Fast Red TR Research Articles

A Study on Evaluation of Alkaline and Acid Phosphatase Isozymes During Different Developmental Stages of New Breeding Lines and Races of Bombyx mori L

It is interesting to note that different silkworm races reared in laboratory offer an important testing ground for the application of biochemical methods to taxonomic problems. Moreover, there is scarcity of knowledge on enzyme studies in new breeding lines and races of silkworm specially Bombyx mori L. Therefore, we designed the present study with the main goal to evaluate the activities of alkaline and acid phosphatases quantitatively during different developmental stages of new breeding lines and races viz. Kalimpong-A (KA), B18, Pure Mysore (PM), evolved R1 and R2 of Bombyx mori L. Quantitative estimations of in alkaline and acid phosphatases were expressed in terms of enzyme activity. Alkaline and acid phosphatase activities during the different developmental stages of KA, NB18, PM, evolved R1 & R2 races were determined using Sodium-1 naphthahyl phosphate as a substrate following the dye-coupling method. The assay mixture included 2 ml of substrate and 0.2 ml of enzyme extract and incubation was made for 30 minutes at 25°c. The reaction was stopped by adding 2 ml of post coupling solution (5 parts of 4% sodium dodecyl sulphate and 2 parts of 0.2% Fast red TR salt) and colorimetric determination were made at 540 nm. Results illustrated that the activity of phosphatases was found to be different and high activity was found in the larval stage, which is feeding stage followed by pupae.

Combination of alkaline phosphatase anti-alkaline phosphatase (APAAP)- and avidin-biotin-alkaline phosphatase complex (ABAP)-techniques for amplification of immunocytochernical staining of human testicular tissue

An amplification procedure was developed for the visualization of antigens in human testis using monoclonal antibodies against desmin and vimentin. The technique combines the high sensitive and specific APAAP- and ABAP-methods. Depending on the quality of the antibodies used and the processing of the material prior to the immunocytochemical staining the amplification technique may be applied either as a single APAAP and ABAP- or as a double APAAP and ABAP-combination. Especially after the double amplification reaction a distinct increase of the staining intensity of the vimentin- (in Sertoli cells, myofibroblasts of the lamina propria, and fibroblasts of the interstitium) and desmin- (in myofibroblasts of the lamina propria and smooth muscle cells of the blood vessels) like immunoreactivity was observed. If different diazonium salts were used for the visualization of the alkaline phosphatase activity (e.g. Fast Red TR Salt, Fast Blue BB Salt) desmin- and vimentin-like immunoreactivity can be demonstrated in the same tissue section in a double sequential staining approach. For double staining, the alkaline phosphatase technique may be combined successfully with a technique or a combination that uses peroxidase as a marker.

The Comparative Nucleophilicity of Naphthoxide Derivatives in Reactions with a Fast-Red TR Dye. A Discovery-Oriented Capstone Project for the Second-Year Organic Laboratory

In this experiment, organic chemistry students perform reactions between three naphthyl acetate derivatives and the diazonium salt Fast-Red TR, under basic conditions. The three naphthyl acetate derivatives used in this study are 2-naphthyl acetate (1a), 6-bromo-2-naphthyl acetate (1b) and 1,6-dibromo-2-naphthyl acetate (1c). The two-step, one-pot reactions (ester hydrolysis followed by reaction with Fast Red TR) are screened for the formation of an orange–red diazo dye both visually and by UV–vis spectroscopy. Students discover that the hydrolysis and electrophilic aromatic substitution is the fastest with ester 1a and the slowest with ester 1c. This allows students to conclude that electrophilic aromatic substitution, rather than ester hydrolysis, is rate-limiting. This experiment is performed over a two-week period: during the first week students synthesize and purify compounds 1b and 1c, which are easily characterized through 1H NMR and IR spectroscopies. The ester hydrolysis and diazotization visual and UV–vis studies are performed during the second week of the experiment.

CD163, a Marker of Perivascular Macrophages, Is Up-Regulated by Microglia in Simian Immunodeficiency Virus Encephalitis after Haptoglobin-Hemoglobin Complex Stimulation and Is Suggestive of Breakdown of the Blood-Brain Barrier

Macrophages and microglia are the major cell types infected by human immunodeficiency virus and simian immunodeficiency virus (SIV) in the central nervous system. Microglia are likely infected in vivo, but evidence of widespread productive infection (ie, presence of viral RNA and protein) is lacking. This conclusion is controversial because, unlike lymphocytes, macrophages and microglia cannot be discreetly immunophenotyped. Of particular interest in the search for additional monocyte/macrophage-lineage cell markers is CD163; this receptor for haptoglobin-hemoglobin (Hp-Hb) complex, which forms in plasma following erythrolysis, is expressed exclusively on cells of monocyte/macrophage lineage. We examined CD163 expression in vitro and in vivo by multiple techniques and at varying times after SIV infection in macaques with or without encephalitis. In normal and acutely SIV-infected animals, and in SIV-infected animals without encephalitis, CD163 expression was detected in cells of monocyte/macrophage lineage, including perivascular macrophages, but not in parenchymal microglia. However, in chronically infected animals with encephalitis, CD163 expression was detected in activated microglia surrounding SIV encephalitis lesions in the presence of Hp-Hb complex, suggesting leakage of the blood-brain barrier. CD163 expression was also induced on microglia in vitro after stimulation with Hp-Hb complex. We conclude that CD163 is a selective marker of perivascular macrophages in normal macaques and during the early phases of SIV infection; however, later in infection in animals with encephalitis, CD163 is also expressed by microglia, which are probably activated as a result of vascular compromise.

Patterns of gene expression in schistosomes: localization by whole mountin situhybridization

As a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mount in situ hybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

Disruption of the Insulin-like Growth Factor Type 1 Receptor in Osteoblasts Enhances Insulin Signaling and Action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.

CHARACTERIZATION OF COUNTERFEIT ARTESUNATE ANTIMALARIAL TABLETS FROM SOUTHEAST ASIA

In southeast Asia, the widespread high prevalence of counterfeits tablets of the vital antimalarial artesunate is of great public health concern. To assess the seriousness of this problem, we quantified the amount of active ingredient present in artesunate tablets by liquid chromatography coupled to mass spectrometry. This method, in conjunction with analysis of the packaging, classified tablets as genuine, substandard, or fake and validated results of the colorimetric Fast Red TR test. Eight (35%) of 23 fake artesunate samples contained the wrong active ingredients, which were identified as different erythromycins and paracetamol. Raman spectroscopy identified calcium carbonate as an excipient in 9 (39%) of 23 fake samples. Multivariate unsupervised pattern recognition results indicated two major clusters of artesunate counterfeits, those with counterfeit foil stickers and containing calcium carbonate, erythromycin, and paracetamol, and those with counterfeit holograms and containing starch but without evidence of erythromycin or paracetamol.

Kinetic Analysis of a Two‐Phase EC Mechanism with Concomitant Lateral Processes

AbstractElectrochemical transfer of azo dye fast red TR across the water/1,2 – dichloroethane (DCE) interface followed by the coupling reaction with 1‐naphthylamine in the organic phase is studied. Pseudo first order rate constants of this reaction were obtained by fitting the experimental Ip−/Ip+ ratio under different experimental conditions with the theoretical values reported by Nicholson and Shain. The occurrence of lateral processes is demonstrated (partition of the azo dye non assisted by potential and electrochemical transfer of the protons generated in the coupling reaction), which constrains the accurate determination of the kinetic parameters.

Continuous monitoring of prostatic acid phosphatase using self-indicating substrates

Background: The continuous measurement of acid phosphatase (EC 3.1.3.2) activity in serum represents an analytical task not yet sufficiently accomplished. Methods: Introducing two novel substrates—2-chloro-4-nitrophenyl phosphate (CNP-P), which was preferred, and 4-nitronaphthyl-1-phosphate (NN-P)—an alternative assay to measure enzymatic activity was developed and compared with a modification of Hillmann's method (azo coupling of released naphth-1-ol with a diazonium compound). Apart from different substrate concentrations of 2-chloro-4-nitrophenyl phosphate, 4 mmol/l, and naphthyl-1-phosphate (N-P), 8 mmol/l (with Fast Red TR, 5 mmol/l), respectively, following identical conditions were selected: Citrate, 50 mmol/l, pH 5.75; pentane-1,5-diol, 150 mmol/l; tartrate, 60 mmol/l; 37 °C. Results: Whereas intensity and stability of the azo dye unpredictably depend on the albumin concentration of the sample, the direct test with 2-chloro-4-nitrophenyl phosphate resisted sample interferences, showed no intrinsic hydrolysis by albumin, relied on stable reagents and proved superior in sensitivity, precision and ease of handling. In measuring prostatic phosphatase, the proposed procedure closely correlated with Hillmann's method. The preliminary 0.95-reference intervals for adults were 1.2–3.9 kU/l and 5.8–14.8 U/l for total activity, respectively. Conclusions: The direct assay of the enzyme is suited as an economic, rapid and robust method for mechanized or manual use.

Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.

Fake artesunate in southeast Asia

Artesunate is a key antimalarial drug in the treatment of multidrug-resistant Plasmodium falciparum malaria in southeast Asia. We investigated the distribution of counterfeit artesunate tablets by use of the validated, simple, and inexpensive Fast Red TR dye technique. We also aimed to identify distinguishing characteristics of the fake drugs. Of 104 shop-bought "artesunate" samples from Cambodia, Laos, Myanmar (Burma), Thailand, and Vietnam, 38% did not contain artesunate. Characteristics such as cost and physical appearance of the tablets and packaging reliably predicted authenticity. The illicit trade in counterfeit antimalarials is a great threat to the lives of patients with malaria. The dye test will assist national malaria control authorities in urgently needed campaigns to stop this murderous trade.

A colorimetric field method to assess the authenticity of drugs sold as the antimalarial artesunate

Artesunate is the most widely used of the artemisinin derivatives. These drugs are being used increasingly throughout the tropical world, and are an essential component of the treatment of multi-drug resistant malaria. The recent and widespread appearance of counterfeit artesunate tablets in several countries in Southeast Asia poses a serious threat to health in this region. We have developed a simple, inexpensive colorimetric test to determine artesunate authenticity in tablets. The test is based on a reaction between an alkali decomposition product of artesunate and a diazonium salt, fast red TR (FRTR). The appearance of a yellow color indicates the presence of artesunate. The specificity of the test is dependent on the pH of the reaction. Among other antimalarials tested, (i.e. artemisinin, artemether, chloroquine, quinine, primaquine, sulfadoxine, and pyrimethamine) only artesunate produced a positive color reaction at pH 4. The assay requires only 1% of the total weight of a standard tablet containing 50 mg of artesunate and can be completed within 10 min. The method was tested on six genuine artesunate tablets and six counterfeit artesunate tablets obtained in Southeast Asia. The average amount of artesunate in the genuine tablets was determined to be 50.8±2.9 mg while the counterfeit tablets were found to contain no artesunate.

Non-destructive measurement of acid phosphatase activity in the rhizosphere using nitrocellulose membranes and image analysis.

We developed a method using nitrocellulose membranes and image analysis to localise and quantify acid phosphatase activity in the rhizosphere of two plant species, one with cluster roots (Dryandra sessilis (Knight) Domin) and another with ectomycorrhizal roots (Pinus taeda L.). Membranes were placed in contact with roots and then treated with a solution of x, α-naphthyl phosphate and Fast Red TR. Acid phosphatase activity was visualised as a red imprint on the membrane. We quantified acid phosphatase activity by image analysis of scanned imprints. The method was used to estimate the spatial distribution of acid phosphatase activity within particular root classes (lateral roots, mycorrhizal roots, root clusters). Over 95% of the acid phosphatase activity of the root system of D. sessilis was associated with cluster roots, and between 20 and 32% of the root surface active. About 26 % of the acid phosphatase activity of the root system of P. taeda was associated with mycorrhizal roots and unsuberised white root tips and less than 10% of the root surface was active, irrespective of root type. This non-destructive method can be used for rapid, semi-quantitative assessment of acid phosphatase activity in the laboratory and in situ.

A sensitive fluorescence in situ hybridization technique for detection of porcine reproductive and respiratory syndrome virus

A sensitive fluorescence in situ hybridization (ISH) for detecting porcine reproductive and respiratory syndrome virus (PRRSV) RNA in viral infected tissue was developed using digoxigenin-labeled RNA probes targeted on the nucleocapsid gene of PRRSV. In situ RNA/RNA hybrids were detected with an anti-digoxigenin antibody–alkaline phosphatase conjugate and further revealed with Fast Red TR salt/naphthol AS-MX phosphate using a fluorescent microscope. Viral nucleic acid was readily demonstrated within macrophages, known to be the major target of PRRSV. In addition, positively stained cells were found in the salivary gland and skin tissues which have not been reported to contain PRRSV infected cells before. In conclusion, the fluorescence ISH used in this study provides a fast and sensitive means for screening virus-infected tissues in which relatively few cells are affected. This advantage will be especially beneficial for studying viral persistence and for routine diagnosis of PRRSV infection.

In Situ Analysis of Community Structure in Activated Sludge with 2-Hydroxy-3-Naphthoic Acid-2-Phenylanilide Phosphate and Fast Red TR In Situ Hybridization.

Fluorescent vital staining and rRNA-targeted in situ hybridization were used to investigate the physiological activity and microbial population structures of two types of activated sludge: fresh returned sludge from a municipal wastewater treatment plant and MITI-standard sludge (a microbial source in a biodegradation test, Modified MITI Test). The ratio of esterase-active bacteria, as determined by double-staining with 4', 6-diamidino-2-phenylindole (DAPI) and 6-carboxyfluorescein diacetate (6CFDA), was 60-80% to total bacterial number in both types of activated sludge; both ratios being much higher than those of the colony forming bacteria. Microbial population structures in both types of activated sludge were determined by 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP) and Fast Red TR in situ hybridization (HNPP-FISH). Approximately 50% of the DAPI-stained bacteria was detected by HNPP-FISH in wastewater treatment plant sludge and 40% in the MITI-standard sludge. The bacterial community structure in wastewater treatment plant sludge was dominated by the Flavobacterium-Cytophaga group. Characteristic coccoid cells, which fluoresced bright yellow under UV excitation after DAPI-staining, were dominant in the MITI-standard sludge.

RRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water.

An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I): the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site, 54-68% of all cells visualized by staining with DAPI (4',6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 39-45% of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes group. At the oligotrophic site, only 26-38% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.

Development of a direct in situ PCR method for detection of specific bacteria in natural environments.

We applied HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis, E. coli cells in polluted river water also were detected.

Identification of arterial and venous segments of blood vessels using alkaline phosphatase staining of ink/gelatin injected tissues.

The present study describes a method for discriminating between the arterial and venous segments of blood vessels in mouse tissues and organs using alkaline phosphatase (ALPase) staining of ink/gelatin injected tissues. Anesthetized mice were injected through the left ventricle with blue ink/gelatin, and various organs and tissues were removed from the body and fixed by immersion in 10% formalin. Sections 50-100 microm thick were incubated for ALPase in a medium containing naphthol AS-BI phosphate and fast red TR by the azocoupling method. In such specimens as the brain and skeletal muscles, ALPase activity was found in arterioles and capillaries on the arterial side, whereas it was absent in capillaries on the venous side and in venules. In the liver, only branches of the hepatic artery were positive. ALPase activity was absent in the vessels of the lung except for a positive reaction in branches of the bronchial arteries. These findings indicate that the ALPase activity is confined to the arterioles and arterial segments of the capillaries in the systemic circuit. Thus, ALPase staining of ink/gelatin injected specimens is a useful method for differentiating the arterial and venous segments of the micro-vascular bed in various organs and tissues in mice.

Fluorescent cytochemistry of acid phosphatase and demonstration of fluid-phase endocytosis using an azo dye method.

The aim of this work was the development of a fluorescent microscopy technique to visualize acid phosphatase activity in living and pre-fixed cells. We have shown that a coupling azo dye method, using naphthol AS-MX phosphate (NP) as substrate and fast red TR (FR) as a diazonium salt coupling agent, gives rise to a fluorescent azo dye reaction product which permits a highly sensitive demonstration of lysosomal acid phosphatase in both living and pre-fixed monolayer cell cultures. The granular staining is prevented by inhibition of acid phosphatase activity using fluoride and/or orthovanadate in both living and pre-fixed preparations. Lysosomal staining in living cells is also abolished by inhibition of fluid-phase endocytosis using low temperatures or inhibition of oxidative phosphorylation. It was shown that whilst NP entered living cells by passive diffusion, occurrence of FR in lysosomes resulted from fluid-phase endocytosis. Spectroscopic analysis of the emission and absorption features of FR, NP, naphthol AS-MX (N), and the N-FR azo dye reaction product in solution corroborated our microscopic results. The differing uptake mechanisms, and the occurrence of lysosomally localized azo dye, were also in keeping with the predictions of quantitative structure-activity relationship models of this system.

Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein.

We report a new technique using fluorescent probes to detect a mRNA and a protein simultaneously in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR was used as a fluorescent substrate for alkaline phosphatase. It was possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. We applied this technique to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. We stained embryos bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA was detectable in the ventral region of the limb primordium, and β-galactosidase protein in the dorsal region. In the middle, both mRNA and protein were detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium.