continuous subculture with nitrate - NR nitrate reductase - PSR Paul's Scarlet rose - PVP polyvinylpyrrolidone - U urea - U+Mo MX₁ medium containing urea instead of nitrate - U-Mo MX₁ medium containing urea instead of nitrate and also purified of Mo     

Optimum concentration and pH of phosphate buffer for assay of cytochrome c oxidase in intact plant mitochondria

For measurement of cytochrome c oxidase activity in intact plant mitochondria the optimum concentration of K-Pi buffer and pH in the reaction was found to be 75 mM and 7.4 respectively. The suitable concentration of K-Pi buffer for suspending and storing mitochondria, however, was found to be 20 mM or lower. These requirements applied equally well for mitochondria from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), maize (Zea mays L.), and snap bean (Phaseolus vulgaris L.).     

Fused heterocyclic compounds bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 1: Design,synthesis and biological evaluation of novel 5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives

In our continuous efforts to identify novel potent HIV-1 NNRTIs, a novel class of 5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives were rationally designed, synthesized and evaluated for their anti-HIV activities in MT4 cell cultures. Biological results showed that most of the tested compounds displayed excellent activity against wild-type HIV-1 with a wide range of EC₅₀ values from 5.98 to 0.07 μM. Among the active compounds, 5a was found to be the most promising analogue with an EC₅₀ of 0.07 μM against wild-type HIV-1 and very high selectivity index (SI, 3999). Compound 5a was more effective than the reference drugs nevirapine (by 2-fold) and delavirdine (by 2-fold). In order to further confirm their binding target, an HIV-1 RT inhibitory assay was also performed. Furthermore, SAR analysis among the newly synthesized compounds was discussed and the binding mode of the active compound 5a was rationalized by molecular modeling studies.     

Establishment of an in vitro sciarid fly larvae assay to study plant resistance

Mechanisms underlying natural plant resistance to herbivorous invertebrates are still poorly understood in comparison with bacterial or fungal interactions. One reason is the difficulty in reliably and reproducibly assessing the effects under controlled conditions. This article describes a newly developed in vitro biological assay system that enables the interactions between sciarid larvae and plants, whose roots they feed on, to be studied under highly controlled conditions. The bioassay eliminates the problems created by the often variable environmental factors by providing an aseptic arena where experimental plants can be germinated and grown on agar within a Petri dish. Sciarid fly eggs are then collected, sterilised and added to the Petri dish. The system allows the eggs to hatch and the larvae to feed on the plant roots. A range of developmental parameters can then be recorded over time which can then be correlated with the experimental plant type. This assay system also allows a simultaneous comparison or 'choice chamber' between two (or more) different genotypes. The assay should greatly help to facilitate the identification of new components involved in insect resistance mediated pathway via the characterisation of mutant plants.     

Design,synthesis and biological evaluation of low molecular weight CXCR4 ligands

The chemokine receptor CXCR4/stromal cell-derived factor-1 (SDF-1: CXCL12) signaling axis represents a crucial drug target due to its relevance to several diseases such as HIV-1 infection, cancer, leukemia, and rheumatoid arthritis. With the aim of enhancing the binding affinity and anti-HIV activity of a potent CXCR4 ligand as a lead, 23 low molecular weight compounds containing dipicolylamine (Dpa) and cyclam cationic moieties with varying spacers and spatial positioning were designed, synthesized and biologically evaluated. All of the synthesized compounds screened at 1.0 μM in the NanoBRET assay system exhibited >70% inhibition of the binding of a competitive probe TAMRA-Ac-TZ14011 (10 nM) to CXCR4 in the presence of zinc (II) ion. Furthermore, selected compounds 3, 8, 9, 19 and 21 with spatial distances between the next carbon to Dpa and the next carbon to cyclam within the range of 6.5–7.5 Å showed potent binding affinity selective for CXCR4 with IC₅₀ values of 1.6, 7.9, 5.7, 3.5 and 4.5 nM, respectively, with corresponding high anti-HIV activity with EC₅₀s of 28, 13, 21, 28 and 61 nM, respectively, in the presence of zinc (II) ion. Some compounds with remarkably more potent CXCR4-binding affinity than that of an initial lead were obtained. These compounds interact with different but overlapping amino acid residues of CXCR4. The present studies have developed new low molecular weight CXCR4 ligands with high CXCR4-binding and anti-HIV activities, which open avenue into the development of more potent CXCR4 ligands.