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PubMed2005

Pharmacokinetics and metabolism of acriflavine in rats following intravenous or intramuscular administration of AG60, a mixture of acriflavine and guanosine, a potential antitumour agent.

Acriflavine (ACF; CAS 8063-24-9), a mixture of trypaflavine (TRF) and proflavine (PRF) at a ratio of 2:1 is being investigated in rodents as an anticancer agent. However, its pharmacokinetics have not been investigated in mammals. Guanosine is known to potentiate the anticancer activity of some compounds. The pharmacokinetics of AG60, a 1:1 mixture of ACF and guanosine, were therefore investigated in rats. Rats were given 2 or 10 mg kg(-1) AG60 by intravenous bolus or 6 or 30 mg kg (-1) intramuscularly. An HPLC-based method was developed to analyse the levels of TRF, PRF, and their metabolites in plasma, bile, urine and tissue homogenates. The plasma concentrations of TRF and PRF decreased rapidly after intravenous administration and more slowly after intramuscular administration. Both TRF and PRF were distributed widely, most notably in the kidney, and were eliminated slowly. Three glucuronosyl conjugate metabolite peaks were tentatively identified in the bile. The intramuscular route leads to a prolongation of TRF or PRF plasma levels, and the systemic exposures for both TRF and PRF were both relatively high. These observations indicate that the intramuscular route may be the best way to administer AG60 for various clinical applications.

Acriflavine↗
PubMed1991

Polyacrylic microspheres as a solid phase for microparticle enhanced nephelometric immunoassay (NEPHELIA (R)) of transferrin.

Polyfunctional hydrophilic microspheres (MS) can be produced by copolymerisation with gamma-irradiation of acrylic monomers. Transferrin (TRF) can be covalently bound to these MS by reaction between aldehyde groups of the MS and primary amino groups of the protein. MS-TRF conjugates thus obtained are agglutinated by specific antiserum and this agglutination is inhibited by free TRF. Agglutination and inhibition are quantified by measurement of the light scattered by MS-TRF conjugate clusters with a specially designed nephelometer, a process designated as microparticle enhanced nephelometric immunoassay (NEPHELIA (R)) for TRF. Recovery, correlation and reproducibility studies, simultaneously performed in three different laboratories, show that this TRF immunoassay is accurate for a large concentration range. NEPHELIA (R) may appear as an alternative method for a large variety of molecules.

Humans↗
PubMed1993

Thermogenic response to food: intra-individual variability and measurement reliability.

The objective of this study was to assess the reliability and variability of the thermogenic response to food (TRF) in healthy adult subjects. Eight healthy adults (4 men, 4 women) participated in three sets of measurements of TRF, evaluated by indirect calorimetry with a ventilated hood system during 6 hours after a standardized meal. The reliability of TRF measurement was estimated using a one-way analysis of variance with repeated measurements. After the 6-hour period of measurement, the within-subject coefficient of variation for TRF was 10.7%, while the between-subject variability was twice as high (24.1%, p < 0.01). The overall reliability estimate of TRF was acceptable (RE > or = 0.80) after a single measurement which lasted at least 6 hours postprandially, while duplicate and triplicate measurements reached a similar degree of reliability in 3 hours. These results suggest that intra-individual variability contributes little to the variability commonly seen in TRF studies, provided that the measurement period is at least 6 hours.

Adult↗
PubMed2003

Electron paramagnetic resonance analysis of transferrin-bound iron in animal models of blunt trauma.

BACKGROUND: Blood iron sequestration is known to be implicated in the systemic acute-phase response to trauma injury. The objective of the present research was to assess the effect of iron sequestration in animal models of blunt trauma by means of electron paramagnetic resonance spectroscopy of iron in complex with transferrin, a main iron-transporting protein in blood, and to correlate this effect with the extent of induced injury. METHODS: Two animal models of blunt trauma were explored in the present study. Blunt trauma in the rat model was produced by exposure of 14 animals to blast overpressure (BOP) (at peak BOP of either 86 +/- 5 kPa or 112 +/- 2 kPa) generated in a shock tube. Blunt trauma in the porcine model was produced by impact of high-speed projectiles made from a rubber-tipped, plastic composite weighing 28.64 +/- 0.12 g (mean +/- SEM, n = 8) with a length of approximately 6 cm and a diameter of approximately 4 cm. The projectiles were propelled by compressed helium onto eight animals at a velocity of 101.8 +/- 3.8 m/s (mean +/- SEM, n = 8) at the point of impact. Each experiment was accompanied by a pathology assessment using an injury scoring system developed for blunt trauma injuries to derive a severity score for whole-body involvement. Amounts of transferrin-bound iron (TRF-[Fe3+]) in whole blood and blood plasma samples were measured using quantitative electron paramagnetic resonance spectroscopy. The observed alterations in the amounts of blood TRF-[Fe3+] were correlated with estimated injury score ratios in each animal. RESULTS: Blunt trauma produced by either BOP exposure of rats or projectile impacts in pigs was accompanied by TRF-[Fe3+] sequestration observed in both blood and blood plasma. The amount of TRF-[Fe3+] in blood was shown to have inverse correlation with the extent of injury (Pearson r = -0.90 in the rat model and r = -0.93 in the porcine model) estimated by injury score ratios and was not dependent on location of the injury (lung, liver, spleen, or jejunum). CONCLUSION: The presented data suggest that assessment of TRF-[Fe3+] in blunt trauma can provide a good deal of information on severity of injury. The response of TRF-[Fe3+] can be considered as a potential surrogate marker of the systemic alterations in blunt trauma and, therefore, warrants further investigation in a human pilot study.

Animals↗
PubMed1984

Transferrin binding by peripheral blood mononuclear cells in human lymphomas, myelomas and leukemias.

Significantly more peripheral blood mononuclear cells (PBM) from 39 patients with lymphoma (p less than 0.001), myeloma (p less than 0.001), or leukemia (p less than 0.001-0.025) bind transferrin (Trf) than those of 23 normals. That Trf was bound to a receptor on these cells was shown by the inhibition of these cells bound to 125I-Trf with unlabeled Trf. The paucity of Trf receptors on PBM from normals and their presence on diseased cells prompt a suggestion that Trf receptors may offer a useful approach to the diagnosis and possible treatment of some blood dyscrasias.

Adult↗
PubMed2000

Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid.

We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.

Central Nervous System Viral Diseases↗
PubMed2003

Rapid identification of bacteria from positive blood cultures by terminal restriction fragment length polymorphism profile analysis of the 16S rRNA gene.

Bacteremia results in significant morbidity and mortality, especially among patient populations that are immunocompromised. Broad-spectrum antibiotics are administered to patients suspected to have bloodstream infections that are awaiting diagnosis that depends on blood culture analysis. Significant delays in identification of pathogens can result, primarily due to the dependence on growth-based identification systems. To address these limitations, we took advantage of terminal restriction fragment (TRF) length polymorphisms (T-RFLP) due to 16S ribosomal DNA (rDNA) sequence diversity to rapidly identify bacterial pathogens directly from positive blood culture. TRF profiles for each organism were determined by sizing fragments from restriction digests of PCR products derived from two sets of 16S rDNA-specific fluorescent dye-labeled primers. In addition, we created a TRF profile database (TRFPD) with 5899 predicted TRF profiles from sequence information representing 2860 different bacterial species. TRF profiles were experimentally determined for 69 reference organisms and 32 clinical isolates and then compared against the predicted profiles in the TRFPD. The predictive value of the profiles was found to be accurate to the species level with most organisms tested. In addition, identification of 10 different genera was possible with profiles comprising two or three TRFs. Although it was possible to identify Enterobacteriaceae by using a profile of three TRFs, the similarity of the TRF profiles of these organisms makes differentiation of species less reliable with the current method. The ability to rapidly (i.e., within approximately 8 h) identify bacteria from blood cultures has potential for reducing unnecessary use of broad-spectrum antibiotics and promoting more timely prescription of appropriate antibiotics.

Bacteria↗
PubMed2005

Cardioprotection with palm tocotrienol: antioxidant activity of tocotrienol is linked with its ability to stabilize proteasomes.

Tocotrienols, isomers of vitamin E, have been found to possess many health benefits. The present study was designed to determine whether tocotrienol has a direct cardioprotective role. Isolated rat hearts were perfused for 15 min with Krebs-Ringer bicarbonate buffer in the absence or presence of palm tocotrienol derived from the tocotrienol-rich fraction (0.035%) of palm oil (TRF). In another group of studies, the hearts were preperfused for 15 min in the presence of a c-Src inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-3,4-d-pyrimidine (PPI). The hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. As expected, ischemia-reperfusion caused ventricular dysfunction, electrical rhythm disturbances, and increased myocardial infarct size. PPI or TRF could reverse the ischemia-reperfusion-mediated cardiac dysfunction. Ischemia-reperfusion also upregulated c-Src expression and phosphorylation. Although TRF only minimally affected c-Src expression, it significantly inhibited the phosphorylation of c-Src. Ischemia-reperfusion reduced 20S and 26S proteasome activities, an effect prevented by TRF pretreatment. PPI exerted a cardioprotective effect that is not mediated by the proteasome but, rather, through direct inhibition of c-Src. The results of this study support a role for c-Src in postischemic cardiac injury and dysfunction and demonstrate direct cardioprotective effects of TRF. The cardioprotective properties of TRF appear to be due to inhibition of c-Src activation and proteasome stabilization.

Animals↗
PubMed1971

Effect of thyrotropin-releasing factor on serum thyroid-stimulating hormone. An approach to distinguishing hypothalamic from pituitary forms of idiopathic hypopituitary dwarfism.

To test the hypothesis that the primary defect in some patients with idiopathic hypopituitary dwarfism is failure to secrete hypothalamic hypophysiotropic-releasing factors, synthetic thyrotropin-releasing factor (TRF), 500 mug, wa given intravenously, and timed venous samples obtained for determination of the concentration of plasma TSH by radioimmunoassay in three groups of subjects: (a) 11 patients without evidence of endocrine or systemic disease, (group I) (b) 8 with isolated growth hormone deficiency and normal thyroid function, (group II) and (c) 9 patients with idiopathic hypopituitary dwarfism and thyroid-stimulating hormone (TSH) deficiency (group III). The mean fasting plasma TSH value was 4.1 muU/ml in group I, and 3.9 muU/ml in group II; in both groups there was a brisk rise in plasma TSH to peak levels of 12-45 muU/ml at 30-45 min, and a fall toward base line levels at 120 min. All children in group III had basal TSH levels of < 1.5 muU/ml; one failed to respond to TRF; eight exhibited a rise in plasma TSH with peak values comparable with those in groups I and II. In four of eight children in group III who responded to TRF, the TSH response was delayed and the initial rise in plasma TSH was not detectable until 10-60 min. In these four patients, plasma TSH levels continued to rise at 120 min. The mean fasting concentration of plasma thyroxine iodide (T(4)) in subjects with normal thyroid function (groups I and II) was 5.6 mug/100 ml, and the mean plasma T(4) level at 120 min was 6.6 mug/100 ml. This difference between fasting and postTRF plasma T(4) was significant (P < 0.001) by paired analysis. Mean fasting plasma T(4) concentration in group III patients was 1.3 mug/100 ml; after TRF a significant rise in T(4) concentration was not detected in this group. The results indicate that TRF test is useful in distinguishing between primary hypothalamic and pituitary forms of TSH deficiency. In light of the evidence of TRF deficiency in eight of nine patients with idiopathic hypopituitary dwarfism, it seems likely that in these patients, other pituitary hormone deficiencies may be attributable to deficiency of their respective releasing factors.

Adolescent↗
PubMed2004

Telomere length correlates with histopathogenesis according to the germinal center in mature B-cell lymphoproliferative disorders.

In this study we investigated telomere restriction fragment (TRF) length in a panel of mature B-cell lymphoproliferative disorders (MBCLDs) and correlated this parameter with histology and histopathogenesis in relation to the germinal center (GC). We assessed 123 MBCLD samples containing 80% or more tumor cells. TRF length was evaluated by Southern blot analysis using a chemiluminescence-based assay. GC status was assessed through screening for stable and ongoing somatic mutations within the immunoglobulin heavy-chain genes. Median TRF length was 6170 bp (range, 1896-11 200 bp) and did not correlate with patient age or sex. TRF length was greater in diffuse large cell lymphoma, Burkitt lymphoma, and follicular lymphoma (medians: 7789 bp, 9471 bp, and 7383 bp, respectively) than in mantle cell lymphoma and chronic lymphocytic leukemia (medians: 3582 bp and 4346 bp, respectively). GC-derived MBCLDs had the longest telomeres, whereas those arising from GC-inexperienced cells had the shortest (P < 10(-9)). We conclude that (1) TRF length in MBCLD is highly heterogeneous; (2) GC-derived tumors have long telomeres, suggesting that minimal telomere erosion occurs during GC-derived lymphomagenesis; and (3) the short TRF lengths of GC-inexperienced MBCLDs indicates that these neoplasms are good candidates for treatment with telomerase inhibitors, a class of molecules currently the subject of extensive preclinical evaluation.

B-Lymphocytes↗
PubMed1976

Abnormal TSH, PRL and GH response to TSH releasing factor in chronic renal failure.

TSH, PRL and GH response to TSH releasing factor as well as basal T4 and T3 were evaluated in a group of patients with chronic renal failure undergoing chronic hemodialysis. Serum T4 and T3 were lower than normal. Basal TSH was normal as compared to control, but did not rise after TRF stimulation. Larger dosages of TRF did not correct this abnormal response. Basal PRL was higher than control and remained at the same level during the test. GH was stimulated by the TRF with a peak occurring 20 min after injection. This abnormal secretion was not blunted by T3 administration. TRF half-life measured in 3 patients was 4 min. These data indicate that 1) there is an abnormal response to TRF in chronic renal failure which does not seem to be due to an altered sensitivity to, or metabolism of TRF; and 2) there is an abnormal TSH secretion which may be responsible for the low T4 and T3 measured in these patients.

Adolescent↗
PubMed2004

Terminal telomere repeats are actually short in telomerase-negative immortal human cells.

Telomerase-negative immortal human cells maintained telomere length by a mechanism called alternative lengthening of telomeres (ALT mechanism). These cells (ALT cells) have two prominent characteristics of long telomere repeats at each chromosome end revealed by Southern blotting (terminal restriction fragments: TRF) and the presence extrachromosomal telomere repeat (ECTR) DNA. We report here that the TRF length of ALT cells revealed by the conventional unidirectional (UD) current or pulse-field (PF) current electrophoresis appeared to be over estimated. The TRF length determined by the pulse inverse-field (PIF) current electrophoresis (2-9 kbp depending upon cell lines) was much smaller than that (ca. 23 kbp) by UD or PF current electrophoresis. These results were in consistent with very weak telomere staining in situ at chromosome ends in ALT cells. When a mixture of HinfI-digested genomic DNA of human diploid fibroblasts and synthetic telomere repeat DNA with similar size of ECTR DNA was electrophoresed using a UD current, the apparent TRF size shifted to larger molecular weight, while the size shift did not occur by PIF current electrophoresis. These results together with other data indicate that the unusually long TRF of ALT cells determined by using conventional electrophoresis is an artifact produced by a complex formed by short TRF and short ECTR DNA.

Cell Line↗
PubMed2004

Time-resolved fluorescence measurements using microlens array and area imaging devices.

Time-resolved fluorescence (TRF) assay formats are frequently used technologies in high-throughput screening. In this article, we have characterised the novel Plate::Vision(2) 96-microlens array reader (Carl Zeiss Jena GmbH, Germany) and compared it to the novel LEADseeker Generation IV multimodality imaging system (LEADseeker Gen IV; Amersham Biosciences UK Ltd., UK) for applications in the TRF mode. In europium measurements using the TRF mode, the Plate::Vision displayed a limit of detection for europium of approximately 3 pM, which was comparable to two established TRF readers, the Discovery and the Victor V (both PerkinElmer Life Sciences Inc., USA). The LEADseeker's limit of detection only extended down to europium concentrations of approximately 10 pM in these experiments. For TRF resonance energy transfer (TR-FRET) experiments, a europium-biotin (Eu-biotin) conjugate was titrated with a streptavidin-allophycocyanin (SA-APC) conjugate. The Plate::Vision produced Z' values larger than 0.5 for the acceptor fluorophor emission with concentrations of Eu-biotin as low as 3 nM combined with 175 pM SA-APC. To achieve Z' values of at least 0.5 with the LEADseeker, concentrations of 10 nM Eu-biotin combined with SA-APC of at least 0.8 nM were required. In a drug screening application using TR-FRET, the energy transfer from a europium-labelled protein X (Eu-protein X) to a complex of biotinylated peptide Y with SA-APC was measured. Using the Plate::Vision, a Z' factor larger than 0.5 for the acceptor fluorophor emission was only obtained for a Eu-protein X concentration of at least 10 nM in combination with biotinylated peptide Y/SA-APC at saturating concentrations. Both the Plate::Vision and the LEADseeker show good quality results for applications in the TRF mode and enable an increased throughput based on their shortened measurement time in comparison to classic photomultiplier tube-based readers.

Biological Assay↗
PubMed2000

Behavioral and emotional problems in Chinese children: teacher reports for ages 6 to 11.

This study examined the applicability of the Chinese Version of Teacher's Report Form (TRF-CV) and estimated the prevalence of behavioral problems in a general population sample of 2,936 children aged 6 through 11 years in the Shandong Province of China. Teachers completed the TRF-CV and the Conners Hyperkinesis Index (CHI). The TRF-CV total scale showed satisfactory 2-week test-retest reliability (r = .83) and internal consistency (Cronbach's alpha = .94). The TRF-CV Total Problems, Attention Problems, Delinquent Behavior, and Aggressive Behavior had acceptable concurrent validity with the CHI (mean r = .62). With the TRF-CV Total Problems score of 26 as a cutoff, an overall correct classification rate of 90% for clinical sample and nonreferral required children was obtained. Exploratory factor analysis yielded six syndromes: Aggressive/Delinquent Behavior, Withdrawn/Depressed, Somatic Complaints, Attention Problems, Social Problems, and Thought Problems, with significant correlations with corresponding American cross-informant syndromes (mean r = .84). The overall prevalence rate of behavioral problems was 15.5% (95% CI = 14.2-16.8%), with a boy-to-girl ratio of 2.0:1 (chi2 = 59.70, p < .001). Younger boys exhibited more externalizing problems. These findings indicate that the TRF-CV is applicable for Chinese children, and the prevalence of behavioral problems shown by it among Chinese children seems comparable to that found in other countries. Although most of the American syndromes were well replicated, the differences in the present subjects, when submitted to principal components analysis, from American samples from whom the original syndromes were derived, could have prevented the study from replicating distinctions between aggressive vs. delinquent and depressed vs. withdrawn syndromes.

Affect↗
PubMedDate not supplied

Correlation of chemosensitivity to anticancer drugs and telomere length, telomerase activity and telomerase RNA expression in human ovarian cancer cells.

BACKGROUND: We examined the correlations among telomere length (TRF), telomerase activity (TA) and the steady-state level of telomerase RNA expression (hTR) of human ovarian cancer cells with different phenotypes and investigated whether the cells' sensitivities to anticancer agents correlate with TRF, TA or hTR. MATERIALS AND METHODS: The TRF, TA and hTR of 11 human ovarian cancer cell lines and 2 cisplatin-resistant ovarian cancer cell lines were determined by genomic Southern blotting, a telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction. The chemosensitivities of the cell lines to cisplatin (CDDP), paclitaxel (TAX), etoposide (ETO), CPT-11 (CPT), cyclophosphamide (CYC), ifomide (IFO) and doxorubicin (DOX) were decided as IC50 values by calorimetric assay. RESULTS: All 11 cell lines presented shorter mean TRFs (5.0 kb) than normal control tissue (8.0 kb); 10 cell lines presented a 3.2-fold higher mean TA than the control and all 11 cell lines expressed hTR. Quantitatively, the steady-state levels of hTR correlated with the TRF (p < 0.05). Significant positive correlations between hTR and CDDP sensitivities (at 24 hours of exposure), ETO (72 hours), CPT (48 hours) and CYC/IFO (24-72 hours) were observed. The same was true for TRF and the CDDP sensitivities (at 24 hours). TAX and DOX did not have any impact on these factors. The TRF, TA and hTR values in the two CDDP-resistant cell lines were generally reduced, compared to their parent cell lines. CONCLUSIONS: Alkylating agents (CDDP, CYC and IFO) and topoisomerase inhibitors (ETO, CPT) may have the potential to influence the structural alteration of hTRs and telomeres and thus, the down-regulation of the TA in ovarian cancer cells.

Antineoplastic Agents↗
PubMedDate not supplied

Prognostic significance of telomeric repeat length alterations in pathological stage I-IIIA non-small cell lung cancer.

This study was performed to evaluate the prognostic significance of alteration in telomere length in pathological stage (p-stage) I-IIIA non-small cell lung cancer (NSCLC). Paired cancer and normal lung tissues were obtained from 72 patients with histologically confirmed p-stage I-IIIA NSCLC. Terminal restriction fragment (TRF) length, which indicates telomere length, was measured by Southern blot analysis. Tumor telomerase activity was also assayed by non-radioactive PCR-ELISA in 55 patients. TRF length (mean +/- SD) in normal tissue was 6.2 +/- 1.1 Kb. Therefore, upper and lower limits of normal range in TRF length was set at 8.4 (mean + 2SD) Kb and 4.0 (mean-2SD) Kb, respectively. A tumor showing TRF length over normal range was defined as positive for the alteration. In 72 patients, 25 (34.7%) with alteration in TRF length had significantly shorter survival durations than those of the others. Telomerase activity did not correlate with survival duration. In multivariate analysis, alteration in TRF length (P = 0.0033) was second to p-stage (P = 0.0004) in importance among the various parameters.

Adult↗
PubMed2001

Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry.

BACKGROUND: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography-mass spectrometry (LC-MS). METHODS: Serum (25 microL) was diluted with 100 microL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. RESULTS: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and interassay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018-0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002-0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170). CONCLUSIONS: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

Animals↗
PubMed2001

Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics.

Terminal Restriction Fragment (TRF) patterns, also known as Terminal Restriction Fragment Length Polymorphisms (T-RFLP), are a recently introduced PCR-based tool for studying microbial community structure and dynamics. Since the first review of TRF methodology (Marsh, 1999. Curr. Op. Microbiol. 2: 323-7), at least 35 new research articles were published that include this powerful tool in some part of their reports. This review covers some of the applications that TRF patterns were used for and provides a discussion of how to create and analyze TRF pattern data. This data has the advantage of being simply and rapidly produced using standard DNA sequencing equipment. The raw data are automatically converted to a digitized form that can be easily analyzed with a variety of multivariate statistical techniques. The identification of specific elements in a TRF pattern is possible by comparison to entries in a good sequence database or by comparison to a clone library. As an added advantage when investigating complex microbial communities such as those in soils and intestines, TRF patterns are recognized as having better resolution than other DNA-based methods for evaluating community structure.

Animals↗