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PubMedDate not supplied

Expression and functional activity of breast cancer resistance protein (BCRP, ABCG2) transporter in the human choriocarcinoma cell line BeWo.

1. Breast cancer resistance protein (BCRP, ABCG2) is a drug efflux transporter that is believed to affect the drug disposition of several drugs and xenobiotics. In the present study, we evaluated the localization and functional expression of BCRP in the human choriocarcinoma cell line BeWo, an in vitro model of the human trophoblast, and compared it with the expression of P-glycoprotein (MDR1, ABCB1) as the most widely studied placental transporter. In addition, the expression of BCRP at the mRNA level was compared with that of MDR1 in the human term placenta. 2. Western blotting analysis revealed high endogenous expression of BCRP protein in BeWo cells. Using indirect immunofluorescence microscopy, we found that the BCRP transporter appears to be localized predominantly at the apical plasma membrane. Functional studies showed a significant effect of the BCRP inhibitors GF120918 (5 micromol/L) and Ko143 (1 micromol/L) on mitoxantrone accumulation and, thus, confirmed efflux activity of BCRP in BeWo cells. 3. Using absolute mRNA quantification with real-time reverse transcription-polymerase chain reaction, we found high expression of BCRP in BeWo cells, whereas no transcript of MDR1 (P-glycoprotein), the most extensively studied drug transporter, was detected. 4. In the human placenta, BCRP was localized predominantly in the syncytiotrophoblast layer; however, immunopositivity for the BXP-21 antibody was also observed in fetal vessels of the chorionic villi. The number of BCRP transcripts in the human term placenta was found to be more than 10-fold higher compared with the expression of MDR1. 5. In conclusion, we suggest that BeWo cells could serve as a suitable in vitro model to study trans-trophoblast transport of BCRP substrates and that placental BCRP can play an important role in preventing the accumulation of potentially toxic xenobiotics in the trophoblast cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders.

Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body (DLB). We have employed a multiplex quantitative proteomics method, iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), in conjunction with multidimensional chromatography, followed by tandem mass spectrometry (MS/MS), to simultaneously measure relative changes in the proteome of cerebrospinal fluid (CSF) obtained from patients with AD, PD, and DLB compared to healthy controls. The diagnosis of AD and DLB was confirmed by autopsy, whereas the diagnosis of PD was based on clinical criteria. The proteomic findings showed quantitative changes in AD, PD, and DLB as compared to controls; among more than 1,500 identified CSF proteins, 136, 72, and 101 of the proteins displayed quantitative changes unique to AD, PD, and DLB, respectively. Eight unique proteins were confirmed by Western blot analysis, and the sensitivity at 95% specificity was calculated for each marker alone and in combination. Several panels of unique makers were capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity. Although these preliminary findings must be validated in a larger and different population of patients, they suggest that a roster of proteins may be generated and developed into specific biomarkers that could eventually assist in clinical diagnosis and monitoring disease progression of AD, PD and DLB.

Alzheimer Disease↗

PubMed1991

Evaluation of two extraction methods for the determination of egg yolk cholesterol.

Controversy concerning egg cholesterol values exists in recent literature due to varying procedures used for cholesterol determination. The purpose of the present study was to investigate the efficacy of direct sample saponification (Method A) versus saponification of a lipid extract (Method B) for analysis of yolk cholesterol. Method A resulted in a value of 19.1 +/- .4 (SE) mg cholesterol/g of yolk for the National Institute of Standards and Technology (NIST) reference (cholesterol in whole egg powder) as compared with the NIST certified value of 19.0 +/- .2 mg/g. Method B resulted in a significantly lower value of 14.6 +/- .5 mg/g. Egg yolk cholesterol values were determined to be 196 +/- 4.2 mg per egg by Method A and 132 +/- 11 mg per egg by Method B. Various amounts (1, .5, .25 g) of yolk cholesterol assayed by either method proportionately decreased cholesterol values as yolk amount decreased; however, Method B consistently resulted in lower yolk cholesterol. These data suggest that both Methods A and B are valid for determining relative differences between treatments; however, the NIST standard data indicate that for quantification of absolute cholesterol values, direct saponification is more accurate. The NIST standard of cholesterol in whole egg powder should be used as a control for comparing cholesterol data regardless of extraction method used.

Animals↗

PubMed2003

[Stunning, hibernation, and heart failure in patients with coronary disease: crucial role of impaired coronary flow reserve].

Hibernating myocardium can be defined as a chronic, but reversible left ventricular dysfunction that may contribute to congestive heart failure in patients with coronary artery disease. The dysfunction can improve after coronary revascularization and therefore its identification and treatment become central in the management of patients with heart failure secondary to coronary artery disease. Hibernating myocardium can be detected by several techniques (echocardiography performed during the infusion of dobutamine, single photon and positron emission tomography-PET, and magnetic resonance imaging), but none of these techniques can be considered unequivocally superior to the others for the identification of hibernating myocardium. As PET technology has advanced, the noninvasive quantification of absolute regional myocardial blood flow has become possible. The measurement of myocardial blood flow by PET has contributed to the demonstration that transmural blood flow in hibernating muscle is generally within the normal range while the coronary flow reserve is invariably and severely impaired. These findings have contributed to a new pathophysiological theory of hibernation where repetitive ischemia and stunning are considered as the initial mechanisms that might start the process of myocardial hibernation.

Animals↗

PubMed1998

Fractal dimension and approximate entropy of heart period and heart rate: awake versus sleep differences and methodological issues.

1. Investigations that assess cardiac autonomic function include non-linear techniques such as fractal dimension and approximate entropy in addition to the common time and frequency domain measures of both heart period and heart rate. This article evaluates the differences in using heart rate versus heart period to estimate fractal dimensions and approximate entropies of these time series.2. Twenty-four-hour ECG was recorded in 23 normal subjects using Holter records. Time series of heart rate and heart period were analysed using fractal dimensions, approximate entropies and spectral analysis for the quantification of absolute and relative heart period variability in bands of ultra low (<0.0033 Hz), very low (0. 0033-0.04 Hz), low (0.04-0.15 Hz) and high (0.15-0.5 Hz) frequency.3. Linear detrending of the time series did not significantly change the fractal dimension or approximate entropy values. We found significant differences in the analyses using heart rate versus heart period between waking up and sleep conditions for fractal dimensions, approximate entropies and absolute spectral powers, especially for the power in the band of 0.0033-0.5 Hz. Log transformation of the data revealed identical fractal dimension values for both heart rate and heart period. Mean heart period correlated significantly better with fractal dimensions and approximate entropies of heart period than did corresponding heart rate measures.4. Studies using heart period measures should take the effect of mean heart period into account even for the analyses of fractal dimension and approximate entropy. As the sleep-awake differences in fractal dimensions and approximate entropies are different between heart rate and heart period, the results should be interpreted accordingly.

Adult↗

PubMed1994

Absolute quantitative proton NMR spectroscopy based on the amplitude of the local water suppression pulse. Quantification of brain water and metabolites.

Quantification in localized proton NMR spectroscopy has been achieved by various methods in recent years. A new method for absolute quantification is described in this paper. The method simultaneously rules out problems with B1 field inhomogeneity and coil loading, utilizing a relation between the locally optimized amplitude of a chemical shift selective water suppression pulse and the acquired signal. Validity and feasibility of quantification using the method of the water suppression pulse is demonstrated. Brain water and cerebral metabolites have been quantified in a study of 12 healthy volunteers. Localized proton NMR spectra were obtained from a region of primarily white matter in the occipital lobe. The observable water content in the NMR spectra was 0.685 +/- 0.025. The absolute metabolite concentrations were: [total choline] = 1.25 +/- 0.21 nM, [total creatine] = 6.71 +/- 0.59 nM and [NAA] = 9.15 +/- 0.74 nM. It is concluded that the quantification method is easily applied in vivo, and that the absolute concentrations obtained are similar to results in other studies except those relying on assumptions of the concentration of an internal reference. The advantage of the quantification method reported here is that it is independent of the assumptions needed for other methods.

Adult↗

PubMed1996

An evaluation of the accuracy of four ELISA methods for measuring natural and recombinant human interferon-g.

We describe an evaluation of four ELISA methods, including three commercial kits, for measuring recombinant and natural human interferon-g (hIFN-g). Using a panel of samples, including well-characterized reference standards, we compared relative quantification between assays, within assays and, where possible, the absolute accuracy of quantification as compared to other analytical methods. The four assays generated markedly different results; up to an almost 60-fold difference between the highest and lowest values for one sample. The differences between assays were not necessarily predictable. No single correction factor could be determined to correct results from one method to another across the panel of samples tested. We conclude that investigators should be diligent to revalidate commercial methods before depending on such methods and resultant data.

Amino Acid Sequence↗

PubMed1999

Quantification of breast cancer blood flow in absolute units using Gjedde-Rutland-Patlak analysis of 99mTc-MIBI uptake.

BACKGROUND: Scintimammography with 99mTc-MIBI has been proven as efficient technique of diagnosis of breast cancer. Nevertheless, quantification of breast carcinoma blood flow (BCBF) in absolute units is not yet developed. To compensate this, we analysed kinetics of 99mTc-MIBI uptake in breast cancer using Gjedde-Rutland-Patlak (GRP) approach. METHODS: If BC is radioactivity in breast cancer quantified by dynamic scintigraphy, C(h)-blood concentration of 99mTc-MIBI and K-transport constant, then, assuming 99mTc-MIBI uptake to breast carcinoma unidirectional for early minutes after injection and subjected to equation d(BC)/dt = K; C(h), classic GRP plot can be obtained from this by integration as BC/C(h)=K x (integral of C(h)(dt))/C(h) + V0 and placing [(integral of C(h)(dt))/C(h)] as X, and (A/Ch) as Y. The K can be then obtained as slope. K is breast cancer clearance equal to product (retention fraction); (blood flow) : K = E; BCBF. K can be calculated from A(t) and Ch(t) as asymptote of 99mTc-MIBI retention function h(t) = F-1[F[A(t)]/F[Ch(t)]], where F depicts Fourier transforms. The BCBF can be then obtained as ratio K/E. We employed the technique in 33 patients with breast carcinoma of stages T(1-3)N(0-3)M(0-1) injecting 99mTc-MIBI (370-510 MBq) as i.v. bolus. In 12 scintigraphy with 99mTc-MAA (370 MBq) injected via catheter intraaortically was performed as validation study. RESULTS: E values were essentially uniform over the population with overall mean 0.58 sd 0.06. Blood clearance curves did not differ between various stages also and were subjected to biexponential approximation. K was in all cases obtained from the slope of initial 3 min part of GRP plot, strongly linear (r > 0.95, p < 0.001) in all cases. 99mTc-MAA validation study revealed significant correlation with 99mTc-MIBI blood flow values (r = 0.94, p < 0.01). The BCBF(as ml/min/100 cm3) was in T1 12.85 sd. 4.76, in T2 15.87 sd. 1.78, in T3 17.35 sd. 2.45, and in T4 23.07 sd. 2.21, expressing tendency to increase with stage. Higher BCBF was significantly associated with metastatic spread and in patients with BCBF over 17 ml/min/100 cm3 distant mets were revealed in all cases. CONCLUSION: Hence, analysis of early kinetics of 99mTc-MIBI in breast carcinoma provides correct estimates of blood flow in the neoplasm and can be applied in clinical studies and for calculation of cytostatic delivery to BC.

Journal Article↗

PubMed1988

Direct immunomagnetic quantification of lymphocyte subsets in blood.

A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies (MoAb) are used for the direct and fast quantification of the absolute number of cells of various lymphocyte subsets in blood. Blood samples were incubated with microspheres coated with a subset specific MoAb. Using a magnet the microsphere-rosetted cells were isolated and washed. Following lysis of the cell walls to detach the microspheres, the cell nuclei were stained with acridine orange and counted in a haemocytometer using an immunofluorescence microscope. With MoAb specific for CD2, CD4, CD8 and CD19, reproducible absolute counts of the corresponding lymphocyte subsets were obtained which correlated closely with those obtained by an indirect quantification method.

Cell Separation↗

PubMed2004

Absolute transcript levels of thioredoxin- and glutathione-dependent redox systems in Saccharomyces cerevisiae: response to stress and modulation with growth.

We report the co-ordinated fine-tune of mRNA molecules that takes place in yeast (Saccharomyces cerevisiae) in response to diverse environmental stimuli. We performed a systematic and refined quantification of the absolute expression patterns of 16 genes coding for thioredoxin- and glutathione-dependent redox system components. Quantifications were performed to examine the response to oxidants, to sudden temperature upshifts and in association with metabolic changes accompanying culture growth and to explore the contribution of mRNA decay rates to the differences observed in basal expression levels. Collectively, these quantifications show (i) vast differences in the steady-state amounts of the investigated transcripts, cTPxI being largely overexpressed compared with GPX1 during the exponential phase and GPX2 beyond this growth stage; (ii) drastic changes in the relative abundance of the transcripts in response to oxidants and heat shock; and (iii) a unique temporal expression profile for each transcript as cells proceed from exponential to stationary growth phase, yet with some general trends such as maximal or near-maximal basal amounts of most mRNA species at early growth stages when glucose concentration is high and cells are actively growing. Moreover, the results indicate that (i) the half-lives of the investigated transcripts are longer and distributed within a narrower range than previously reported global mRNA half-lives and (ii) transcriptional initiation may play an important role in modulating the significant alterations that most mRNAs exhibit in their steady-state levels along with culture growth.

Gene Expression Regulation, Fungal↗

PubMedDate not supplied

Quality control in combinatorial chemistry: determinations of amounts and comparison of the "purity" of LC-MS-purified samples by NMR, LC-UV and CLND.

The absolute purities of 20 purified samples from a combinatorial library have been determined by a new method that uses the DMSO sidebands [1J[13C-1H]] as an internal standard for quantification. The obtained absolute amounts are compared with the amounts of sample obtained by weighing, with the calculated weights obtained by chemiluminescent nitrogen detection (CLND) chromatography and with the relative purities obtained by LC-UV chromatography.

Chromatography↗

PubMed2000

In vitro flow quantification with contrast power Doppler imaging.

To evaluate the effectiveness of contrast harmonic (power Doppler imaging) as an ultrasonic modality to quantify flow, an in vitro model of perfusion was studied using Optison, a second-generation ultrasound (US) contrast agent. The in vitro model was made of two dialysis cartridges placed parallel and allowed absolute and relative flow quantification on both tube (entry lines) and tissue (cartridges) simulations. Video intensity curves were generated using intermittent harmonic power Doppler imaging after bolus injection of contrast. Correlation between flow and different parameters extracted from time-intensity curves and previously defined as indicators of flow was established for both tissue and entry lines, for flow rates ranging from 0 to 400 mL/min. Single-compartment equations were also tested on the model. A good correlation for the tissue model was observed between absolute flow and onset time (O), time to maximal enhancement (TME), peak intensity (P), area under the curve (AUC), and maximal ascending slope (S) parameters, with a r = 0.94, 0.94, 0.91, 0.92 and 0.92, respectively. The correlation for O, TME, P and AUC parameters was r = 0.86, 0.90, 0.78 and 0.82, respectively for entry lines. The correlation for tissue model and entry line was slightly improved when comparing flow ratios with peak ratios (P1/P2) and slope ratios (S1/S2) (r = 0.95 and 0.94). Flow calculation using the gradient-relationship method also showed a good correlation (r = 0.88) with the experimental flow. The results obtained indicated that absolute and relative quantification of flow using PDI is feasible in tube and tissue models. Several clinical applications, namely in myocardial, hepatic and renal artery studies, could be derived from these results.

Albumins↗

PubMed2006

Novel methods of quantitative real-time PCR data analysis in a murine Helicobacter pylori vaccine model.

Monitoring of Helicobacter pylori in the stomach is important to assess the efficacy of new vaccines against the pathogen. To realise the full potential of quantitative real-time PCR (q-PCR), this technology has to offer accurate and easy models of post-PCR data analysis. In this work, we used a variety of absolute and relative approaches of q-PCR data analysis to monitor the H. pylori infection in the stomach of immunized mice. Relative quantification was performed with Ct-based methods, with the DART program, and with two methods based on the mathematical analysis of raw fluorescence kinetics, the LinReg program and the Sigmoidal Curve Fitting Method. The different calculation methods were validated in mice immunized with cell lysates of Lactococcus lactis expressing the H. pylori urease subunit B in combination with cholera toxin. The H. pylori load was found to be reduced in immunized mice by a factor of 50-144, depending on the calculation method employed. We found that relative quantification using DART, LinReg and Sigmoidal Curve Fitting methods generated similar results (infection ratios of 54-58) with absolute quantification results (54-65). Results were very different to those using relative quantification Ct-based methods without a correction for PCR efficiency (ratio of 92-144) and with results based on conventional culture method (ratio of 34). Overall, this study demonstrates that q-PCR associated with a relative quantification analysis is a powerful tool for the monitoring of microorganisms in tissue. It could be used as an alternative to standard curve approach especially for the investigation of microbial load in vaccine models.

Animals↗

PubMed2005

A microarray configuration to quantify expression levels and relative abundance of splice variants.

Over the past decade, alternative RNA splicing has raised a great interest appearing to be of high importance in the generation of expression diversity. This regulatory process plays a critical role in the normal development and its impact on the initiation and development of human disorders as well as on the pharmacological properties of drugs is increasingly being recognized. Only few studies describe specific alternative splicing expression profiling. Microarray strategies have been conceived to address alternative splicing events but with very few experimental data related to their abilities to provide true quantification values. We have developed a specific microarray configuration relying on a few, well optimized probes per splice event. Basically, five probes of 24mer are used to fully characterize a splice event. These probes are of two types, exon probes and junction probes, and are either specific to a splice event or not. The performances of such a 'splice array' were validated on synthetic model systems and on complex biological materials. The results indicate that DNA chips based on this design combining exon and junction derived probes enable the detection and, absolute and relative quantification of splice variants. In addition, this strategy is compatible with all the microarrays that use oligonucleotide probes.

Alternative Splicing↗

PubMed1996

Quantification of circulating 1,25-dihydroxyvitamin D by radioimmunoassay with 125I-labeled tracer.

We report here the first RIA for 1,25-dihydroxyvitamin D utilizing a radioiodinated (125I) tracer. This is also the first validated RIA for 1,25-dihydroxyvitamin D [1,25(OH)2D] that does not require sample prepurification by HPLC before the binding assay. The assay involves acetonitrile extraction, treatment of the crude extract supernate with sodium periodate, extraction and purification of endogenous 1,25(OH)2D by solid-phase chromatography, and finally, quantification by RIA. Calibrators were prepared in stripped human serum and processed exactly the same as samples, eliminating the need for internal control for procedural losses of endogenous 1,25(OH)2D. The assay consists of a 2-h room temperature incubation with the primary antibody, a 20-min incubation with a second antibody, and separation of bound from free by centrifugation. Assay results can be in hand with 5 h. The detection limit of the assay is 2.4 ng/L 1,25-dihydroxyvitamin D3. Results compare well with those from an accepted radioreceptor assay. Sample pretreatment with sodium periodate is absolutely essential before quantification by RIA; otherwise, concentrations of endogenous 1,25(OH)2D may be greatly overestimated.

Acetonitriles↗

PubMed1997

Nutrient regulation of the intestinal Na+/glucose co-transporter (SGLT1) gene expression.

It is known that dietary carbohydrates regulate the activity of the intestinal SGLT1. We have demonstrated that modifications in SGLT1 activity are due to alterations in SGLT1 expression in response to the sugar content of the diet. To assess the correlation between changes in the activity of SGLT1 and the abundance of SGLT1 protein, we have employed a method for the quantitative measurement of immunoreactive proteins. A calibration curve has been constructed using either a nonadecapeptide (amino acids 402-420), or a recombinant protein corresponding to amino acids 554-640 of the SGLT1 sequence. Immunoblotting the protein samples concurrently with specific quantities of either the peptide or recombinant standard, using antibodies raised against these antigens, enabled accurate quantification of the absolute amounts of immunoreactive protein in the samples. The amount of SGLT1 protein correlates well with measurements of SGLT1 activity. The modulation of the activity of SGLT1 in response to lumenal sugars is due to corresponding changes in the absolute levels of SGLT1 protein.

Animals↗

PubMed1994

Distance from symmetry: a three-dimensional evaluation of facial asymmetry.

PURPOSE: A new method for the quantification of facial asymmetry has been developed and applied to a group of 80 young, healthy, white adults with no craniofacial, dental, or mandibular disorders. The method calculates an asymmetry vector (distance from the symmetry) that allows both the quantification of the absolute degree of asymmetry and its three-dimensional direction and verse. METHODS: For each subject, the three-dimensional coordinates of 16 standardized soft-tissue facial landmarks (trichion, nasion, pronasale, subnasale, B point, pogonion, eye lateral canthi, nasal ala, labial commissure, tragus, gonion) were automatically collected using a noninvasive instrument. The coordinates underwent a four-step analysis: 1) calculation of the plane of symmetry, the facial centers of gravity, and the asymmetry vector in each subject; 2) calculation of the mean absolute asymmetry in each sample; 3) calculation of the mean asymmetry vector (resultant vector) in the population; and 4) calculation of the separate contributions of the single facial structures to the facial asymmetry. RESULTS: A certain degree of soft-tissue facial asymmetry was found both in the individuals and in the global population, and it was evident especially in the middle (tragus) and lower (gonion) thirds of the face. The right side of face was larger than the left side.

Adult↗

PubMed1996

Assessment of the contribution of the spleen to granulocytopoiesis and erythropoiesis of the mid-gestation human fetus.

Several current textbooks of hematology describe the spleen of the mid-gestation human fetus as a granulocytopoietic and an erythropoietic organ. Although studies in fetal rodents support this view, a convincing demonstration of such in normal human fetuses is lacking. We tested the hypothesis that the human mid-gestation fetal spleen is normally (1) a site of active granulocytopoiesis and erythropoiesis, or (2) a site of production of specific granulocytopoietic or erythropoietic growth factors. This was accomplished using the spleens, livers, and long-bones of 18 human fetuses, 13-22 weeks gestation, immediately following elective, induced, pregnancy terminations. Organs of some of the abortuses were placed directly into formalin for histologic evaluation. Organs from others were subjected to RNA extraction for subsequent probing for specific hematopoietic growth factor mRNA. Cell suspensions were created from the organs of other abortuses for quantification of the absolute number of neutrophils and erythrocytes and their precursors and progenitors. Evidence of active hematopoiesis was present in marrow and liver but not spleen. Transcripts for granulocyte colony-stimulating factor (G-CSF) were detected in the marrow but not the spleen or liver, and transcripts for erythropoietin (Epo) were detected in the liver but not the spleen or marrow. The populations of hematopoietic progenitor cells and normoblasts in the fetal spleen cell were similar to those in fetal blood. Thus, it is likely that the hematopoietic progenitors recovered from fetal spleen cell suspensions are the result of blood within the spleen, rather than from hematopoiesis within the organ. The spleen of mid-gestation human fetuses, unlike the spleen of fetal rats, does not normally function as an active site of granulocytopoiesis or erythropoiesis, nor is it an active site for production of G-CSF or Epo.

Abortion, Induced↗