Dot-enzyme-linked immunosorbent assay (dot-ELISA) for the rapid diagnosis of human fascioliasis

A dot-enzyme-linked immunosorbent assay (dot-ELISA) was developed as a fast and field applicable antibody detection tool for the diagnosis of human fascioliasis. The assay is performed using partially purified antigens from a species of Fasciola at 180 ng protein/dot (2 microliters) and serum samples at 1:20 dilution (1 microliter). Dot-ELISA results completely agreed with those of micro-ELISA. Antigen-coated nitrocellulose sheets stored for 3 mo at -20 C showed results identical to fresh sheets. Sera from patients with fascioliasis (n = 30) and other parasitic or viral infections (n = 120) were compared with sera from healthy controls (n = 14). Ninety samples can be tested within 90 min. The sensitivity, specificity, and speed of the assay may justify its use in laboratory and field studies.     

Triple labeling with two-color immunofluorescence using one light source: a useful approach for the analysis of cells positive for one label and negative for the other two

Many laboratories do not have access to a flow cytometer allowing three-color immunofluorescence analysis through the use of multiple light sources. In view of the usefulness of such analyses in the dissection of cell parameters, we describe an approach permitting the study of three labels by using one light source and the two-color immunofluorescence assay. It is useful for the enumeration of cell subpopulations positive for one label and negative for two or more others as well as for qualitative analysis concerning the expression of these labels. This approach is simple and rapid; it does not require additional material and technical steps other than that used in the two-color immunofluorescence assay. Briefly, it consists of the use of a label coupled to a dye (PE or FITC or instance) and two different labels coupled to the other dye. An argon ion laser, operating at 488 nm and 60 mW, excites both fluorescein and phycoerythrin conjugated antibodies. We provided a general example, using three hypothetical labels (X, Y, and Z), and four practical applications: CD3+CD4CD8- and CD8+CD16-CD3- peripheral blood lymphocytes, CD2+CD16-CD3- and CD56+CD16-CD3- peripheral blood, and decidual infiltrating lymphocytes.     

Changes in growth,osmotic potential and cell permeability of wheat cultivars under salt stress

10-d-old wheat seedlings were grown hydroponically in presence and absence of 100 mM NaCl for 7 d. Salt stress decreased growth of shoots and roots of both cultivars; fresh mass of sensitive cultivar being more affected. NaCl increased membrane permeability to urea, methylurea and ethylurea and decreased membrane partiality in root cortex cells of sensitive cultivar. Neither parameter changed by NaCl in resistant cultivar. NaCl treatment decreased water permeability and osmotic potential in both cultivars; sensitive cultivar was more affected. The results extends our previous data that cell membrane properties are different in salt sensitive and resistant genotypes and so cell permeability could be a potential trait indicating salt tolerance.     

Changes in cell membrane permeability and lipid content of wheat root cortex cells induced by NaCl

Wheat seedlings were grown hydroponically in absence and presence of 100 mM NaCl for 7 d. Cell membrane permeability to nonelectrolytes and water was determined by the plasmometric method for individual intact cells. NaCl increased membrane permeability to urea, methylurea and ethylurea and decreased permeability to water. Membrane lipid partiality was decreased by NaCl. The effects of NaCl on cell permeability parallel changes in the lipid composition of the plasma membranes induced by NaCl stress suggesting that nonelectrolyte permeability is a useful tool to probe alterations in the lipid matrix of the membrane.     

Effect of perfusate pH on coronary flow and adenosine release in isolated rabbit heart

This study was undertaken in an attempt to further understand the relationship between adenosine and H+ ion. Using Langendorff hearts from male rabbits, the perfusion fluid pH was lowered from 7.4 to 7.1 and 6.8 with CO2. A 31 and 86% increase in coronary flow with a simultaneous increase in the release of adenosine by 61 and 128% was observed at pH 7.1 and 6.8, respectively. A direct relationship between adenosine release and coronary flow with a correlation coefficient of 0.99 was found at pH values of 7.4, 7.1, and 6.8. The degradation products of adenosine namely inosine and hypoxanthine were unchanged at 7.1 and 6.8 from 7.4. These data support a role for adenosine in the regulation of coronary flow and suggest a relationship between adenosine and H+ ion.     

Effect of treatment with azathioprine on the responses of rat lymphocytes to phytohaemagglutinin.

The proliferative responses of rat peripheral blood lymphocytes (PBL) and spleen cells to phytohaemagglutinin (PHA) were studied after single or multiple (daily for 4 days) injections of azathioprine (AZ). Lymphopenia developed within 4 h of a single dose (78 mg/kg) of AZ and persisted for at least 72 h. There was no lymphopenia 24 h after the last of 4 daily injections. In vitro, PBL were more sensitive than spleen cells to the inhibitory effect of AZ. Likewise, the responses of PBL were relatively more depressed than those of spleen cells after single or multiple injections of AZ. The degree of depression was less than was expected from the effect of AZ in vitro. Multiple small doses were more depressive than multiple large doses. Serum from treated rats, used at 20% concentration, was more depressive than normal. Thus, rat lymphocytes are quite sensitive to AZ in vitro, but appear to be relatively resistant in vivo, this resistance resembling the resistance of the primary antibody response to AZ treatment.     

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.     

Synthesis of (+/−)–1′-Aza-carbocyclic-pyrimidine-2′,3′-dideoxynucleoside analogues as potential anti-HIV agents

1′-Aza-carbocyclic-2′, 3′-dideoxyuridine, 3′-deoxythymidine and 2′, 3′-dideoxycytidine were synthesized from 1-aminopyrrolidine intermediate 13 and evaluated as anti-HIV agents in MT-4 cells.     

A 31P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.