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PubMed2006

Quantitative proteomic analysis of the budding yeast cell cycle using acid-cleavable isotope-coded affinity tag reagents.

Quantitative profiling of proteins, the direct effectors of nearly all biological functions, will undoubtedly complement technologies for the measurement of mRNA. Systematic proteomic measurement of the cell cycle is now possible by using stable isotopic labeling with isotope-coded affinity tag reagents and software tools for high-throughput analysis of LC-MS/MS data. We provide here the first such study achieving quantitative, global proteomic measurement of a time-course gene expression experiment in a model eukaryote, the budding yeast Saccharomyces cerevisiae, during the cell cycle. We sampled 48% of all predicted ORFs, and provide the data, including identifications, quantitations, and statistical measures of certainty, to the community in a sortable matrix. We do not detect significant concordance in the dynamics of the system over the time-course tested between our proteomic measurements and microarray measures collected from similarly treated yeast cultures. Our proteomic dataset therefore provides a necessary and complementary measure of eukaryotic gene expression, establishes a rich database for the functional analysis of S. cerevisiae proteins, and will enable further development of technologies for global proteomic analysis of higher eukaryotes.

Carbon Isotopes↗
PubMed1998

Recent advances in noninvasive techniques to monitor hormone-behavior interactions.

This paper reviews recent advances in field endocrinology, a focus as well as a method in primatology and behavioral ecology that permits the examination of social behavior and life history through hormonal investigations in natural settings. Endocrine data complements the traditional behavioral data collected by field scientists by providing quantitative measures for the examination of adaptive tradeoffs, costs of social strategies, and reproductive and social significance of mating events. Further, investigations of the physiological mechanisms of reproductive constraint provide tests of the adaptive significance of reproductive skew in cooperative and competitive breeders. Hormone data also can provide insights into the costs of competition and aggression and the role of temperament in individual reproductive success and the evolution of social systems. New, noninvasive methods for the collection of this information have augmented and expanded field endocrinology through the use of techniques that do not require potentially confounding physical or physiological manipulations. Specifically, urine and fecal samples can be collected from free-ranging animals and contain gonadal and adrenal hormones that parallel profiles of serum hormones. Sampling, preservation, extraction, and assay methods for the analysis of excreted steroids are reviewed along with the species and questions to which these methods have been applied.

Animals↗
PubMed2002

Chromosome V loss due to centromere knockout or MAD2-deletion is immediately followed by restitution of homozygous diploidy in Saccharomyces cerevisiae.

To investigate the possibility of inducing specific chromosome loss by centromere deletion in eukaryotic cells, the yeast diploid strain ZG1, carrying three pairs of heterozygous marker genes (CAN1(S)/can1(R), URA3/Deltaura3, hphMX4/HIS1), widely spread on both arms of chromosome V, was constructed. One of the two centromeres V of ZG1 was replaced by the LEU2 gene via the well-established PCR-mediated knockout technique. After DNA transformation, putative yeast colonies that showed loss of heterozygosity (LOH) for the three markers of chromosome V (CAN1(S) URA3 hphMX4) were identified among the colonies selected for leucine prototrophy. Phenotypic tests, colony-PCR and Southern blot analysis of these cells demonstrated the physical loss of the CAN1(s), URA3, and hphMX4 marker genes from the genome. Further tetrad analysis results were consistent with this conclusion; however, four-spore viability indicated a normal chromosome number of these transformants. To verify the diploidy of the selected chromosome V, the HIS1 gene was deleted with a standard KanMX4 knockout DNA cassette. The resulting heterogeneity of the HIS1/KanMX4 markers, together with quantitative PCR and densitometric analysis on chromosome V, confirmed its diploid complement, thereby indicating that an endoreduplication event had taken place. Restitution of diploidy also occurred in MAD2-deleted strains undergoing higher rates of spontaneous chromosome V loss, indicating a more general phenomenon that is undetectable by phenotypic analysis alone.

Calcium-Binding Proteins↗
PubMed1978

Spectrophotometric determination of lymphocyte mediated sheep red blood cell hemolysis in vitro.

An assay has been developed in which the degree of in vitro spleen cell-mediated sheep red blood cell (SRBC) hemolysis is quantitated spectrophotometrically (QHS). Immune mouse spleen cells, SRBC and guinea pig complement were incubated at 37 degrees C for 1 h in buffer, centrifuged, and the supernatant solutions read at 413 nm. The greatest degree of cell-mediated SRBC hemolysis was observed in day +5 harvested spleens following a day 0, 5% SRBC i.p. injection. In the assay system, it was found that a stimulator SRBC dose of 0.2% resulted in maximal stimulation with little non-specific hemolysis. This assay is presented as an alternative to the well established plaque forming and rosette assays.

Animals↗
PubMed1994

Assays of oxidative DNA damage biomarkers 8-oxo-2'-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection.

High-performance liquid chromatography with electrochemical detection is a highly sensitive and selective method for detecting oxo8dG and oxo8Gua, biomarkers of oxidative DNA damage. When employed together with the DNA isolation and monoclonal antibody-based immunoaffinity purification methods described, oxo8dG and oxo8Gua in DNA and urine can be readily detected and quantitated, offering a powerful approach for assessing oxidative DNA damage in vivo. Application of the technique to the detection of oxo8dG from DNA permits quantitation of the steady-state levels of this oxidatively modified deoxynucleoside and overcomes the detection problems associated with the extremely low levels present in DNA. In addition, the selectivity gained by this detection method eliminates the problem of separating the signal for oxo8dG from those of normal deoxynucleosides. The quantitation of oxo8dG and oxo8Gua in biological fluids is noninvasive and complements the measurement of oxo8dG in DNA by estimating the rate of oxidative DNA damage occurring within the body or in a population of cells. This analytical approach may allow one to estimate oxidative DNA damage in an animal or individual exposed to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, or environmental toxins. Furthermore, these assays may allow one to assess the potentially beneficial effects of intervention strategies that protect DNA from such damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1985

Quantitative microcomplement fixation tests using chicken anti-viral antibody extracted from egg yolk.

Yolk immunoglobulins obtained from hens immunized with human rotavirus and two plant virus antigens were used in quantitative microcomplement fixation tests. Difficulties inherent in the use of avian complement were overcome by utilizing a mixture of the C1 component of chicken complement and guinea-pig complement devoid of its own C1. The method is suitable for detecting small quantities of viral antigen and antibody and for detecting antigenic activity present on peptide fragments of viral proteins.

Animals↗
PubMed2003

Magnetization transfer MRI of mouse brain reveals areas of high neural density.

Extending applications of magnetization transfer contrast (MTC) in magnetic resonance imaging (MRI) of the human central nervous system, this work quantitatively describes MTC of the murine brain. As a novel finding, complementing T(1)- and T(2)-weighted MRI, MTC allows for the distinction of densely packed gray matter from normal gray and white matter. Examples include the Purkinje cell layer and the granular cell layer in the mouse cerebellum as well as the delineation of the CA3 subfield of the hippocampus relative to surrounding hippocampal gray matter and white matter tracts such as the hippocampal fimbria. Using a kainate lesion model, the CA3 hyperintensities in MTC and T(1)-weighted MRI are assigned to the densely packed somata of pyramidal cells.

Animals↗
PubMed1983

Immunonephelometric determination of proteins in cerebrospinal fluid in various neurological disorders.

The purpose of this investigation was to examine the diagnostic value of nine specific proteins in differentiating multiple sclerosis from other neurological disorders. The quantitative determination of albumin, transferrin, haptoglobin, IgG, IgA, IgM, orosomucoid, complement-C3 and prealbumin was carried out using an automated immunonephelometric method. Using a combination of IgG-index ((Sp-IgG/S-IgG)/(Sp-albumin/S-albumin)) and haptoglobin-index ((Sp-haptoglobin/S-haptoglobin)/(Sp-albumin/S-albumin)) it is possible to increase the diagnostic value. Using a modified index it is possible to obtain the same diagnostic value by combining modified IgG-index ((Sp-IgG/S-IgG)/Sp-albumin) and modified haptoglobin-index ((Sp-haptoglobin/S-haptoglobin)/Sp-albumin). The diagnostic specificity and sensitivity for the modified combined index was 0.60 and 1.00, respectively.

Albumins↗
PubMed1995

Hind III genomic polymorphism of the C3b receptor (CR1) in patients with SLE: low erythrocyte CR1 expression is an acquired phenomenon.

Expression of the human erythrocyte C3b receptor (CR1-CD35) and its Hind III RFLP was studied in a group of 37 patients with SLE, 15 consanguineous relatives of the patients and 48 healthy normal subjects. The CR1 number on erythrocytes was quantitated by ELISA using a mAb to CR1. Serum levels of complement proteins (C3, C4, C3d) and circulating immune complexes (CIC) were estimated simultaneously in controls and relatives. The patients were followed up during the course of the treatment. The CR1/erythrocyte (CR1/E) in patients were found to be significantly low in comparison to controls. The gene frequencies for the alleles H and L (7.4 and 6.9 kb Hind III restriction fragments) in the patients were 0.75 and 0.25, respectively, which did not differ significantly from the controls (0.77 and 0.23 in normal subjects and 0.79 and 0.21 in consanguineous relatives of the patients). However, patients expressed fewer CR1/E within each genotype than their relatives and healthy subjects. CR/E was found to be stable in consecutive samples in controls. In patients, the numbers varied between low and high during the course of the treatment. The variation in the numbers was significantly correlated with C3d and CIC as well as with the severity of the disease. Our results suggest that low levels of CR1 on erythrocytes in SLE patients are required during the course of the disease and that the 6.9 kb restriction fragment does not play a role in causing susceptibility to the disease.

Adolescent↗
PubMed2002

Assessment of left atrial appendage wall velocities by transesophageal tissue Doppler echocardiography: a clinical study in patients with sinus rhythm.

To assess left atrial appendage (LAA) wall velocities, 42 patients in sinus rhythm underwent tissue Doppler interrogation during a clinically indicated transesophageal echocardiography. Color Doppler (B-mode and M-mode) and pulsed Doppler of LAA walls were obtained and analyzed in all patients. Color-coded tissue Doppler rendered a qualitative assessment of LAA wall, depicting both the timing and the sequence of LAA contraction. With pulsed Doppler interrogation, a triphasic signal was recorded in all patients, consisting of a positive wave (D1), followed by a biphasic wave (positive D2 and negative D3). Peak velocities of D1, D2, and D3 were 6.1 +/- 2, 20.1 +/- 7, and 16.1 +/- 5 cm/s, respectively. Mean coefficient of variation of LAA wall velocities was 6%, significantly lower than that of LAA percentage area change (29%). Compared with patients with abnormal relaxation, patients with normal mitral inflow had higher D1 peak velocities (7.3 +/- 1.2 vs 4.3 +/- 1 cm/s, respectively; P <.0001). Time sequence of ECG, LAA flow, and mitral inflow indicates that D1 component of LAA wall occurs in early diastole and is likely due to the upward movement of the mitral ring toward the base of the LAA wall. In conclusion, evaluation of LAA wall using tissue Doppler is feasible and reproducible. Although color tissue Doppler analysis allows a qualitative assessment, pulsed Doppler gives new quantitative insights for the comprehensive assessment of LAA wall dynamics, which complements the information obtained with flow interrogation.

Atrial Appendage↗
PubMed1975

C1q deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum.

This report describes a new, rapid, sensitive, and quantitative method for the detection of immune complexes, endotoxins, and other complement activating materials in patients sera utilizing the ability of these substances to react with isolated C1q. The procedure is based on the inhibition of radiolabeled C1q binding to sensitized sheep erythrocytes by C1q-reactive substances in pathological sera. The C1q deviation test may be performed on 50 mu1 of serum, using 1 mug of radiolabeled C1q per sample. The procedure may be completed in 1.5-2 h, it is capable of detecting 5 mug of aggregated human IgG per ml of serum, and its coefficient of variation is 4.2%. Application of the test to the study of 193 sera from 43 patients with Dengue hemorrhagic fever showed a positive correlation between degree of C1q deviation and severity of disease.

Antigen-Antibody Complex↗
PubMed1984

Herpes gestationis with transient bullous lesions in the newborn.

A case of severe bullous dermatosis in the newborn son of a mother with herpes gestationis is reported. Bullous lesions appeared in the child about 2 hours after delivery and regressed completely without specific treatment by the end of the first month, leaving numerous epidermal cysts. The immunologic phenomena were both qualitatively and quantitatively comparable to those obtained in the mother, i.e., deposition of complement (C3) and smaller amounts of IgG at the dermo-epidermal junction of the affected skin. A transient increase of the total serum IgE was demonstrated in the mother, while the serum IgE level in the newborn was less than 5 U/ml. The pathogenetic role of IgG autoantibodies in herpes gestationis and, more generally, in autoimmune diseases is discussed.

Complement C3↗
PubMed1987

Common mechanism of ampC beta-lactamase induction in enterobacteria: regulation of the cloned Enterobacter cloacae P99 beta-lactamase gene.

Expression of the chromosomal beta-lactamase from the ampC gene in inducible in both Enterobacter cloacae and Citrobacter freundii. Cloning of ampC as well as its regulatory gene, ampR, from E. cloacae P99 revealed a gene organization indentical to that of C. freundii in the corresponding region. Although almost no similarities could be found between the restriction maps of ampC and ampR in the two species, the genes cross-hybridize. Also, both ampR gene products have a size of about 31,000. The regulatory features of E. cloacae beta-lactamase induction are very similar to those in C. freundii, i.e., beta-lactamase synthesis is repressed by AmpR in the absence, and stimulated in the presence, of inducer. The AmpR function can be transcomplemented between the two species, but there are quantitative regulatory aberrations in such hybrids, in contrast to the total complementation obtained within each system. These results suggest that the mechanism of beta-lactamase induction is the same in E. cloacae, C. freundii, and other gram-negative bacteria with inducible chromosomal beta-lactamase expression.

Cloning, Molecular↗
PubMed2004

Proteomics: current techniques and potential applications to lung disease.

Proteomics aims to study the whole protein content of a biological sample in one set of experiments. Such an approach has the potential value to acquire an understanding of the complex responses of an organism to a stimulus. The large vascular and air space surface area of the lung expose it to a multitude of stimuli that can trigger a variety of responses by many different cell types. This complexity makes the lung a promising, but also challenging, target for proteomics. Important steps made in the last decade have increased the potential value of the results of proteomics studies for the clinical scientist. Advances in protein separation and staining techniques have improved protein identification to include the least abundant proteins. The evolution in mass spectrometry has led to the identification of a large part of the proteins of interest rather than just describing changes in patterns of protein spots. Protein profiling techniques allow the rapid comparison of complex samples and the direct investigation of tissue specimens. In addition, proteomics has been complemented by the analysis of posttranslational modifications and techniques for the quantitative comparison of different proteomes. These methodologies have made the application of proteomics on the study of specific diseases or biological processes under clinically relevant conditions possible. The quantity of data that is acquired with these new techniques places new challenges on data processing and analysis. This article provides a brief review of the most promising proteomics methods and some of their applications to pulmonary research.

Animals↗
PubMed1983

Ecto-5'-nucleotidase activity in human T cell subsets. Decreased numbers of ecto-5'-nucleotidase positive cells from both OKT4+ and OKT8+ cells in patients with hypogammaglobulinemia.

T lymphocytes from control subjects were separated into subsets using monoclonal antibodies of the OKT series and complement lysis and analyzed for ecto-5'-nucleotidase activity both by quantitative radiochemical assay and a histochemical stain. T cells from 15 control subjects contained 54+/-4% OKT4(+) (helper/inducer) cells and 32+/-3% OKT8(+) (cytotoxic/suppressor) cells. Total T cell ecto-5'-nucleotidase activity was 10.9+/-2.1 nmol/h per 10(6) cells with 25+/-7% positive by histochemical stain. Ecto-5'-nucleotidase activity in OKT4-enriched populations was 5.43+/-1.8 nmol/h per 10(6) cells with 14+/-2% positive by histochemical stain; that in OKT8-enriched populations was 17.1+/-5.9 nmol/h per 10(6) cells with 35+/-8% positive by histochemical stain. Two of four patients with congenital agammaglobulinemia and four of seven patients with common variable immunodeficiency had decreased proportions of OKT4(+) T cells with corresponding increases in the proportions of OKT8(+) T cells (OKT4/OKT8 = 0.60 to 1.0 as compared with 1.7+/-0.2 for control subjects). All four patients with congenital agammaglobulinemia, and three of seven patients with common variable immunodeficiency also had low T cell ecto-5'-nucleotidase activity (<5.5 nmol/h per 10(6) cells). Ecto-5'-nucleotidase activity in OKT4- enriched populations isolated from four patients with low total T cell activity was 2.85+/-0.90 nmol/h per 10(6) cells with 10+/-4% positive by histochemical stain; that in OKT8-enriched populations was 6.82+/-1.7 nmol/h per 10(6) cells with 7.5+/-3% positive by histochemical stain. Thus, the number of ecto-5'-nucleotidase positive cells is decreased, especially in the OKT8(+) subpopulation, and the low total T cell ecto-5'-nucleotidase activity seen in these patients is due to fewer positive cells rather than to substantially less activity per cell. Our data indicate that ecto-5'-nucleotidase activity defines two subpopulations of T lymphocytes (ecto-5'-nucleotidase positive and negative), the proportions of which are markedly altered in many patients with hypogammaglobulinemia. In preliminary studies with seven patients, increased numbers of ecto-5'-nucleotidase negative T cells appeared to correlate with increased suppressor T cell activity toward in vitro immunoglobulin synthesis. Therefore, ecto-5'-nucleotidase may be a useful cell surface marker in the study of imbalances of regulatory T cell subsets in patients with antibody synthesis disorders.

Adenosine Deaminase↗
PubMed1994

Quantitative polymerase chain reaction (PCR) to analyze messenger-RNA expression in tissue obtained by directional atherectomy.

There is a widely recognized need to evaluate gene expression in human vascular plaque tissue. Directional atherectomy made it possible to sample plaque tissue from primary stenoses and subsequently from restenoses from one individual. Conventional approaches to analyze mRNA content of lesions, such as Northern blot analysis, and slot blot analysis are not sensitive enough to evaluate mRNA levels in atherectomy specimens limited by low cell number or low copy number per cell. The purpose of this study was to investigate whether gene expression, as reflected in mRNA copy number, in atherectomy biopsies, could be sufficiently analyzed by quantitative polymerase chain reaction (PCR). The authors applied PCR to detect platelet-derived growth factor-A mRNA expression in 12 human lesions sampled percutaneously by directional atherectomy. After reverse transcription, specific amplification of the resulting cDNA was performed. This was successful with cDNA from less than 0.5 microgram of total cellular RNA. To quantitate mRNA content of specimens, the authors coamplified cDNA copies of mRNA from lesions and from a synthetic reference RNA in the same reaction vessel. Quantitative PCR is best applied if tissue is more than 45 mg in weight and of high cellularity with low calcification. This method allows quantitation of mRNA in human primary and restenotic lesions, and it complements histochemical approaches and in situ hybridization of coronary and peripheral atherectomy specimens.

Aorta↗
PubMed2006

Wild-type and central DNA flap defective HIV-1 lentiviral vector genomes: intracellular visualization at ultrastructural resolution levels.

HIV-1 and other lentiviruses have the unique ability among retroviruses to efficiently replicate in non-dividing cells as a result of the active nuclear import of their DNA genome across an interphasic nuclear membrane. Previous work has shown that a three-stranded DNA structure synthesized during HIV-1 reverse transcription, called the central DNA flap, acts as a cis-determinant of HIV-1 genome nuclear import. Concordantly, DNA Flap re-insertion in lentiviral-derived gene therapy vectors stimulates gene transfer efficiencies and complements the level of nuclear import to wild-type levels quantitatively indistinguishable from wild-type virus in all cell types and tissues examined so far. In order to define the precise nature of the replicative defect of DNA flap mutant viruses, we carried out in situ DNA hybridization experiments with electron microscopy to determine the subcellular localization of DNA flap mutant and wild-type HIV-1 genomes. We found that Flap defective DNA genomes accumulate at the cytoplasmic face of the nuclear membrane with no overlap across the nuclear membrane, whereas wild-type genomes localize throughout the nuclear compartment. These data provide an unequivocal confirmation of the role of the DNA flap in HIV-1 nuclear import and further establish that the DNA flap controls a step that immediately precedes translocation through the nuclear pore. Further, the widespread distribution of wild-type genomes within the open chromatin confirms the recent genome-wide mapping of HIV-1 cDNA integration sites and points to an as-yet poorly understood step of intranuclear transport of HIV-1 pre-integration complexes.

Active Transport, Cell Nucleus↗
PubMed2006

Surface plasmon resonance mass spectrometry for protein analysis.

The combination of surface plasmon pesonance (SPR) and mass spectrometry (MS) provides a unique methodology for studying proteins and their interactions. SPR is utilized to assess protein quantitative variations and the kinetic aspects of protein interactions, whereas MS complements the analysis by providing an exclusive look at the structural features of the interacting proteins via measurement of their mass. Thus, intrinsic protein structural modifications that go unregistered via the SPR detection can readily be assessed from the MS data. The purpose of this chapter is dissemination of the procedures and protocols for successful SPR-MS analysis. The individual steps of the complete SPRMS process are illustrated via analysis of cardiac troponin I (cTnI).

Biosensing Techniques↗