Novel comprehensive approach for accessible biomarker identification and absolute quantification from precious human tissues

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies.     

The growth of children in relation to the timing of obesity development

Objective: To examine whether there is an association between the timing of the development of obesity and children's growth. Research Methods and Procedures: This study investigated 141 prepubertal obese children (76 girls) and 72 healthy non‐obese children (39 girls). The target height standard deviation score (SDS), the percentage weight for height, and the height SDS (H‐SDS) at presentation and at the age of 2 years were calculated. Patients were classified, according to whether obesity developed before or after the age of 3 years, as presenting with early‐onset or late‐onset obesity, respectively. Results: Mean age (±SD) at presentation was 9.4 (2.1) years. At the age of 2 years, the H‐SDS of the children with early‐onset obesity was 1.3 (1.0) vs. 0.9 (1.3) for the late‐onset obese (p > 0.5) and 0.4 (1.0) for controls (p < 0.001), and the children with late‐onset obesity were also significantly taller than controls (p < 0.005). At presentation, children with early‐onset obesity were significantly taller than children with late‐onset obesity [1.1 (0.8) vs. 0.6 (1.0); p < 0.001] and controls [0.2 (0.8); p < 0.001]. There was no increase in H‐SDS after the age of 2 years in the late‐onset obese children (p > 0.05). H‐SDS values were below average in 21% of the children with late‐onset obesity and in only 4% of the children with early‐onset obesity. Discussion: These findings indicate that late development of obesity is not associated with increased stature in prepubertal children; however, it may be preceded by growth acceleration in the early years of life. Growth acceleration in early life may be a predictor for future obesity.     

Simple method for developing percentile growth curves for height and weight

The present paper demonstrates the ease of use of method I by Preece and Baine ([1978] Ann Hum Biol 5:1-24) in generating smoothed growth curves for both height and weight. Using the National Center for Health Statistics (NCHS) growth curve data, smoothed curves were developed and compared to those produced using the least-squares-cubic-spline method. Based on the lower sum of squares and better fit of shape as indicated by residual examination, it was concluded that the method I curve fitting procedure by Preece and Baine ([1978] Ann Hum Biol 5:1-24) fit centile growth curves for height and weight in 2-18-year-old male and female children as well as, if not better than, the least-squares-cubic-spline method used in developing the 1979 NCHS growth curves. Further, as this paper demonstrates, smoothed curves can be generated on a desktop computer using readily available software (the SOLVER function within Microsoft EXCEL).     

Growth patterns among seminomadic pastoralists (Datoga) of Tanzania

Anthropometric measurements made on 470 individual children (age 0–18 years) from a seminomadic population of Datoga pastoralists living in northern Tanzania were used to describe patterns of child growth. Comparisons with reference growth curves derived from American samples suggest that pastoral Datoga children grow poorly in this region. Body compositional changes with age differed markedly from the reference population. There were negligible fat gains through childhood, even among females. Comparison with data on other East African pastoralists showed that population growth performance is intermediate between that of nomadic and settled pastoralists. Little catch-up growth occurs during childhood, and adolescence appears to be delayed among males. The results contribute to the growing database on health indicators for African pastoralists and suggest a need for further research to investigate mechanisms for growth stunting in these populations. Am J Phys Anthropol 109:187–209, 1999. © 1999 Wiley-Liss, Inc.     

A colony lift immunoassay for the specific identification and quantification of Listeria monocytogenes

A colony lift immunoassay (CLI) has been developed to detect Listeria monocytogenes after the organisms have been cultured on filter membranes or agar plates. Polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA), used in the CLI, were prewet with methanol and used to imprint colonies that were grown on the filter or agar plates. A positive control was applied to the edge of each membrane. The imprinted membranes were subsequently air dried, peroxidase neutralized, blocked, and reacted for 20 min with a 2-microg/ml unconjugated Mab EM-7G1 solution. The membranes were washed briefly and reacted for 30 min with a 1:2000 dilution of a commercially prepared peroxidase-labeled goat anti-mouse secondary antibody (Kirkegaard and Perry Laboratories (KPL), Gaithersburg, MD). After a second wash step, the membranes were exposed to a 3,3',5, 5'-tetramethylbenzidine membrane substrate (KPL), rinsed in deionized water, and allowed to dry. Colonies of L. monocytogenes were identified by a blue color reaction on the membrane, which could be used to reference the colonies either on the filter membranes or agar plates. The CLI was tested against a wide range of Listeria species as well as several non-Listeria species and was shown to have a high degree of sensitivity (96%) and specificity (90%). We have shown that it is useful as a simple and rapid method to detect and identify L. monocytogenes.     

A high-throughput method for quantification of glycoprotein sialylation

Sialic acid can improve qualities of therapeutic glycoproteins such as circulatory half-life, biological activity, and solubility. In production of therapeutic glycoproteins, a high-throughput method is required for process monitoring and optimization to ensure consistent and optimal sialic acid content. Current methods for quantifying sialic acid, however, require chromatographic separation that is time-consuming and cannot rapidly analyze many samples in parallel. Here we present a novel high-throughput method for quantifying glycoprotein sialylation. Using chemical reduction, enzymatic release of sialic acid, and chemical derivatization of the sialic acid, the method can accurately, rapidly (15 min), and specifically analyze many samples in parallel. It requires only 45 μl of sample and has a quantitation limit of 2 μM sialic acid. It has also been validated for monitoring sialylation of recombinant interferon gamma (IFN-γ) produced in Chinese hamster ovary (CHO) cell culture. This method is useful for various applications in upstream and downstream bioprocesses.     

A direct method for quantification of non-transferrin-bound iron

A direct method for quantification of non-transferrin-bound iron has been developed. This assay relies on the use of a large excess of a low affinity ligand (nitrilotriacetic acid, NTA) which removes and complexes all low molecular weight iron and iron nonspecifically bound to serum proteins. Iron bound to transferrin, ferritin, desferrioxamine, and its metabolites is unaffected. The Fe-NTA complex present in the serum ultrafiltrate is then quantified using an automated HPLC procedure where on-column derivatization with a high affinity iron chelator (3-hydroxy-1-propyl-2-methyl-pyridin-4-one) takes place. The iron complexes of desferrioxamine and its metabolites are unaffected by the above-derivatization procedure. With minor modifications, this method is equally applicable for the quantification of low molecular weight iron in other biological fluids.     

A PCR-based method for detection and quantification of small RNAs

Recent cloning efforts have identified hundreds of thousands of small RNAs including micro RNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). These non-coding small RNAs need to be further validated and characterized by detecting and quantifying their expression in different tissues and during different developmental courses. A simple, accurate, and sensitive method for small RNA expression profiling is in high demand. Here, we report such a PCR-based method.     

A method for the quantification ofFrankia by estimation of hyphal length

Summary The cellular mass ofFrankia, a filamentous actinomycete, was readily quantified by estimating hyphal length, using a modification of Tennant's method for the estimation of root length. Each sample ofFrankia was stained with Coomassie Brilliant blue G 250, dispersed well, and suspended in a 0.5% agar solution. One drop of the suspension was placed in a Petroff-Hausser counting chamber with 0.05 by 0.05mm grid squares. The number of intersections betweenFrankia hyphae and the grid lines in a standard area were counted under a microscope and converted to hyphal length. Using the formula: hyphal length (HL) in mm equals (11/14) times the number of intersections (n) times the grid dimension (0.05 mm). The validity of the line intersection method was tested by comparison with total protein estimates of replicate aliquots ofFrankia culture. Correlations between total protein and hyphal length estimates were strong (r² from 0.76 to 0.95; standard errors of 3 to 9% of estimated length). These results show that line intersection counts may be a satisfactory routine method for quantifyingFrankia in culture and may be especially suitable for detecting small amounts of livingFrankia in less time than with other methods.Dedicated to the memory of Professor John G. Torrey     

A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIII-tubulin represented 3.2% of the total HeLa β-tubulin.     

A kinetic method for quantification of aspartate aminotransferase isoenzymes

A spectrophotometric assay is proposed to determine the levels of aspartate aminotransferase (AAT) isoenzymes from chicken liver by a steady-state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of substrate 2-oxoglutarate at pH 6.2. The use of a standard curve permits the determination of the percentage of chicken liver c-AAT and m-AAT isoenzymes. This method yields results in good correlation with those achieved by different extent adipate inhibition and by differential centrifugation.     

A new method for the quantification of chitin and chitosan in edible mushrooms

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.     

A novel method for characterisation and quantification of plant root exudates

A microcosm unit is described which readily allows manipulation of experimental conditions to enable the subsequent impact on root exudation release to be monitored with time. Festuca ovina and Plantago lanceolata seedlings were grown in this microcosm unit over a 34 day experimental period under conditions of high (3.75 mol m^–3 N) or low (1.25 mol m^–3 N) nitrate-nitrogen treatment. At the end of the experimental period the seedlings in the microcosms were labelled with [¹⁴C]-CO₂ and the fate of the label within the plant and its release by the roots monitored. Total organic carbon (TOC) content of the collected exudate material was measured throughout the experimental period as well as during the ¹⁴C-chase period and comparison of plant C budgets using these two measurements is discussed. Nitrogen treatment as found to have a greater effect on exudate release by F. ovina than by P. lanceolata seedlings as indicated by both the total organic carbon and ¹⁴C results. The use and applications of the microcosm unit are discussed.     

A robust and selective method for the quantification of glycosylphosphatidylinositols in biological samples

We have developed an assay for the quantification of glycosylphosphatidylinositol (GPI)-anchored glycoconjugates. The method is based on nitrous acid deamination and sodium borodeuteride reduction of the glucosamine residue, common to all GPI structures, to yield [1-2H]-2,5-anhydromannitol. Following acid methanolysis and trimethylsilyl derivatization, detection is by selected ion monitoring gas chromatography-mass spectrometry. The unnatural inositol isomer scyllo-inositol is used as an internal standard and the [1-2H]-2,5-anhydromannitol trimethylsilyl derivative is detected by following a characteristic electron-impact fragment ion at m/z 273. This method is more selective for GPIs than assays based on measuring myo-inositol content, which are often confounded by contaminating inositol-phospholipids. We show that the method can be applied to measure total GPI content in crude total lipid extracts and even in whole trypanosome ghosts. The method was applied to whole cell lysates of wild-type, GPI-deficient, and glycosaminoglycan-deficient CHO cells. The data revealed that proteoglycans did not interfere with total glucosamine estimates but that there are background levels of non-GPI and nonproteoglycan glucosamine-containing material in CHO cells.     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.     

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.