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PubMed1980

The application of differential pulse voltammetry at the glassy carbon electrode to multivitamin analysis.

Differential pulse voltammetry at the glassy carbon electrode has been applied to the determination of ascorbic acid, pyridoxine and folic acid in a multivitamin preparation. The individual vitamins all gave well-defined peaks in the anodic region with a linear response of peak current to concentration. The water-soluble vitamins were extracted into aqueous solution and folic acid into dibasic potassium phosphate solution. Before determination of pyridoxine, the ascorbic acid peak was depressed by reaction with formaldehyde. Iron (II) present in the multivitamin preparation did not interfere with the analysis. The voltammetric method compared favourably in terms of accuracy and precision with official methods: it was found to be much simpler, with sample manipulation kept to a minimum. The method was found to be generally applicable to the determination of the vitamins in several multivitamin preparations, or, in simplified form, to the determination of the individual vitamin preparations.

Ascorbic Acid↗
PubMed2001

Final steps of natural killer cell maturation: a model for type 1-type 2 differentiation?

Analysis of cytokine and differentiation antigen expression in human natural killer (NK) cells revealed that interleukin 13 (IL-13) and interferon gamma (IFN-gamma) are produced at sequential stages during irreversible IL-12-induced differentiation. In human NK cell clones, polyclonal CD3-CD161+CD56- cells and peripheral lymphocytes, IL-4 induced the proliferation of both IL-13+ NK and T cells, whereas IL-12 allowed a proliferation-independent accumulation of IFN-gamma+ cells. These data disproved the NK1-NK2 hypothesis and challenge the current T helper 1 (TH1)-TH2 paradigm. We propose that the cytokine environment regulates a type 2-->0-->1 developmental progression, with IL-12 needed for terminal differentiation and IL-4 delaying this process, rather than a type 1 versus type 2 decision of a type 0 cell.

Antigens, Differentiation↗
PubMed1998

Differentiation in an olfactory cell line. Analysis via differential display.

The olfactory epithelium is a unique system, in which new neurons are continually generated throughout adult life. Olfactory neurons are derived from stem cells that lie adjacent to the basal lamina of the olfactory epithelium; these stem cells divide several times and their progeny differentiate into mature sensory neurons. In our laboratory immortalized cell lines have been derived from these dividing cells. The morphology of these cell lines and their expression of neuronal markers varies with culture conditions. When grown in low serum medium one of these cells lines, OLF 442, differentiates by extending long neurites and increasing its expression of neurofilament and B50/GAP43 proteins at the same time reducing expression of glial fibrillary acidic protein (GFAP). Identification of differentially expressed mRNA in cell lines has previously relied on both screening for known markers, and the use of subtractive techniques for identification of unique mRNA species. The differential display technique allows simultaneous detection of differentially expressed mRNA at different time periods and growth conditions. A modified Liang and Pardee differential display technique was used to screen OLF 442 over a number of time intervals in serum-depleted media, and compared with OLF 442 grown in complete media. The differentially displayed fragments were cloned and sequenced, leading to the identification of a number of sequences, both known and unknown. The known sequences include SPARC (encoding a Ca2+ binding secreted Protein which is Acidic and Rich in Cysteine), which is reported to function as a modulator of the cell matrix, and RHAMM, the receptor for hyaluronan-mediated motility. Both the known and the unknown sequences are being studied further to provide insight into the differentiation of olfactory neurons.

Animals↗
PubMed2001

Identification of a polymorphic mucin-like gene expressed in the midgut of the mosquito, Aedes aegypti, using an integrated bulked segregant and differential display analysis.

The identification of putative differentially expressed genes within genome regions containing QTL determining susceptibility of the mosquito, Aedes aegypti, to the malarial parasite, Plasmodium gallinaceum, was investigated using an integrated, targeted approach based on bulked segregant and differential display analysis. A mosquito F2 population was obtained from pairwise matings between the parasite-susceptible RED strain and the resistant MOYO-R substrain. DNA from female carcasses was used to genotype individuals at RFLP markers of known chromosomal position around the major QTL (pgs 1). Midguts, dissected 48 hr after an infected blood meal, were used to prepare two RNA bulks, each representing one of the parental genotypes at the QTL interval. The RNA bulks were compared by differential display PCR. A mucin-like protein gene (AeIMUC1) was isolated and characterized. The gene maps within the pgs 1 QTL interval and is expressed in the adult female midgut. AeIMUC1 RNA abundance decreased with time after blood meal ingestion. No differential expression was observed between the two mosquito strains but three different alleles with inter- and intrastrain allelic polymorphisms including indels and SNPs were characterized. The AeIMUC1 gene chromosome location and allelic polymorphisms raise the possibility that the protein might be involved in parasite-mosquito interactions.

Aedes↗
PubMed1997

New Rhizobium leguminosarum flavonoid-induced proteins revealed by proteome analysis of differentially displayed proteins.

Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium. R. leguminosarum bv. trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4'-dihydroxyflavone. Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output. Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins. The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed. The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base. The identity of 12 proteins, including NodE and NodB, was established. NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth. Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.

Acyltransferases↗
PubMed2003

HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain.

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H(2)N-(His)(6)-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO(2)H, which consists of four functional elements: i) an affinity ligand (His(6)-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d(4)) or no (d(0)) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d(4)) or (d(0)) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d(4) co-elute with their d(0)-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni(2+) affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of (13)C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.

Amino Acid Sequence↗
PubMed2002

Differential display analysis of gene expression in invertebrates.

Screening for differentially expressed genes is a straightforward approach to study the molecular basis for changes in gene expression. Differential display analysis has been used by investigators in diverse fields of research since it was developed. Differential display has also been the approach of choice to investigate changes in gene expression in response to various biological challenges in invertebrates. We review the application of differential display analysis of gene expression in invertebrates, and provide a specific example using this technique for novel gene discovery in the nematode Caenorhabditis elegans.

Animals↗
PubMed1995

Search for life on Mars: evaluation of techniques.

An important question for exobiology is, did life evolve on Mars? To answer this question, experiments must be conducted on the martian surface. Given current mission constraints on mass, power, and volume, these experiments can only be performed using proposed analytical techniques such as: electron microscopy, X-ray fluorescence, X-ray diffraction, alpha-proton backscatter, gamma-ray spectrometry, differential thermal analysis, differential scanning calorimetry, pyrolysis gas chromatography, mass spectrometry, and specific element detectors. Using prepared test samples consisting of 1% organic matter (bovine serum albumin) in palagonite and a mixture of palagonite, clays, iron oxides, and evaporites, it was determined that a combination of X-ray diffraction and differential thermal analysis coupled with gas chromatography provides the best insight into the chemistry, mineralogy, and geological history of the samples.

Aluminum Silicates↗
PubMed1983

Differential inactivation analysis of diploid yeast exposed to radiation of various LET. I. Computerized single-cell observation and preliminary application to X-ray-treated Saccharomyces cerevisiae.

This series of investigations was designed to observe growth and division of single, diploid yeast cells within the first four generations after irradiation with ionizing radiation. Evidence exists that cell reactions important for the final cell fate occur during this period, and therefore the analysis of cell kinetics and of stationary forms of inactivated cells can be performed. A large number of experiments is necessary to obtain statistically confirmed results of single-cell observation. An automatically steered microphotographic registration device has been developed to facilitate the collection of large numbers of observations. Optical data scanned by a TV camera and digitally stored in a computer are processed by pattern recognizing programs to achieve the correct correlation of newly built cells to existing ones and to deliver a pedigree over four generations of at least eight cells for every irradiated single cell. The pooled data of many pedigrees of this kind allow the analysis of the differential behavior of a total population. From the analysis of X-irradiated cells one can conclude that a single cell that produces at least a microcolony of five cells is eventually able to form a microcolony and thus can be considered a survivor. That means the division probability of cells to go from generation zero to three corresponds to the survival curve of the colony-forming ability test. Therefore this method is suitable for the differential description of the important phenomenological cell reactions after irradiation.

Cell Division↗
PubMed1987

Control of the adipogenic differentiation of 3T3-F442A cells by retinoic acid, dexamethasone, and insulin: a topographic analysis.

Differentiation of 3T3-F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3-F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 microM) potentiates about 50-fold the inhibitory effect of retinoic acid (10 microM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3-F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high-serum medium containing isobutyl methyl xanthine and dexamethasone to a serum-free, hormone-supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.

1-Methyl-3-isobutylxanthine↗
PubMed2002

Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line.

We have used an approach using 2-dimensional gel electrophoresis with mass spectrometry analysis combined with oligonucleotide chip hybridization for a comprehensive and quantitative study of the temporal patterns of protein and mRNA expression during myeloid development in the MPRO murine cell line. This global analysis detected 123 known proteins and 29 "new" proteins out of 220 protein spots identified by tandem mass spectroscopy, including proteins in 12 functional categories such as transcription factors and cytokines. Bioinformatic analysis of these proteins revealed clusters with functional importance to myeloid differentiation. Previous analyses have found that for a substantial number of genes the absolute amount of protein in the cell is not strongly correlated to the amount of mRNA. These conclusions were based on simultaneous measurement of mRNA and protein at just a single time point. Here, however, we are able to investigate the relationship between mRNA and protein in terms of simultaneous changes in their levels over multiple time points. This is the first time such a relationship has been studied, and we find that it gives a much stronger correlation, consistent with the hypothesis that a substantial proportion of protein change is a consequence of changed mRNA levels, rather than posttranscriptional effects. Cycloheximide inhibition also showed that most of the proteins detected by gel electrophoresis were relatively stable. Specific investigation of transcription factor mRNA representation showed considerable similarity to those of mature human neutrophils and highlighted several transcription factors and other functional nuclear proteins whose mRNA levels change prominently during MPRO differentiation but which have not been investigated previously in the context of myeloid development. Data are available online at http://bioinfo.mbb.yale.edu/expression/myelopoiesis.

Animals↗
PubMed2003

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli.

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.

Bacterial Proteins↗
PubMed1988

Thermal studies on the interaction of water and microcrystalline cellulose.

The interaction between water and microcrystalline cellulose in the absence and presence of lactose has been studied by thermogravimetric analysis, differential thermal analysis and immersional calorimetry. The results indicate that most of the water held within a system used for the preparation of spherical granules by extrusion/spheronization is present as free water which may be readily lost by evaporation. There is approximately 0.856 mol of water per 100 g of microcrystalline cellulose which appears to be absorbed as structured water. Microcrystalline cellulose may therefore be described as a 'molecular sponge'. The enthalpy of fusion of the 'free' water offers a simple method of estimating the effective surface area of the microcrystalline cellulose.

Calorimetry↗
PubMed2004

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.

Blotting, Northern↗
PubMed2001

Identification of Candida dubliniensis, based on ribosomal DNA sequence analysis.

Differentiation of Candida albicans and the recently described C. dubliniensis has proven difficult due to the high degree of phenotypic similarity of these species. The present study examines sequence variations in the ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions of C. albicans (n = 5) and C. dubliniensis (n = 7) strains, with a view to identifying sequence differences that would enable consistent differentiation of these two species by restriction fragment length polymorphism (RFLP) analysis. The ITS1 and ITS2 regions, together with the entire 5.8S rRNA gene of the strains, were amplified by the polymerase chain reaction (PCR), using primers ITS1 and ITS4, PCR products from both C. albicans and C. dubliniensis were of similar size (around 540 bp); however, sequence analysis revealed over 20 consistent base differences between the products of the two species. On the basis of sequence variation, the restriction enzyme MspA1 I was selected and used to differentiate the PCR products of C. albicans and C. dubliniensis by RFLP analysis. MspA1 I yielded two discernible fragments from C. albicans PCR products, whilst those from C. dubliniensis appeared undigested, thereby providing an approach to differentiate the two species.

Candida↗
PubMed1982

The use of differential scanning calorimetry and differential thermal analysis in studies of model and biological membranes.

Differential scanning calorimetry (DSC), and to a lesser extent differential thermal analysis (DTA), are powerful yet relatively rapid and inexpensive thermodynamic techniques for studying the thermotropic phase behavior of lipids in model and biological membranes, without the introduction of exogenous probe molecules. In this review the principles as well as the scope and limitations of DSC and DTA are discussed first. The application of these techniques to the study of the thermotropic phase behavior of aqueous dispersions of various single synthetic phospholipids are then summarized, and the effects of cholesterol, free fatty acids, lysophospholipids, drugs, anesthetics and proteins on the gel to liquid-crystalline phase transitions exhibited by these model systems are discussed. The phase mixing properties of model membranes consisting of mixtures of two or more synthetic or natural phospholipids are considered next. Finally, the thermotropic phase behavior of prokaryotic plasma membranes and of the plasma, microsomal and mitochondrial membranes of eukaryotic cells are reviewed, and the applications of DSC and DTA to study the thermal behavior of specific membrane proteins, as well as the physical properties of the membrane lipid phase, are summarized.

Acholeplasma laidlawii↗
PubMed1995

Analysis of differential gene expression in the kidney by differential cDNA screening, subtractive cloning, and mRNA differential display.

It is becoming increasingly evident that significant changes in gene expression occur during the course of disease progression in both genetic and nongenetic kidney diseases. Knowledge of the differentially expressed genes may yield information about the abnormal biochemical events that occur in the initiation and pathogenesis of these diseases. The purpose of this review is to provide an overview of some of the current approaches for identifying and analyzing differentially expressed genes. The power of these techniques lies in their their ability to detect differences in the levels of specific mRNAs in the diseased compared to the nondiseased kidney without prior knowledge of their identity. The three basic techniques considered are differential cDNA library screening, subtracted cDNA libraries, and PCR-based differential display. Emphasis is placed on cDNA library construction and differential screening. Also reviewed are the analysis of differentially expressed cDNAs by Southern and Northern blot hybridization, S1-protection, RT-PCR, DNA sequencing, and DNA sequence analysis.

Base Sequence↗