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PubMedDate not supplied

Polymerase chain reaction amplification of Guthrie card deoxyribonucleic acid: extraction of nucleic acid from filter matrices.

This study evaluated two deoxyribonucleic acid (DNA) extraction methods for Guthrie card bloodspots. Our water-based extraction technique was compared with a commercial kit (GFX extraction system). In contrast to our nondenaturing method, the GFX system utilizes chaotropes to extract DNA. Extracted DNA is subsequently purified by selective elution from a glass fiber matrix. To evaluate DNA extraction, polymerase chain reaction (PCR) for a 491 bp region encoding the cystic fibrosis delta F508 mutation was performed and PCR products electrophoresed on polyacrylamide gels and stained with ethidium bromide. Amplification of GFX-extracted DNA required low sample volume (1 to 5 microL) indicating the presence of residual PCR inhibitors. The GFX volumes (1/20 to 1/4 punch) were comparable to our standard conditions and represented 0.16-0.80 microL whole blood (20 to 40 ng DNA). The GFX-extracted DNA was found to be stable (6 mo, -15 degrees C). Performance time for the GFX method was less than our standard water-based extraction (about 30 min), but more costly. The GFX extraction kit was adaptable for limited sample (i.e., extraction of a 3 mm punch yields 20 amplifications). The GFX extraction kit provides a useful means to standardize Guthrie card DNA extraction.

Blood Chemical Analysis↗
PubMed2006

High-throughput DNA methylation profiling using universal bead arrays.

We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

Base Sequence↗
PubMed1995

[Blood pressure and hormonal regulation in active and passive orthostasis].

UNLABELLED: The procedure using a tilt-table is far better standardized and less dependent on the patient's compliance compared to the active manoeuvre of standing up. The object of this study is to detect to what extent results are comparable concerning hormonal regulation and the adaptation of blood pressure. In 20 males (59.1 +/- 10.9 years), without any evidence of central nervous disorder, we measured the systolic, diastolic and the median blood pressure, heart frequency and hormone levels (noradrenaline, aldosterone, renin) at rest and over 10 minutes in the upright position after actively or passively (by tilt-table) assuming an orthostatic posture. The blood pressure showed little change after the patient sat upright himself, whereas the passive manoeuvre caused a significant decline of systolic blood pressure lasting over the entire sampling period. The adaptation of hormone levels and heart frequencies, however, showed no significant differences. Renin and aldosterone rose in both examinations. Noradrenaline showed an increase of 100% after active assumption of orthostatic posture and 80% using the tilt-table. Hence in patients who are passively brought into the upright position there is no increased noradrenaline output to stabilize blood pressure. This points to the fact that orthostatic circulatory mechanisms are not sufficiently brought into play after the non-physiological passive manoeuvre. IN CONCLUSION: (1.) the tilt-table compared to active orthostatic posture provides better standardized conditions; (2.) there are no significant differences in hormone levels; and (3.) maneuvering patients into an upright position provides a greater risk of fainting due to a pathological fall in blood pressure.

Aged↗
PubMed1989

Determination of quinonoid dihydrobiopterin by high-performance liquid chromatography and electrochemical detection.

Sodium bisulphite is shown to react with quinonoid dihydrobiopterin to form a stable adduct. Sodium bisulphite does not react with tetrahydrobiopterin. Quinonoid dihydrobiopterin reacts with dithioerythritol to form tetrahydrobiopterin, whereas the quinonoid dihydrobiopterin bisulphite adduct does not. Using these properties we have developed an indirect method for the quantitative measurement of quinonoid dihydrobiopterin. The method requires division of a sample into two. Dithioerythritol is added to one half (a). This converts quinonoid dihydrobiopterin to tetrahydrobiopterin and prevents the oxidation of tetrahydrobiopterin. Measurement of the tetrahydrobiopterin content of this sample by electrochemistry following high-performance liquid chromatographic separation (with dithioerythritol present in the mobile phase to prevent autoxidation of the tetrahydrobiopterin on column), therefore provides a total value of the tetrahydrobiopterin plus quinonoid dihydrobiopterin present within the original sample. Sodium bisulphite is added to the other portion of the sample (b), followed immediately by dithioerythritol which prevents autoxidation of the remaining tetrahydrobiopterin. The bisulphite reacts with the quinonoid dihydrobiopterin present and the quinonoid dihydrobiopterin-bisulphite adduct is no longer detected by electrochemistry at the retention time of tetrahydrobiopterin. Using reversed-phase high-performance liquid chromatography and redox electrochemical detection, measurement of tetrahydrobiopterin in the absence (a) and presence (b) of bisulphite enables the concentration of quinonoid dihydrobiopterin to be calculated by subtraction (a - b). This method is shown to be quantitative and preliminary experiments demonstrate that it can be adapted for biological samples.

Biopterins↗
PubMedDate not supplied

Determination of alpha-amylase activity in serum and dialysate from patients using icodextrin-based peritoneal dialysis fluid.

OBJECTIVE: Low serum activity of alpha-amylase has been reported in peritoneal dialysis (PD) patients following treatment with icodextrin-based peritoneal dialysis fluid (IPDF). However, these results have been questioned because icodextrin interferes with the polysaccharide reagent included in the assay as a substrate for alpha-amylase in the sample. DESIGN: We adapted a routine method using p-nitrophenol maltoheptaoside as substrate for the analysis of total alpha-amylase in serum and dialysate from 27 patients using IPDF. Serum from 12 healthy volunteers and serum and dialysate from 19 PD patients using glucose-based peritoneal dialysis fluid (GPDF) were used as controls. For the PD patients, time on dialysis ranged from 1 to 24 months (mean 5.7 months) and time of exposure to IPDF ranged from 1 to 52 weeks. RESULTS: To test for interference and recovery, and thus to validate the alpha-amylase assay, samples were spiked with IPDF and synthetic alpha-amylase. This revealed that addition of up to 75% IPDF did not interfere with the assay. Furthermore, alpha-amylase was fully recovered when spiked in serum from patients treated with IPDF. We show that total alpha-amylase activity is considerably lower in the serum of IPDF patients (20.3 +/- 16.5 U/L, p < 0.001) than GPDF patients (85.5 +/- 51.7 U/L) and healthy persons (55.1 +/- 13.6 U/L). CONCLUSIONS: We have shown that the IL method (ILTest; Instrumentation Laboratory, Lexington, MA, USA) measures alpha-amylase activity in samples containing icodextrin metabolites. The clinical significance of reduced plasma alpha-amylase activity, as well as the relative importance of pancreatic versus salivary and tissue-bound alpha-amylase, in PD patients using IPDF is not known.

Aged↗
PubMedDate not supplied

Simultaneous in-situ monitoring of parallel polymerization reactions using light scattering; a new tool for high-throughput screening.

A recently introduced technique, simultaneous multiple sample light scattering (SMSLS), was used to monitor parallel polymerization reactions in situ. SMSLS is designed for real-time, high-throughput screening and provides a time-dependent light scattering signature for each reaction, which contains both qualitative and semiquantitative information. Qualitatively, the signature immediately indicates whether the reaction occurs or not, whether there is an initial lag period, and how long the reaction takes until it stops. The signature also provides estimates of the reaction rate and weight average molecular mass M(w), and its shape can help identify mechanistic aspects, for example, controlled versus free radical polymerization, presence of impurities, etc. The method is inherently adapted to small sample volumes and requires no special sample preparation or postpolymerization characterization. The demonstration here involved the free radical polymerization of acrylamide under varying conditions and should be readily applicable to a wide variety of other reactions. Results were cross-checked with multi-detector gel permeation chromatography.

Journal Article↗
PubMed2000

High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC).

One of the critical steps in the positional cloning of a complex disease gene involves association analysis between a phenotype and a set of densely spaced diallelic markers, typically single nucleotide repeats (SNPs), covering the region of interest. However, the effort and cost of detecting sufficient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunction with denaturing high performance liquid chromatography (DHPLC), as a possible strategy for augmenting the efficiency, economy, and throughput of SNP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e.g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples. We have adapted this technology to identify variants in pooled DNA. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0%-50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-terminator sequencing. Using DHPLC, we could consistently detect SNPs at lower than 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that DNA pooling of samples for DHPLC analysis is an effective way to increase throughput efficiency of SNP detection.

Alleles↗
PubMed2000

Perceived versus measured height. Which is the stronger predictor of psychosocial functioning?

The relationship between perceptions versus measured height and children's psychosocial adaptation in a sample of medically referred youth with short stature was investigated. All children referred for a growth evaluation to one regional pediatric endocrinology clinic received a psychosocial screening assessment as a routine component of their initial visit. Data were collected for patients ages 4-18 years (n = 620) with heights ranging from -4.0 to -1.1 SD for age- and gender-adjusted population norms. Patients (8 years and older) and in all cases a parent/guardian served as informant through paper-and-pencil questionnaires. Both children and parents overestimated the child's height. Overestimations of height were associated with greater patient and parent satisfaction with stature. Perceived height was more strongly associated with psychosocial adaptation than was measured height. Clinical management decisions designed to enhance patient quality of life by increasing projected adult height through hormonal interventions should take into account both measured and perceived patient height.

Adaptation, Psychological↗
PubMed1998

Serological and genetic characterisation of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaption and passages in cell culture or calves.

Danish isolates of bovine respiratory syncytial virus (BRSV) were characterised by nucleotide sequencing of the G glycoprotein and by their reactivity with a panel of monoclonal antibodies (MAbs). Among the six Danish isolates, the overall sequence divergence ranged between 0 and 3% at the nucleotide level and between 0 and 5% at the amino acid level. Sequence divergences of 7-8%, 8-9% and 2-3% (nucleotide) and 9-11%, 12-16% and 4-6% (amino acid) were obtained in the comparison made between the group of Danish isolates and the previously sequenced 391-2USA, 127UK and 220-69Bel isolates, respectively. Phylogenetic analysis showed that the Danish isolates formed three lineages within a separate branch of the phylogenetic tree. Nevertheless, the Danish isolates were closely related to the 220-69Bel isolate, the prototype of the intermediate antigenic subgroup. The sequencing of the extracellular part of the G gene of additional 11 field BRSV viruses, processed directly from lung samples without prior adaption to cell culture growth, revealed sequence variabilities in the range obtained with the propagated virus. In addition, several passages in cell culture and in calves had no major impact on the nucleotide sequence of the G protein. These findings indicated that the previously established variabilities of the G protein of RS virus isolates were not attributable to mutations induced during the propagation of the virus. The reactivity of the Danish isolates with G protein-specific MAbs were similar to that of the 220-69Bel isolate. Furthermore, the sequence of the immunodominant region was completely conserved among the Danish isolates on one side and the 220-69Bel isolate on the other. When combined, these data strongly suggested that the Danish isolates belong to the intermediate subgroup.

Amino Acid Sequence↗
PubMed1980

Biochemical and histochemical alterations in skeletal muscle in man during a period of reduced activity.

This study investigated the hypothesis that the adaptations in skeletal muscle to prolonged exercise overload, involving high levels of adenosine triphosphate (ATP) resynthesis, result in a preferential adaptation to pathways involved in energy metabolism. The change in selected properties of skeletal muscle during a period of reduced activity was used as an indication of training-induced adaptations. Muscle biopsy samples from the vastus lateralis were analyzed 6 weeks and 18 weeks after a 5-month, intense, intermittent training program. Significant reductions occurred (p less than 0.05) in enzyme activities representative of the citric acid cycle (succinic dehydrogenase, SDH), beta oxidation of free fatty acids (3-hydroxyacyl CoA dehydrogenase, HADH), glycogenolysis (total phosphorylase, PHOSP), and glycolysis (phosphofructokinase, PFK). In addition, reductions in concentration (p less than 0.05) were also found for ATP, creatine phosphate (CP), and glycogen. With the exception of PFK, all enzyme changes and the high energy phosphates reached new stable levels by at least the 6th week of detraining. The absence of changes in muscle cell type and size during the detraining period supports the hypothesis that adaptations in energy potential of the muscle cell predominate in this type of high intensity overload situation.

Adenosine Triphosphate↗
PubMed1997

Time headway in car following and operational performance during unexpected braking.

The relation between car-following behaviour and braking performance was studied in a driving simulator. The theoretical perspective was that individual differences in tactical car-driving behaviour may be related to skills on the operational level of the driving task via a process of adaptation. In a sample of 16 young and middle-aged experienced drivers independent assessments were made of preferred time headway during car following and of braking skill. Starting from modern theories of visual-motor learning, braking performance was analyzed in terms of a reaction time component, an open-loop visual-motor component, and a closed-loop visual-motor component involving the precise adjustment of braking (timing and force) to the situation. The efficiency of the visual-motor component of braking was a strong and significant predictor of choice of time headway to the lead vehicle in such a way that less efficient braking indicated a preference for a longer time headway. This result supports the theory of adaptation on the individual level.

Adult↗
PubMed2002

Influence of dietary phosphorus deficiency with or without addition of fumaric acid to a diet in pigs on bone parameters.

The purpose of this study was to examine if substantial bone loss occurs in weaned pigs by feeding a phosphorus-deficient diet with or without fumaric acid. Eighteen weaned pigs were used. The animals were assigned to three groups: group C (control; 0.65% P on DM basis), group LP (low phosphorus; 0.37% P on DM basis) and group LPF (low phosphorus plus fumaric acid; 0.35% P on DM basis plus 2% fumaric acid). These three diets were fed to the groups for a period of four weeks after a two-week adaptation period. Blood samples were collected once a week. Carboxyterminal telopeptide of type I collagen (ICTP) in serum was used as a bone resorption marker. Osteocalcin (OC) and bone-specific alkaline phosphatase (bAP) were used as bone formation markers. Bone mineral density (BMD) and content (BMC) were determined by peripheral quantitative computer tomography. BAP activities significantly increased (24%) in group LPF, and at the last sampling day group LPF had significantly increased activities in comparison to group C. In contrast, ICTP concentrations significantly increased with time in group LP and LPF, and at the last sampling day group LPF had significantly increased activities in comparison to group C. BMD and BMC in femur and tibia significantly decreased in group LP and LPF. The results show that P-deficient diets induce a bone loss. Fumaric acid did not influence the degree of bone loss. With a better understanding of its effect on bone, dietary phosphorus requirements in pigs could be more precisely defined.

Adaptation, Physiological↗
PubMed2004

Phosphate binding protein as the biorecognition element in a biosensor for phosphate.

This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins.

Amino Acid Substitution↗
PubMed1993

Myosin light chain gene expression associated with disease states of the human heart.

During development of the human heart, the atrial isoform of alkali myosin light chain (MLC1A) is expressed in the ventricle. With maturation of the heart, MLC1A expression is completely replaced by that of the adult ventricular myosin light chain, MLC1V. We have evaluated the re-expression of MLC1A as a marker of different disease states of the human ventricle. RNA was isolated from the ventricles of patients with idiopathic dilated cardiomyopathy (CM) and severe congenital cardiac defects (CCD). Northern blot analysis was used to measure the mRNA levels of MLC1A and MLC1V in these samples. As a control, the level of regulatory MLC2V mRNA was also measured. We find that the level of MLC2V mRNA per microgram total ventricular RNA is very similar in CM, CCD and normal human samples. In contrast, we find that MLC1V mRNA levels tend to be reduced in both CM and CCD samples. In the case of the CCD samples, this apparent drop in MLC1V is compensated by expression of the developmental MLC1A isoform. However, in CM patients in end-stage failure, expression of MLC1A is barely detectable. The expression of MLC1A in CCD samples may reflect an adaptive mechanism in response to cardiac overload. The failure to detect substantial MLC1A expression in the CM samples may reflect the failure of such an adaptive mechanism.

Adult↗
PubMed1999

The prognostic importance of volume-weighted mean nuclear volume, mitotic index, and other stereologically measured quantitative parameters in supraglottic laryngeal carcinoma.

BACKGROUND: Stereologically measured mean nuclear volume has been proven to have prognostic importance in several types of cancer, such as malignant melanoma and carcinomas of the breast, oral region, bladder, and uterine cervix. The main purpose of the current study was to investigate the possible prognostic importance of mean nuclear volume and mitotic index in carcinoma of the supraglottic larynx. METHODS: The study was performed with a stratified, random sample of 113 patients from a well-defined group of 386 patients with supraglottic laryngeal carcinoma treated with radiotherapy at the Finsen Institute in Copenhagen. Histologic sections from pretreatment biopsies were used to estimate the following parameters: mean nuclear volume (Vv(3)(0)), mitotic index (MI), number of nuclei per mm(2) (QA), mean nuclear profile area (Anuc), and the area fraction of nuclei in cancer tissue (AA). The geometric means of the parameters were used as cutoff points in a single factor and in a multivariate survival analysis with relapse free survival as the primary endpoint. RESULTS: The geometric means of the measured parameters were (Vv(3)(0)) = 480 micro(3), QA = 3630 nuclei/micro(2) cancer tissue, MI = 0.48 mitosis/100 nuclei, AA = 0.21, and Anuc = 57.9 micro(2). CONCLUSIONS: None of the stereologically estimated parameters proved to have prognostic importance, whereas tumor size, and lymph node status did. The method of adaptive, stratified, random sampling used in this study can save a great deal of work and is highly recommended by the authors.

Aged↗
PubMed1979

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements.

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.

Bacteriorhodopsins↗
PubMed2004

The development and psychometric evaluation of the Brief Resilient Coping Scale.

This article introduces the Brief Resilient Coping Scale (BRCS), a 4-item measure designed to capture tendencies to cope with stress in a highly adaptive manner. Two samples of individuals with rheumatoid arthritis (ns = 90 and 140) provide evidence for the reliability and validity of the BRCS. The BRCS has adequate internal consistency and test-retest reliability. Convergent validity of the scale is demonstrated by predictable correlations with measures of personal coping resources (e.g., optimism, helplessness, self-efficacy), pain coping behaviors, and psychological well-being. Resilient coping, as assessed by the BRCS, also buffers the effects of high levels of arthritis-related and non-arthritis-related stressors on depressive symptoms. The sensitivity of the BRCS to changes associated with a cognitive-behavioral intervention is also demonstrated. The BCRS may be useful for identifying individuals in need of interventions designed to enhance resilient coping skills.

Adaptation, Psychological↗
PubMed2006

Attachment and psychological adaptation in high exposure survivors of the September 11th attack on the World Trade Center.

This study examined the relationship between individual differences in adult attachment and psychological adaptation in a sample of high-exposure survivors of the terrorist attacks on the World Trade Center on September 11, 2001. Symptoms of posttraumatic stress disorder (PTSD) and depression were assessed via self-report 7 and 18 months after the attacks. In addition, friends and relatives were asked to provide evaluations of participants' adjustment before and after the attacks. Findings indicate that securely attached individuals exhibited fewer symptoms of PTSD and depression than insecurely attached individuals and were viewed by friends and relatives as showing an increase in adjustment following the attacks. Highly dismissing adults were viewed by their friends and family as showing neither increments nor decrements in adjustment, despite the fact that highly dismissing people self-reported relatively high levels of PTSD and depression.

Adaptation, Psychological↗