Analytical method for determination of nitric oxide in zebrafish larvae: Toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coil lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae. (C) 2011 Elsevier Inc. All rights reserved.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu2+) to cuprous ions (Cu1+), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

Use of S1 nuclease and formamide in combination for the reassociation studies on GC-rich DNA

The optimal parameters in the use of nuclease S1 in DNA reassociation kinetics in the presence of formamide have been determined. The conditions are especially suitable for the study of DNA rich in mole percent GC. A 10-fold dilution of the reassociation samples leading to a decrease in both NaCl and formamide concentrations, consequently resulting in a lowering of Tm by only 1.5°C, and the S1 digestion at temperatures identical to the reassociation assay in order to retain the stability of the duplex, are two important aspects of this system. Under these conditions, the kinetics of reassociation followed the theoretically predicted pattern, while the earlier reported methods have shown lower values.

A simple procedure for purification of N-carbamoylputrescine: Application to assays of putrescine transcarbamoylase and agmatine iminohydrolase activities

The observation that N-carbamoylputrescine is quantitatively excluded on O-(carboxymethyl)-cellulose columns with simultaneous retention of putrescine and agmatine has been utilized to develop a sensitive radiometric assay for putrescine transcarbamoylase and a colorimetric assay for agmatine iminohydrolase. A simple procedure for obtaining bulk amounts of pure synthetic N-carbamoylputrescine by separation from putrescine and dicarbamoylputrescine on Dowex 50 (Na+) resin is described.

Isolation of hen's egg yolk very low density lipoproteins by DEAE-cellulose chromatography

A procedure has been developed for the isolation of very low density lipoproteins from hen's egg yolk plasma using DEAE-cellulose chromatography. This procedure is rapid and does not require ultracentrifugation and should, therefore, serve as a useful procedure for use in laboratories where this facility does not exist.

A sensitive radioimmunoassay for isepentenyladenosine

A sensitive radioimmunoassay for the nucleoside isopentenyladenosine (iA) by using iA-specific antibodies and a nitrocellulose membrane filtration technique is described. The reliability of the method is demonstrated by using specific tRNAs of known structures for the estimation of iA in their digests. This assay can be used to quantitate minute amounts of iA in the presence of a large excess of other nucleosides.

Separation of sulfur-containing components of transfer ribonucleic acid on Bio-Gel P-2 and Sephadex G-10 columns

S-Labeled nucleosides of E. coli tRNA and some of the derivatives of thionucleosides were separated on Bio-Gel P-2 and Sephadex G-10 columns employing buffers of low salt concentration and high pH.

A simple method for large-scale preparation of chick embryo tRNA

A simple method for preparing bulk quantities of tRNA from chick embryo has been developed. In this method chick embryos were homogenized in a buffer of pH 4.5, followed by deproteinization with phenol. The aqueous layer was allowed to separate under gravity. The resulting aqueous layer, after two more phenol treatments, was directly passed through a DEAE-cellulose column and the tRNA eluted therefrom with 1 Image NaCl. The tRNA prepared by this method was as active as the one prepared at neutral pH.

New colorimetric method for the estimation of resorcinol

A new, rapid, simple, and sensitive colorimetric method for the estimation of resorcinol in microgram amounts is described.

Artifactual staining of proteins on polyacrylamide gels by nitrobluetetrazolium chloride and phenazine methosulfate

Different purified proteins were shown to give purple formazan bands corresponding to the protein stain following electrophoresis on polyacrylamide gels, in the presence of nitrobluetetrazolium (NBT) and phenazine methosulfate (PMS). Both PMS and NBT are needed for formazan production which has a favorable pH at 8.5. Sulfhydryl blockers in the incubation medium inhibited this color development to different extents. While proteins with free SH groups like bovine serum albumin, ovalbumin, and urease showed this pyridine nucleotide independent artifact, nonthiol proteins, viz., bovine pancreatic ribonuclease A, and riboflavin-binding protein from chicken egg white failed to do so. The nonenzymatic formazan formation observed with different proteins could also be shown in an in vitro assay system. It is clear that the “nothing dehydrogenase” phenomenon observed in several cases may be due to the thiol group-mediated artifactual staining of proteins.

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.

New colorimetric method for the estimation of aldehyde derivatives of vitamin A

A new colorimetric method for the estimation of aldehyde derivatives of vitamin A is described. The assay depends upon the formation of colored derivatives of the vitamin A aldehydes by reaction with p-aminobenzoic acid or p-aminosalicylic acid in the presence of 2.0 N hydrochloric acid.

Disc electrophoresis of sorghum seed proteins in polyacrylamide gels

Electrophoretic analyses of sorghum flour protein by disc electrophoresis in polyacrylamide gels containing urea have been described. The albumin, globulin, and prolamin fractions of sorghum endosperm meal have been investigated, using pH 9.5 and 4.3 gel systems with four different buffers. Highly complex patterns were observed for all three protein fractions. It has been suggested that this method can provide a convenient tool for the analyses of seed proteins which are relatively insoluble in aqueous buffers.

A colorimetric method for the estimation of phloroglucinol

1. 1. A simple method has been devised for the estimation of phloroglucinol based on the formation of an intense colored compound with a modified Ehrlich reagent in the presence of trichloroacetic acid. Some factors affecting the formation of color have been studied. 2. 2. Careful regulation of trichloroacetic acid content in the system permits its estimation in 1–15 μg in the micro range and in 10–50 μg in the macro range. Phloroglucinol lends itself to ready separation by paper chromatography and estimation after clution from paper.