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PubMed1994

A novel method to quantitate methylation of specific genomic regions.

A new solid-phase primer extension method has been developed for the quantitation of methylation differences and is described here. The method is less cumbersome than Southern blot analysis, expresses the results in a numerical format, can be adapted to a microtitration well format, and thus allows the analysis of a large series of samples. The model gene analyzed here is the calcitonin gene, but the method can be adapted to the analysis of methylation alterations in any area of the genome. The primer extension method clearly differentiated hypermethylated samples from normally methylated samples and a range for normal values could be determined. In quantitation experiments the method showed linearity in a range from 2% to 100% malignant blasts diluted with normal leukocytes.

Bone Marrow↗
PubMed2004

Two-stage adaptive strategy for superiority and non-inferiority hypotheses in active controlled clinical trials.

In active controlled trials without a placebo arm, there are usually two study objectives: to test a superiority hypothesis that the experimental treatment is more effective than the active control therapy, and to test a non-inferiority hypothesis that the experimental treatment is therapeutically no worse than the active control within a defined margin. For a two-stage adaptive design, it is not necessary to give a fixed sample size calculation at the planning stage of the study when treatment effect information is often insufficient. Instead, decision and estimation of the design specifications can be made more reliably after the first stage when interim results are available. We propose the use of conditional power approach to determine the sample size and critical values for testing the superiority and non-inferiority hypotheses for the second stage based on the observed result of the first stage. The proposed adaptive procedure preserves the overall type I error rate for both superiority and non-inferiority, and has the flexibility of early termination of the study (for futility or efficacy) or extending the study by appropriate sample size.

Controlled Clinical Trials as Topic↗
PubMed1998

Phenotypic expression of autosomal dominant retinitis pigmentosa in a Swedish family expressing a Phe-211-Leu variant of peripherin/RDS.

PURPOSE: To characterize the clinical phenotype, with emphasis on electrophysiology, of members of a Swedish family with autosomal dominant retinitis pigmentosa due to a novel mutation, F211L, in the peripherin/RDS gene. METHODS: Nine patients with autosomal dominant retinitis pigmentosa and two healthy family members underwent a full clinical evaluation including kinetic visual field testing, measurement of dark adaptation threshold, and full-field electroretinography. Blood samples were collected and DNA analysis was performed using denaturing gradient gel electrophoresis (DGGE). RESULTS: The grandfather, six of seven siblings from the middle generation, and two young boys carried the mutation F211L in the peripherin/RDS gene. The mutation segregated with the clinical presentation of disease. Fundus examination revealed mainly macular atrophy. All assessed parameters of retinal function (visual acuity, dark adaptation threshold, visual fields, and full-field electroretinograms) demonstrated a successive reduction with increasing age. Full-field electroretinograms showed a diminished rod response in all affected individuals and a reduction of the cone b-wave amplitudes with increasing age, indicating retinitis pigmentosa. In the affected family members, the disease seems to progress at a similar rate with increasing age. CONCLUSIONS: The peripherin/RDS gene mutation F211L is associated with a clinical phenotype and includes early loss of rod function and successive reduction of cone function with increasing age, but impressively well-preserved visual acuity and visual fields in young and middle-aged patients and moderately reduced vision in the old patient. Compared to previously described phenotypes segregating with mutations in the peripherin/RDS gene, the present family demonstrates a more benign clinical phenotype, which is concordant within the family.

Adolescent↗
PubMed2006

Rapid estimation of microbial numbers in water using bulk fluorescence.

Enumeration of microbial cells without culturing is an essential technique for microbial ecology and water quality evaluation. Here we show that bulk fluorescence using the SYBR Gold DNA stain can be used to rapidly quantify microbial cells per millilitre in fresh, marine and estuarine waters. The bulk fluorescence method is comparable to estimating cell concentrations in cultures using optical density; however, this enhanced method enables the user to estimate microbial numbers at lower concentration (> 10(5) cells ml(-1)) found in environmental samples. The technique worked in both single-cell and 96-well plate fluorescent spectrophotometers. Differences of approximately 10(5) cells per millilitre were discernible and the precision of the bulk fluorescence was higher than direct counting by epifluorescent microscopy. Treatment with DNase I increased sensitivity by lowering background noise attributed to free DNA. This technique is simple, rapid, inexpensive and adaptable for automatically estimating microbial numbers in water samples.

California↗
PubMedDate not supplied

Long-term effects of pediatric intensive care unit hospitalization on families with young children.

OBJECTIVE: The purpose of this study was to prospectively examine the long-term effects of the pediatric intensive care unit experience on parents and on family adaptation. DESIGN: A three-group prospective, comparison, convenience sample was used in this study. SETTING: The study took place in a midwestern university-affiliated tertiary pediatric medical center. SUBJECTS: Three groups of parents (parents with children in the pediatric intensive care unit, parents with children in a general care unit, and parents with nonhospitalized ill children) were studied. The children were ages 5 years or younger. OUTCOME MEASURES: Indicators of family adaptation included parental stress, stress symptoms, family functioning, and life events. RESULTS: The mothers' stress symptoms in all groups were more prevalent than a normative sample, and they perceived their families as dysfunctional after discharge from the hospital. Slope analysis was used to examine the patterns of stress symptoms and family functioning and indicated little directional change over time for the mothers. CONCLUSION: Despite the smaller than expected sample sizes over time, results suggest that parents are still having stress-related symptoms and difficulties with family functioning as long as 6 months after a child's illness event.

Adaptation, Psychological↗
PubMed2006

In vivo and in vitro evaluation of marginal fit of class II ceromer inlays.

The aim of this study was to evaluate the marginal adaptation of class II ceromer (Targis, Vivadent) indirect inlay restorations under both in vivo and in vitro conditions. Twenty Targis inlays were produced for class II inlay cavities (13 mandibular and seven maxillary teeth) in 20 patients. The in vivo adaptation of the inlay to the tooth cavity was evaluated by means of silicone replica technique. For in vitro evaluation, 20 mesio-occluso-distal (MOD) Targis inlays were made in extracted upper molars. Half of the inlays were cemented with Variolink high-viscosity resin cement while the other half was cemented with Variolink Ultra. The replica specimens and in vitro samples were sectioned buccolingually and mesiodistally, and marginal adaptation was evaluated at both proximal and occlusal margins at 200x magnification under a light microscope. The data was analyzed with anova (P < 0.05). The in vivo mean film thickness values for occlusal and proximal locations were recorded as 73 and 132 microm respectively. In vitro mean marginal fit values were observed as 48 and 67 microm for occlusal and proximal margins of inlays luted with Variolink II high viscosity. The marginal fit values recorded under in vivo conditions were higher in magnitude than the measurements obtained under in vitro conditions. The use of a highly filled resin luting agent with an ultrasonic insertion technique did not cause an increase in marginal gap width of the inlay.

Acrylic Resins↗
PubMed2005

[Identification of psychrotrophs SYP-A2-3 producing cold-adapted protease from the No. 1 Glacier of China and study on its fermentation conditions].

The psychrotrophs SYP-A2-3 producing the cold-adapted protease has been isolated from the bacterial samples collected from the No. 1 Glacier of China and identified as Bacillus cereus according to its morphological and physiochemical characteristics and 16s rDNA gene sequence analysis. It could grow between 0 degree C and 38 degrees C while its optimal growth temperature was 25 degrees C and the optimal temperature for its protease production was 15 degrees C. The cold-adapted protease was identified as neutral metallo-protease, the molecular weight was 34.2 kD shown by SDS-PAGE, the optimal pH and temperature for activity was 7.0-8.5 and 42 degrees C, respectively. Various fermentation conditions of its protease production were also investigated. The results showed that casein was the best nitrogen source while glucose and starch were suitable carbon source for its protease production. The initial pH of fermentation broth ranged from 6.5 to 7.0 was optimal. Under optimized conditions, the protease activity produced by SYP-A2-3 could reach 3800 U/mL and 4800 U/mL conducted in shaking flask and 5 L stirred jar experiment, respectively.

Bacillus cereus↗
PubMed2006

Aerodynamic mass spectrometry interfacing of microdevices without electrospray tips.

A new concept for electrospray coupling of microfluidic devices with mass spectrometry was developed. The sampling orifice of the time-of-flight mass spectrometer was modified with an external adapter assisting in formation and transport of the electrosprayed plume from the multichannel polycarbonate microdevice. The compact disk sized microdevice was designed with radial channels extending to the circumference of the disk. The electrospray exit ports were formed by the channel openings on the surface of the disk rim. No additional tips at the channel exits were used. Electrospray was initiated directly from the channel openings by applying high voltage between sample wells and the entrance of the external adapter. The formation of the spatially unstable droplet at the electrospray openings was eliminated by air suction provided by a pump connected to the external adapter. Compared with the air intake through the original mass spectrometer sampling orifice, more than an order of magnitude higher flow rate was achieved for efficient transport of the electrospray plume into the mass spectrometer. Additional experiments with electric potentials applied between the entrance sections of the external adapter and the mass spectrometer indicated that the air flow was the dominant transport mechanism. Basic properties of the system were tested using mathematical modeling and characterized using ESI/TOF-MS measurements of peptide and protein samples.

Journal Article↗
PubMed1989

A simple and rapid microplate assay for glycoprotein-processing glycosidases.

A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates [( 3H]glucose for glucosidases and [3H]mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported (B. Saunier et al. (1982) J. Biol. Chem. 257, 14155-14161; T. Szumilo and A. D. Elbein (1985) Anal. Biochem. 151, 32-40). These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. This procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of alpha-glucosidase I.

Animals↗
PubMed1995

High resolution, standard format two-dimensional protein electrophoresis using disposable glass micropipettes and non-dedicated equipment.

A method for high resolution, two-dimensional polyacrylamide gel electrophoresis (PAGE) of proteins on a standard format that utilizes non-dedicated electrophoretic equipment is described. This method combines key features of various previous protocols into an improved approach which can readily be applied by persons experienced only in sodium dodecyl sulfate (SDS)-PAGE. The method yields high-quality two-dimensional protein separations that are reliably reproducible and uniform between different analyses. High protein resolution is attained and preserved primarily by facilitating the execution of the first isoelectric focusing (IEF) dimension and streamlining the initiation of the second (SDS-PAGE) dimension. Important features of this method include the use of piperazine diacrylate cross-linker, disposable glass micropipettes, and conical sample chambers in the IEF dimension. In addition, the equilibration of the first-dimensional gels is accomplished at the same time the gels are loaded onto the second dimension. Loss of protein resolution due to diffusion after the end of the first dimension is reduced by minimizing the time required to load the SDS-PAGE dimension. The method is versatile and adaptable to the analysis of one or many samples. The high degree of resolution, reproducibility and uniformity attained by this method are sufficient to abrogate the need for dedicated equipment in most applications.

Amnion↗
PubMed1997

[Consumption of psychoactive substances by school-age adolescents of Ribeirão Preto, SP (Brazil). II--Distribution of consumption by social levels].

INTRODUCTION: Many of the epidemiological studies on the consumption of legal and illegal psychoactive substances have included the evaluation of the influence of social context on the levels of prevalence of this consumption using indirect social indicators such as family income, and educational and housing levels in an attempt to identify individuals or groups in different social contexts. The present study investigates the distribution of consumption of psychoactive substances according to social class in a sample of teenage pupils in Ribeirão Preto, SP, Southeastern Brazil. MATERIAL AND METHOD: A self-applicable questionnaire duly adapted and submitted to a reliability test was applied to a proportional sample of 1,025 teenagers enrolled in the 8th, 9th, 10th and 12th grades in public and private city schools. The questionnaires contained questions about the use of ten classes of psychoactive substances, demographic questions and validation information, as well as questions about the perception and intrinsic behavior related to drug consumption. The adaptation of a model that identifies 5 social class strata (business middle class, managerial middle class, lower middle class, proletariat and subproletariat) on the basis of indicators that situate the individuals within the social relations of production, was used. RESULTS: The 3 middle class strata were more often represented, whereas the proletariat and subproletariat were less frequently represented in this teenage pupil population than in the population in general. There was no difference in alcohol or tobacco consumption according to social class, although prevalence tended to be higher at the two extremes of the social ladder. In contrast, the consumption of illegal substances was higher in the middle class and lower in the proletariat. CONCLUSION: Although the consumption of legal substances did not differ among social classes, the higher consumption of illegal substances by the wealthier teenagers was probably due to the higher cost of these products as compared those of alcohol and tobacco.

Adolescent↗
PubMed1992

Responses of neurons in the gracile nucleus of cats to innocuous and noxious stimuli: basic characterization and antidromic activation from the thalamus.

1. Responses to innocuous and noxious mechanical and thermal stimuli were recorded from 90 neurons in the gracile nucleus of anesthetized cats. Cells were tested by antidromic activation for projections to the contralateral ventrobasal thalamus. 2. Cells were characterized broadly by their responses to mechanical stimuli as 1) responding only to tapping (16%), 2) fast-adapting to low-intensity mechanical stimuli (33%), or 3) slowly adapting (51%; most with a fast-adapting component to their responses). All fast-adapting cells and those slowly adapting cells that were tested with noxious heat were further categorized on the basis of their patterns of firing and responses to stimuli. These plus the tap-responsive cells comprised a more restricted sample of 76 categorized cells. 3. Many (22) slowly adapting cells responded to noxious heat (69% of tested slowly adapting cells; 29% of all categorized cells), either on the first application (9 cells) or after sensitization (13 cells), indicating input originating in nociceptors. Nearly all of these (21) responded more to intense pressure than to innocuous pressure. The majority of slowly adapting cells not responsive to noxious heat (5 of 8) or not tested with it (8 of 12) also responded more to intense than to innocuous pressure, suggesting possible input originating in nociceptors. Most cells that responded to noxious heat also had both rapidly and slowly adapting responses with low thresholds. Many were recorded in the range of the cluster region of the gracile nucleus. 4. Cells antidromically activated from the thalamus projected to the rostral part of the ventral posterior lateral nucleus, regardless of their physiological category, and included many with nociceptive input. Latencies of antidromic activation were shorter at more caudal locations in the gracile nucleus, indicating higher conduction velocities to the thalamus. Responses of antidromically activated cells to low-intensity phasic stimuli tended to be greater than those of cells not antidromically activated. 5. Background activity of the neurons was low, most firing at less than one spike/s. Antidromically activated cells had higher background activity than cells not antidromically activated. 6. The results indicate a greater proportion and more widespread distribution of cells with nociceptive input in the cat gracile nucleus than has been previously recognized. Many of these projected to the ventrobasal thalamus, showing that information originating in nociceptors can reach the thalamus through a dorsal column-medial lemniscal pathway in cats.

Adaptation, Physiological↗
PubMed2005

Comparing artificial and natural selection in rate of adaptation to genetic stress in Aspergillus nidulans.

In an experimental study of adaptation to negative pleiotropic effects of a major fungicide resistance mutation in the filamentous fungus Aspergillus nidulans we have investigated the relative effectiveness of artificial selection vs. natural selection on the rate of compensatory evolution. Using mycelial growth rate as a fitness measure, artificial selection involved the weekly transfer of the fastest growing sector onto a fresh plate. Natural selection was approximated by transferring random samples of all the spores produced by the mycelium. Fungicide resistant and fungicide sensitive haploid and diploid strains were used in an evolution experiment over 10 weekly transfers, which is equivalent to 1200 cell cycles. Two different environmental conditions were applied: a constant fungicide-free environment and a weekly alternation between presence and absence of fungicide. Results show that for all strains and conditions used the transfer of a random sample of all spores leads to more rapid adaptation than the transfer of the visually 'fittest' sector. The rates of compensatory evolution in the constant and the alternating environment did not differ. Moreover, haploid strains tend to have a higher rate of adaptation than isogenic diploid strains.

Adaptation, Physiological↗
PubMed2005

Quantitative chemiluminescent immunoassay for NF-kappaB-DNA binding activity.

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous redox-sensitive transcription factor involved in the pro-inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF-kappaB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. The conventional method for detection of NF-kappaB -DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time-consuming and non-quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF-kappaB DNA-binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF-kappaB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3-5h post sample preparation, (2) Greater sensitivity: 10pg NF-kappaB/well, (3) Quantitative: linear range: 10-1000pg NF-kappaB; r2 = 0.999 (4) High throughput adaptability: 96-well plate format can analyze up to 40 samples in duplicate, (5) SAFETY: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF-KB which is translocated into the nucleus and total (bound + unbound) NF-kappaB present in the cytosol/cell.

Animals↗
PubMed2005

Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics.

BACKGROUND: Measuring plasma unbound bilirubin concentration by the peroxidase test is useful in the management of jaundiced newborns. However, the commercially available peroxidase technology is manual, and the unbound bilirubin may be seriously underestimated at the 42-fold sample dilution and single peroxidase concentration used. We investigated improving the test by adapting it to Zone Fluidics, which is a system for automating reactant handling that requires small sample volumes and dilution. METHODS: A computer-directed Zone Fluidics system was constructed using small diameter tubing to connect in series a water-surfactant reservoir, a bi-directional pump, a multiport selection valve to which peroxidase test reactants (45 mul of sample) are attached with one port open to air, and a spectrophotometer flow cell. Test reactants and air are sequentially aspirated through the valve into the tubing connecting the pump and valve to form a reactant "zone" surrounded by air. The zone is advanced to the spectrophotometer flow cell where total and unbound bilirubin are determined (37 degrees C) from the absorbance at 460 nm at a 2-fold sample dilution and 4 peroxidase concentrations. Imprecision was assessed in artificial controls and newborn plasma. Plasma results were compared with those obtained using the commercial method. RESULTS: The CV for unbound bilirubin in the various controls ranged from 11% to 38% (within day) and 12% to 27% (between days). Triplicate CV measurements for newborn plasma measurements ranged from 0.6% to 31% (mean 11%, n=47). Mean unbound bilirubin by Zone Fluidics was 5-fold higher than that by the commercial method. CONCLUSION: Zone Fluidics can be used to automate the peroxidase test and overcome many of the limitations of the commercially available peroxidase technology.

Bilirubin↗
PubMed1989

Family adaptation during a pediatric intensive care hospitalization.

The purpose of this study was to investigate characteristics of families, specifically their adaptability, cohesion, and coping mechanisms during a child's intensive care hospitalization. The sample consisted of 30 families with children hospitalized in a pediatric intensive care unit. The Family Adaptability and Cohesion Evaluation Scales (FACES-III) and the Family Crisis-Oriented Personal Evaluation Scales (F-COPES) were used to collect data. Results indicate that the families were, as a whole, healthy in their levels of functioning; their dimensions of adaptability and cohesion were balanced. The families studied utilized a variety of coping mechanisms, and specific characteristics were identified that enabled the families to adjust to the child's intensive care hospitalization.

Adaptation, Psychological↗
PubMed2000

Adaptive response in patients treated with 131I.

UNLABELLED: The aim of this study was to investigate whether an adaptive response (defined as the induction of radiation tolerance after a small dose of radiation) could be observed in peripheral blood lymphocytes of patients treated with 1311 for thyroid disease. METHODS: For each patient, blood samples were taken immediately before and 1 wk after 131I administration. Each blood sample was divided into 3 fractions and the fractions were subsequently irradiated in vitro with 0, 0.5, and 1.0 Gy 60Co gamma-rays. After blood culture for 70 h, cells were harvested and stained with Romanowsky-Giemsa and micronuclei were counted in 1000 binucleated cells. The increase in micronuclei by the in vitro irradiation of the blood samples taken before and after therapy was compared. In this setup, an adaptive response is represented by a significant decrease of the in vitro induced micronucleus yield after therapy compared with that before therapy. The iodine therapy can be considered as an in vivo adaptation dose, after which the subsequent in vitro irradiation acts as a challenge dose. To investigate the reproducibility of the method, 2 subsequent blood samples of healthy volunteers were taken 7 d apart. Irradiation and cell culture were performed as described. RESULTS: In 8 of 20 patients, a significant (P = 0.0002) decrease was found in the in vitro induced micronucleus yield in the blood sample taken 1 wk after 1311 administration compared with that of the blood sample taken before therapy. No significant (P > 0.1) differences were observed between these 8 patients and the other patients when the number of micronuclei induced in vivo by the iodine treatment and the resulting equivalent total body dose were compared. None of the control subjects showed a significant change in micronucleus yield after in vitro irradiation between both blood samples taken 1 wk apart. CONCLUSION: The iodine treatment can act as an in vivo adaptation dose and can induce an adaptive response that is observed by a decrease of the cytogenetic damage in peripheral blood lymphocytes after in vitro irradiation as a challenge dose. A large interindividual difference was observed.

Adaptation, Physiological↗
PubMed1991

A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites.

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.

Amino Acid Sequence↗