Orcinol Reaction Research Articles

De novo sequencing and transcriptome analysis of Stichopus horrens to reveal genes related to biosynthesis of triterpenoids

As the widespread species in the tropical Indo-Pacific area, the sea cucumber Stichopus horrens is characterized by the presence of appreciable amounts of triterpene glycosides. However, genes involved in biosynthesis of the triterpenoids in sea cucumber have rarely been researched. Here, we aim to explore the triterpenoid biosynthesis of S. horrens collected in the south sea of China. In this study, the de novo sequencing of S. horrens was carried out by Illumina HiSeq 2000. A total of 99,854,932 high quality sequence reads were obtained. These raw data were assembled into 168,151 contigs, and 78,024 unigenes were left after redundance removing. The transcriptomic data clearly showed that triterpenoid biosynthesis was via the MVA pathway in S. horrens. The content of total triterpene glycosides was examined by method of orcinol reaction, and 0.116 ± 0.012 mg/g and 0.016 ± 0.003 mg/g in intestine and body wall, respectively. qRTPCR showed that both oxidosqualene cyclases OSC1 and OSC2 are highly expressed in intestine, suggesting their commitment to triterpenoid biosynthesis. We also reported some potential genes encoding Cytochrome P450 and glycosylations enzymes, which might be involved in the late step of biosynthesis of triterpenoid glycosides. Overall, the present study provided the basic data for future molecular studies on biosynthesis of triterpene glycosides in sea cucumber.

Effects of azithromycin on Pseudomonas aeruginosa isolates from catheter-associated urinary tract infection.

Pseudomonas aeruginosa is a common pathogenic bacterium in urinary tract infections (UTIs), particularly catheter-associated UTIs. The aim of this study was to investigate the effect of azithromycin (AZM) on P. aeruginosa isolated from UTIs. Isolates were identified by biochemical assays and the Vitek system. Antimicrobial susceptibility was determined using the disk diffusion assay. Biofilm formation and adhesion were assayed using a crystal violet staining method. The swimming motility was assayed on agar plates. The elastase activity and rhamnolipid production were determined by the elastin-Congo red method and orcinol reaction, respectively. A total of 32 bacterial isolates were collected from 159 urinary catheters and eight of them were P. aeruginosa isolates. The results showed that the P. aeruginosa isolates had stronger biofilm formation capability and the biofilms were thicker than those of P. aeruginosa PAO1. AZM inhibited biofilm formation and adhesion on urinary catheters, and also decreased swimming motility and the production of virulence factors. The results of this study indicated that AZM is potentially a good choice for use in the treatment of UTIs.

Identification of orcinol reactive substance in pleural fluid cell lysate—A new parameter for classification of pleural effusion

Background Cell-free DNA is observed to be more in exudative pleural effusions . Based on this fact development of a clinical chemistry test for classification of pleural effusion will require DNA extraction followed by PCR amplification and electrophoresis. These procedures may not be cost effective for the purpose for classification of pleural effusion as already established parameters are popular for the purpose which can be estimated by comparatively low cost colorimetric procedures. Therefore development of a simple colorimetric test for the classification of pleural fluid based on nucleic acid identification test can be attempted. The aim of this work is to develop such colorimetric test for classification of pleural effusion using only pleural fluid sample. Methods Cell pellet is obtained from 5 ml pleural fluid which is lysed and subjected to DNA extraction, followed by identification under UV-transilluminator after electrophoresis and orcinol and diphenylamine reaction. Result Exudates show extractable DNA from 5 ml biofluid ( n = 52) which are not observed from transudate ( n = 32). Orcinol reaction is significantly positive in exudates ( n = 52) compared to the transudates ( n = 32). Diphenylamine test cannot differentiate exudate from transudate. Conclusion Orcinol reaction of cell lysate obtained from pleural fluid can classify pleural fluid sample into exudate or transudate.

Ganglioside from eel serum high density lipoprotein (HDL) and its role as a ligand for HDL binding protein

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.

Efficacy of a potentized homeopathic drug (Arsenicum-Album-30) in reducing cytotoxic effects produced by arsenic trioxide in mice: IV. Pathological changes, protein profiles, and content of DNA and RNA

Objective: To examine if the potentized homeopathic drug Arsenicum Album-30 can help restore the damage produced in protein profiles, DNA and RNA contents in liver and testis as a result of arsenic treatment in mice. Design: Sets of mice were injected with arsenic trioxide, one set was fed with Ars. Alb-30, another with Alcohol-30 and the final set was fed neither. The gel electrophoretic protein profiles and DNA and RNA contents in these three sets were studied. Methods: Protein profiles were studied by SDS-PAGE method; the DNA and RNA contents were assayed by the standard methods through diphenylamine and orcinol reactions respectively. Results: arsenic trioxide injection produced some pathological conditions, drastic changes (mainly reduction of protein bands) in protein sub-fractions, reduced DNA and RNA contents in both liver and testis; Ars. Alb-30-fed arsenic-intoxicated mice showed revival and restoration in both liver and testis as revealed by gel patterns and quantitative assay of DNA and RNA. Conclusion: Efficacy of the homeopathic drug Ars. Alb-30 in reducing arsenic-induced damage to protein and nucleic acids is substantiated and the mechanism of action of the homeopathic drug through expression of regulatory genes inferred.

Purification and Properties of Japanese Iris Necrotic Ring Virus, a Possible New Carmovirus.

Japanese iris necrotic ring virus (JINRV) particles were isometric about 35nm in diameter and showed rounded outlines with rough surfaces when negatively stained in uranyl acetate. Purified virus preparations contained single component with a sedimentation coefficient of 118 S. In isopycnic centrifugation in CsCl, the particles formed a single band of buoyant density 1.353g/cm3. Nucleic acid extracted from the purified virus particles was identified as single stranded RNA by the following properties: positive orcinol reaction; susceptible to RNase A both in 1×SSC or 0.1×SSC; increase of about 18% in maximum absorption and shift of the absorption maximum to higher wavelengths by 4nm after treatment with formaldehyde (1.8%). Polyacrylamide gel electrophoresis demonstrated one nucleic acid and one coat protein with estimated molecular weights of 1.28×106 and 38, 000, respectively. An antiserum against purified virus particles with a homologous titer of 512 in Ouchterlony double diffusion tests was prepared. The virus was serologically unrelated to 10 other single component viruses with isometric particles, including tomato bushy stunt, carnation mottle, and several members of the tombusvirus and carmovirus groups. However, its other properties were similar to those of carmoviruses.

A comparison of substance P peptide and preprotachykinin mRNA levels during development of rat medullary raphe and neostriatum

Substance P (SP) and the mRNA coding for its precursor peptide, preprotachykinin (PPT), were measured in medullary raphe nuclei (MRN) and neostriatum (NS) over development in order to determine (1) whether PPT mRNA levels correlate with peptide development, and (2) whether changes in PPT mRNA might help to account for the apparent decline in SP seen immunohistochemically in certain brain areas postnatally. Total RNA was quantified in dissected tissue pieces using the sensitive orcinol reaction. When MRN PPT mRNA levels measured by Northern blot analysis were adjusted to total RNA levels, PPT mRNA per MRN increased over development with a profile similar to that seen for SP peptide. Moreover, both peptide and mRNA levels exhibited a similar decline after postnatal day 15. Therefore, developmental regulation of SP biosynthesis in the MRN may, in part, explain previous evidence documenting a postnatal decline in SP there.In the NS, SP peptide and PPT mRNA increased with a similar profile from E18 through the first post-natal week. Thereafter, SP increased less rapidly than its mRNA, indicating incongruities in prohormone message and processed peptide in the NS.

Intracellular appearance, morphological features and properties of oyster mushroom virus

Viruses in Agaricus hisporus and Lentinula edodes are well-known but have not previously been reported in Pleurotus spp. Two kinds of virus were found in sporophores of Pleurotus sapidus in this study. One occurred as spherical particles 24 nm diam and the other as flexuous rods. The virus preparation of P. sapidus had a typical nucleoprotein ultraviolet spectrum and three peaks were obtained during ISCO sucrose density gradient centrifugation. The nucleic acid derived from the spherical virus was shown to be double-stranded ribonucleic acid (dsRNA) by a positive orcinol reaction and RNase treatment. Four major bands appeared during Polyacrylamide gel electrophoresis, and the molecular weight was determined at 1·3, 1·10, 0·98, and 0·85 × 10°6 using CMV dsRNA and virus dsRNA of Aspergillus niger as markers. Two major components of the virus capsid polypeptide with molecular weights 44 and 22 kDa, respectively, were obtained using discontinuous SDS-PAGE. Immune diffusion tests showed that the anti-serum to P. sapidus virus reacted with P. ostreahis virus and P. florida virus. Ultrathin sections of the septal pore apparatus of P. ostreatus revealed the regular arrangement of the pore cap perforation and the openings. Virus particles appear to be able to pass through the septal dolipore. These results provide new evidence for the translocation of viruses in basidiomycetes.

Some properties of pea stem necrosis virus isolated fro pea in Japan.

Pea stem necrosis virus (PSNV) was readily purified from infected pea plants by a chloroformbutanol procedure with yields of about 200mg/kg leaf tissues. Purified virus preparations contained a single sedimenting component with a sedimentation coefficient of 118S, buoyant density in cesium chloride of 1.35g/cm3 and nucleic acid content of 17%. Polyacrylamide gel electrophoresis demonstrated single nucleic acid component and single coat protein with estimated molecular weights of 1.53×106 and 38, 000 daltons, respectively. Nucleic acid extracted from the virus preparations was identified as single-stranded RNA by the following properties: positive orcinol reaction; susceptibility to RNase A both in 1×SSC or 0.1×SSC; increase of about 21% in maximum absorption and shift both the absorption maximum and minimum to higher wave-lengths by 4 nm on treatment with formaldehyde (1.8%). Preparations of purified particles of PSNV reacted with homologous antiserum (gel diffusion titer, 1:1, 024) but not with any antisera to 8 different plant viruses with isometric particles including tomato bushy stunt, galinsoga mosaic, carnation mottle or melon necrotic spot viruses. However, physicochemical properties were similar to those of carnation mottle and allied viruses.

A Method for Enzymic Extraction and the Measurement of Chloroplast RNA

A method is described for measuring RNA associated with chloroplast thylakoid membranes. Washed thylakoids are incubated with ribonucleases A and T(1), under low Mg(2+) conditions, to release hydrolyzed RNA into solution. After removing the membranes by centrifugation, the mono- and oligonucleotides are adsorbed by Dowex 1-X2 in miniature columns made from Pasteur pipettes, and then eluted with 2 n HCl. RNA is estimated from the absorbance of the eluate at 260 nanometers, with corresponding values obtained by the orcinol reaction for pentose. Polyacrylamide gel electrophoresis patterns of extracted RNA indicate that our current procedures for preparation of thylakoids results in material containing variable and often significant levels of RNA from 80S ribosomes. Thus values for total RNA cannot be used as a valid estimate for the level of 70S ribosomes associated with these membranes, unless an additional procedure is used to estimate the per cent contamination by 80S ribosomes.Recoveries of digested RNA from the Dowex resin of 94 to 98% were obtained with 2 milliliters of HCl eluant, making possible the analysis of thylakoid samples with as little as 4 micrograms of RNA. The procedure involves small columns and only one centrifugation, so that it is useful for obtaining reliable measurements from multiple samples.

Glycosaminoglycans are integral constituents of renal glomerular basement membrane

Basement membranes from canine renal glomeruli were isolated following osmotic lysis and sequential detergent treatment. Substantial amounts of uronic acid in unfractionated membranes were demonstrated with the carbazole and orcinol reactions. About 10–15% of basement membrane uronic acid was solubilized with neutral salt solutions. Denaturation in 8M urea solubilized ⋍70% of the uronic acid but only ⋍10% of basement membrane hydroxyproline; the latter was solubilized after reduction and alkylation. Uronic acid containing glycoprotein isolated by denaturation did not bind to carboxymethylcellulose and migrated as a high molecular weight band on SDS-gel electrophoresis. The ability of isolated rat glomeruli to incorporate radioactive sulfate in vitro was demonstrated. These findings indicate that sulfated glycosaminoglycans are integral components of glomerular basement membrane.

Requirement of alkaline hydrolysis for estimating chloroplast RNA using the orcinol reaction

Considerable amounts of orcinol-reacting material other than RNA-ribose were extracted with hot PCA from isolated spinach and broad bean chloroplasts, while cold PCA only incompletely extracted the chloroplast RNA. The procedure utilizing alkaline hydrolysis followed by an orcinol test was found to be appropriate for measuring the chloroplast RNA.

Glycoproteins released by Leishmania donovani: immunologic relationships with host and bacterial antigens and preliminary biochemical analysis.

The antigenically active glycoproteins (AAGP) released by Leishmania donovani strain 3S promatigotes into growth media and by amastigotes of this strain into the tissue, e.g. blood, of infected hamsters was found to consist of 6 to 7 antigenically distinct components. The antigenic activity of these glycoproteins was resistant to freeze-thawing, protease treatment, and purification by column chromatography using Sephadex G-100. This activity, however, was destroyed by Na periodate and altered by boiling; AAGP adhered firmly to Amicon filter (UM2). The antigenically active substances absorbed UV at 230, 260, 280 nm and gave positive Folin phenol, phenol sulfuric acid, and orcinol reactions. By gel diffusion, the component glycoproteins were found to form lines with concanavalin A and to give reactions to identity and partial identity with human red cells and Mycobacterium butyricum. The possible involvement of the antigenically active glycoproteins in pathogenesis of kala azar is discussed.

Biochemical studies during the development of the chick embryo. VI. On a deoxyribosyl compound in the ribonucleic acid preparation.

Ribonucleic acid prepared according to modified Kirby’s phenol method from 14 day old chick embryos was fractionated into three peaks by MAK column chromatography. To the peak eluted in the range from 0.65 to 0.7 m NaCl, the name “Peak II” was given tentatively. Purified Peak II was homogeneous upon ultracentrifugation, polyacrylamide gel electrophoresis and elution patterns of chromatographies.The peak gave a positive result with the orcinol reaction. Furthermore, the hydrolyzate by DNase and snake venom was active in promoting the growth of L. leichmannii 7830. The peak was not disrupted by treatment with RNase, but was with DNase. Peak II treated with heat gave the same fractionation profile as the native peak by gel filtration on Sephadex G–200.By treatment with 0.3 n KOH at 37°C for 18 hr, Peak II was resolved into two peaks, Peak II–1 and Peak II–2. It was found that Peak II consisted of the susceptible and the non-susceptible components to the alkali. From the susceptible component (Peak II–2) the ...

Some properties of RNA from Fiji disease subviral particles

Nucleic acid isolated from subviral particles of Fiji disease virus (FDV) was identified as double-stranded (ds)-RNA by the following properties: (1) Positive orcinol reaction; (2) resistance to ribonuclease (RNase) in 1 × SSC (sodium chloride-sodium citrate buffer) but not in 0.1 × SSC; (3) susceptibility to RNase in 1 × SSC after thermal denaturation; (4) sharp thermal denaturation curve with a melting temperature of 76° in 0.01 × SSC; (5) buoyant density of 1.60 g/cm 3 in Cs 2SO 4; and (6) no increase in ultraviolet absorption on treatment with formaldehyde at 37°. On electrophoresis in polyacrylamide gel, FDV-RNA separated into nine RNA segments with a total molecular weight of 15.3 × 10 6.

A simple chemical method for the determination of dermatan sulfate in the presence of chondroitin 4-sulfate and chondroitin 6-sulfate.

A simple chemical method for the determination of individual mucopolysaccharides in mixtures of dermatan sulfate and chondroitin sulfates by means of a single reagent was established, utilizing the difference in reaction rates of these polysaccharides with orcinol. To each 1 ml of a sample mixture of standard dermatan sulfate and standard chondroitin sulfate (either 4- or 6-sulfate) was added 3 ml of orcinol reagent and the resulting solution was heated in a boiling-water bath. After 20 and 60 min reaction, absorbances at 660 nm were measured and the concentrations of individual mucopolysaccharides were calculated. High reproducibility was observed for the determination of dermatan sulfate in the presence of chondroitin sulfates. In addition, orcinol reaction for 90 min employing D-glucuronolactone as a standard appeared to be of practical value in the estimation of the uronic acid content of these mucopolysaccharides.

Fruit Abscission in Cowpea,Vigna unguiculata(L.) Walp.

Fruit Abscission in Cowpea,<i>Vigna unguiculata</i>(L.) Walp.

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.

Estimation of pentose 5-phosphate in the presence of heptulose and hexose phosphates by the orcinol method

1. 1. The basic orcinol reaction with pentose, described by Mejbaum in 1939, has been modified. 2. 2. In the present method the temperature of the reaction has been reduced from 100° C to 50° C. This eliminates the reactivity of heptulose and hexose phosphates with the orcinol reagent. 3. 3. If the reaction time is extended to 2.5 hours at 50° C, ribose 5-phosphate only reacts to give 76–78 % of the maximum absorbance which is obtained by heating for 30 minutes at 100° C.

Quantitative thin-layer chromatography of glycolipids in animal tissues

A method is described for quantitative analysis of four glycolipids in brain. The combination of a thin-layer chromatographic system, which makes the previous separation of glycolipids by column chromatography unnecessary, with the orcinol reaction carried out in the presence of silica gel, permitted a rapid and simple determination of cerebrosides, sulphatides and monogalactosyl diglyceride with as little as 50 mg of brain tissue. Somewhat modified procedure was applied for a semi-quantitative determination of glycolipids in liver.