A rapid micro method for counting cells “in situ” using a fluorogenic alkaline phosphatase enzyme assay

Summary A new method has been developed to count cells “in situ”, based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10⁴ to 5×10⁵ for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays. Editor's Statement This paper describes a quick method for quantitation of cell number in microcultures. Such procedures are valuable for the many situations in which minimizing cells and medium volume is desirable, although somewhat specialized equipment is required for the procedure. An alternative procedure for quantitation of cells in microtiter culture appeared previously in this journal (McCaffrey, et al., 24∶247–252).     

Human β-endorphin: Development of tolerance and behavioral activity in rats

Human β-endorphin produced a potent antinociceptive response as estimated by the tail-flick test in rats after intraventricular injection. On a molar basis, the peptide was 21 times more potent than morphine and in addition, the peptide produced morphine-like catatonia and hypothermia. These responses were blocked by naloxone. Repeated injections of the peptide induced tolerance to analgesic response, catatonia and hypothermia. Cross tolerance to morphine was also observed.     

Exploration of disease mechanism in acute kidney injury using a multiplex bead array assay: a nested case–control pilot study

Background: Acute kidney injury (AKI) following cardiac surgery with cardiopulmonary bypass (CPB) causes increased morbidity and mortality.

Objective: To evaluate the plasma profile of biomarkers potentially involved in AKI development following CPB.

Methods: In a nested case–control study, plasma levels of 27 biomarkers in 11 AKI cases were compared with 25 controls.

Results: Pre-CPB, plasma levels of epidermal growth factor and macrophage inflammatory protein-1β, 2?h following CPB, soluble vascular cell adhesion molecule-1 (sVCAM-1), fractalkine and macrophage inflammatory protein-1α, and at later time points, sVCAM-1 and interleukin-6 were associated with AKI.

Conclusion: Biomarkers associated with AKI following CPB may merit further study.     

Serotonergic mechanism in the control of β-endorphin and acth release in male rats

The role of the serotonergic mechanism in the regulation of β-endorphin (β-EP) and adrenocorticotropin (ACTH)-like immunoreactivity in plasma was investigated. Increases in β-EP and ACTH-LI produced by quipazine maleate (QPZ), a serotonergic agonist, 1 hr after injection could be completely prevented by the serotonin (5-HT) antagonist, cinanserin (CIN), which when injected alone, decreased basal plasma concentrations of both β-EP-LI and ACTH-LI. Concurrent injections of L-5-HTP with the 5-HT reuptake inhibitor, fluoxetine, produced an additive increase in plasma β-EP-LI 1 hr after injection. Injection of the 5-HT antagonist, cyproheptadine, significantly decreased plasma β-EP-LI. Stress by immobilization for 30 min or exposing the rats to 40° ± 1°C for 30 min produced an approximate 4-fold increase in plasma β-EP-LI and ACTH-LI, which was potentiated by I.P. injections of fluoxetine. Furthermore, the stress induced increases in plasma concentrations of β-EP-LI and ACTH-LI were significantly reduced by the serotonin antagonists metergoline and cinanserin. These results suggest that 5-HT is a potent stimulator of both β-EP and ACTH release and the increase in plasma concentrations of ACTH and β-EP induced by stress are probably mediated, at least in part, by central serotonergic mechanisms.     

Effect of β-endorphin on pulsatile luteinizing hormone release in conscious castrated rats

The effect of intraventricular administration of β-endorphin on pulsatile LH release in castrated conscious rats was studied. The administration of 1 μg of β-endorphin into the lateral ventricle inhibited pulsatile discharge of LH secretion. Intravenous administration of naloxone blocked the suppressive effect of β-endorphin on LH release. These results suggest a possible role of β-endorphin, in addition to Met⁵-enkephalin, in the control of LH release in male rats.     

LEC–BiFC: a new method for rapid assay of protein interaction

Abstract

Protein–protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein–protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC–BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl–X_L and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC–BiFC can be applied easily in any lab equipped with a fluorescence microscope.     

Cross sectional study on cytokine production (TNF-α, IL-8) in German coalminers with progressive massive fibrosis and in control miners using a rapid wholeblood assay

Whole-blood release of tumour necrosis factor (TNF-α) and interleukin 8 (IL-8) was studied in 26 German ex-coalminers with progressive massive fibrosis (≥A, ILO 1980; cases) and 26 ex-miners free of pneumoconiosis (≤ 0/1; controls) using a simple wholeblood assay. Cases and controls were matched individually by age and duration of coalmine dust exposure (5-year window). Whole-blood cytokine release was determined (blinded to case control status) in incubations without additions (spontaneous) and with endotoxin (LPS, 3 ng ml^-1) or with coalmine dust (CMD; 5 mg ml^-1). CMD-stimulated TNF-α release was significantly increased and LPS-induced IL-8 release was significantly decreased in cases (matched t-tests: p < 0.01). No effect of duration of exposure was detectable in an unmatched analysis. No clear relationship with lung function parameters independent from case/control-status was observed, although a possible positive association with central airway resistance was indicated by multiple regression for both CMD stimulated TNF-α and LPS-stimulated IL-8. This study on individually matched coalminers validates previous findings on monocyte TNF release as a marker for pneumoconiosis using a method (whole-blood assay) that is more feasible for epidemiological studies. The different response of TNF-α and IL-8 may be useful in studying the occurrence of different endpoints like pneumoconiosis and lung function decrease.     

Understanding the effect of different assay formats on agonist parameters: a study using the µ-opioid receptor

The correct interpretation of data is fundamental to the study of G-protein-coupled receptor pharmacology. Often, new assay technologies are assimilated into the drug discovery environment without full consideration of the data generated. In this study, the authors look at μ-opioid receptor agonists in three different assays: (1) [(35)S]GTPγS binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) β-arrestin recruitment. Agonist-concentration effect curves were performed before and after treatment with the irreversible antagonist β-funaltrexamine, and where appropriate, these data were fitted to the operational model of agonism. The Z' value was highest in the β-arrestin assay, followed by the [(35)S]GTPγS and cAMP assays. The cAMP data fitted well to the operational model, as did the [(35)S]GTPγS data, but the [(35)S]GTPγS assay led to an apparent overestimation of K(A) values. However, in the β-arrestin assay, data did not fit the operational model, as treatment with β-funaltrexamine reduced the Emax proportionally to receptor number, with no change in EC(50). In addition, the EC(50) values generated correlated well with affinity values. In conclusion, the β-arrestin recruitment assay does not fit with traditional pharmacological theory but is of great utility as the EC(50) value generated is a good approximation of affinity.     

High-performance liquid chromatographic assay of l-α-glycerophosphorylcholine using a two-step enzymic conversion

A high-performance liquid chromatographic method using an enzymic reactor for determination of l-α-glycerophosphorylcholine in pharmaceutical forms is described. The procedure includes incubation of l-α-glycerophosphorylcholine with glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2), giving choline and glycerophosphate, and subsequent chromatography of choline with a post-column enzymic reactor and electrochemical detection. The results obtained show a close linearity of the whole assay from 2 to 150 nmol/ml l-α-glycerophosphorylcholine, the sensitivity being 2 pmol per 20 μl of injected sample. The precision of the method in the analysis of l-α-glycerophosphorylcholine in pharmaceutical forms, ampoules and capsules, was 1.34 and 1.21%, respectively.     

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C₁₈ cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.     

Simultaneous assay of muscarinic and β-adrenergic receptors using a double isotope technique

Muscarinic receptor and β-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [³H]-L-quinuclidinyl benzilate and [¹²⁵I]-(?)iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,l-propranolol on [³H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of l-quinuclidinyl benzilate on [¹²⁵I]-(?)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.     

Desipramine administered chronically inhibits lipopolysaccharide-stimulated production of IL-1β in the brain and plasma of rats

Nowadays, it is assumed that therapeutic efficacy of antidepressants depends, at least partly, on their anti-inflammatory properties. The present study investigated for the first time the effect of 21-day oral administration of desipramine on the lipopolysaccharide (LPS)-stimulated IL-1β concentration in the olfactory bulb, hypothalamus, frontal cortex, hippocampus and plasma of rats, and on the LPS-induced IL-1β mRNA level in the olfactory bulb. Desipramine (15 mg/kg/day) reduced significantly the LPS (250 μg/kg i.p.)-induced IL-1β concentration in the olfactory bulb, hypothalamus and in plasma, and diminished the LPS effect on IL-1β mRNA in the olfactory bulb. Plasma concentration of desipramine was comparable to its therapeutic range. By using the α1/α2-adrenoceptor antagonist prazosin and the unspecific β-adrenoceptor antagonist propranolol given prior to LPS, we found that the effect of desipramine on LPS-induced IL-1β production was partially mediated by both adrenoceptors in the olfactory bulb and plasma, and that β-adrenoceptors contributed also to its effect on the stimulated IL-1β concentration in the hypothalamus. The effect of LPS on the cerebral IL-1β levels was, in part, mediated by β-adrenoceptors and, in a region-specific manner, by α1/α2-adrenoceptors. The findings provide evidence for central and peripheral anti-inflammatory activity of desipramine and confirm the impact of the noradrenergic system on IL-1β production induced by an immunostimulatory challenge.     

Changes of macromolecular organizations in nonjunctional sarcolemmas after cross-innervation──a study offast-and slow-twitch muscle fibres in rats

INTRODUCTIONMa1n11la1iaIlskeletal1llusclefibresare(listiIlgl1ishedint()severaltypesacc()rdillgtodifferentclassificatiollscl1e111es.Eachtypeofmusc1efibresl1asasetofspecificcharacteristics.Ollewell-kllowl1waytoclassifymusclefibresistodividethemintotwobroadtypestslow-twitcI1ortypeIfibres,andfast-twitchortypelIfibres.ThefOrmerllormallyappearsreda11dthelaterappearswhite.Theypossesswidedifferellceswithrespectt()physiol()gical,biochemical,andmorpho1ogica1pl1ellotypiccharacteristics,suchasisom…     

BACs-on-Beads™ assay,a rapid aneuploidy test,improves the diagnostic yield of conventional karyotyping

Molecular Biology Reports - BACs-on-Beads (BoBs?) assay is a rapid aneuploidy test (RAT) that detects numerical chromosomal aneuploidies and multiple microdeletion/microduplication syndromes....     

Development and validation of a sensitive and rapid non-aqueous LC–ESI-MS/MS method for measurement of diosgenin in the plasma of normal and hyperlipidemic rats: A comparative study

A sensitive and specific electrospray ionization liquid chromatography–tandem mass spectrometry method was developed to detect diosgenin in the plasma of normal and hyperlipidemic rats. Diosgenin was extracted with n-hexane–ethyl acetate (9:1, v/v) using sarsasapogenin as an internal standard. With multiple reaction monitoring modes, linear calibration curves were obtained in the range 10–1500 ng/mL (r ≥ 0.9979) and the limit of quantification was 10 ng/mL. Intra- and inter-assay variabilities were within 7.74%, and accuracies were between ?5.33% and 1.50%. The assay was successfully applied to study pharmacokinetics in rats after oral administration of diosgenin. Significantly different pharmacokinetics between normal and hyperlipidemic rats were observed, which would be beneficial for the clinical use of diosgenin.     

Designing a simple multiplex ligation-dependent probe amplification （MLPA） assay for rapid detection of copy number variants in the genome

Copy number variants （CNVs） are pervasive in the human genome and are responsible for many Mendelian diseases and genomic disorders. The detection of CNVs is an essential element of a complete mutation screening strategy. Many techniques have been developed for gene dosage testing. Multiplex ligation-dependent probe amplification （MLPA） is a robust, easy and flexible technique that can detect both deletions and duplications for more than 40 loci in one assay. It has been widely used in research and diagnostic laboratories. We routinely develop our own MLPA assays for quick validation of array comparative genomic hybridization （CGH） findings. Here we discuss the general principles and critical aspects of MLPA assay development and validation using all synthetic MLPA probes. We believe that MLPA will play important roles in the rapid detection of genomic disorders associated with genomic imbalances, the confirmation of pathogenic mutations involving exonic deletions/duplications, CNV genotyping and population frequency analysis of CNVs.     

Determination of bisphenol A in red wine using a double vortex–ultrasound-assisted microextraction assay: Role of the interfacial properties

Bisphenol A (BPA) is a synthetic compound broadly used in medical devices as well as in packaging of food and drinks. Recently, BPA toxicity has become of concern to environmental public health. Red wine that is susceptible to BPA contamination is an alcoholic beverage made from yeast fermentation of grapes in the presence of grape skins so as to extract phenolic compounds. The aim of this study was to validate an efficient, low cost, and time-saving method for BPA determination in red-wine beverage. To this end, a rapid and simple microextraction method is here proposed consisting in liquid–liquid separation assisted by a vortex–ultrasound–vortex procedure combined with gas chromatographic analysis (GC-Fid or GC-IT/MS). By means of a comparative study between real red-wine matrix and synthetic hydroalcoholic solutions, different parameters related to the microextraction steps were investigated. The minimal amount of extraction solvent for a given volume of sample was calculated for both the systems. It was demonstrated that for red-wine matrix, the extent of phase separation is strongly affected by some wine constituents and that separation can be tuned by varying the amount of the extraction solvent. This double vortex–ultrasound-assisted method achieved high recovery of BPA and enrichment factor compared with other microextraction methods.