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PubMed1999

Microspectrophotometric analysis of respiratory pigments using a novel fibre optic dip probe in microsamples.

A microspectrophotometer system to monitor the reduction of mitochondrial respiratory pigments in cell extracts and permeabilized cells has been developed. The novel optical fibre set-up uses visible spectrophotometry to measure the reduction of mitochondrial electron carriers. The basis of the system is an Ocean Optics S1000 spectrometer, a broadband tungsten based light source, input and output coupling fibre optics and a fibre optic dip-probe which requires less than 20 microl of sample for analysis. The spectral range of the system is from 250 to 850 nm with a spectral resolution of 0.5 nm. Data are presented for the reduction of purified cytochrome c by the reducing agent sodium dithionite and the reduction of cytochrome c by isolated mitochondria using sodium succinate as substrate. Reduction of cytochrome c by digitonin permeabilized cultured mouse cells, C2C12, is also shown. The effect of temperature on cytochrome c reduction in these assays is also demonstrated. The optical design of the probe system is optimized to maintain maximum light throughput and spectral resolution. The key features of the system are small sample size, front-end adaptability, high sensitivity and fast multispectral acquisition which are essential for observing these biological reactions in vivo.

Animals↗
PubMed1998

Highly active antiretroviral treatment in HIV infection: benefits for neuropsychological function.

OBJECTIVES: To determine whether highly active antiretroviral therapy (HAART) is associated with reduced HIV-associated neuropsychological impairment. DESIGN: Cross-sectional analysis in a natural history study of adaptation to HIV/AIDS. METHOD: A sample of 130 homo-/bisexual men with HIV/AIDS (mean age, 41 years; 42% non-white) were evaluated with a neuropsychological battery assessing attention, concentration, psychomotor speed, learning, memory and executive function. Subjects taking HAART were compared with those not taking HAART on demographics, CD4 cell count, viral load, scores on individual neuropsychological tests and proportion with neuropsychological impairment. RESULTS: Sixty-nine (53%) subjects were taking HAART, and 48 (37%) were neuropsychologically impaired. Subjects taking HAART had lower mean CD4 cell counts than those not taking HAART (254 versus 342 x 10(6)/l; P < 0.05), although they were more likely to have undetectable viral load (42 versus 20%; P < 0.01) and were less likely to be neuropsychologically impaired (22 versus 54%; P < 0.0001). Subjects taking HAART performed significantly better on tests of attention, concentration, learning, memory, and psychomotor speed. After excluding subjects with potential non-HIV confounders of neuropsychological function, those without neuropsychological impairment had significantly lower mean viral load levels and were more likely to have undetectable viral load than those with impairment. CONCLUSION: These preliminary findings suggest that HAART benefits neuropsychological function through the reduction of viral load.

Adult↗
PubMed2004

New home telehealth toolkit available.

A new Home Telehealth Toolkit is available for those interested in establishing a new home telehealth program or improving an existing one. Developed by members of the American Telemedicine Association's (ATA) Home Telehealth Special Interest Group (SIG) contains guidelines, procedures, and sample protocols that can be adapted for all phases of a home care telehealth program.

Community Health Nursing↗
PubMed1993

A sensitive high-performance liquid chromatographic method for the determination of 6-mercaptopurine in plasma using precolumn derivatization and fluorescence detection.

A sensitive high-performance liquid chromatographic (HPLC) method for measuring plasma concentrations of 6-mercaptopurine (6-MP) is described. After protein precipitation with 5-sulfosalicylic acid, samples are subjected to precolumn derivatization using the thiol-reactive fluorophore monobromobimane (mBrB). The drug-mBrB adduct is then resolved by isocratic elution from a C18 reversed-phase support and quantified by fluorescence detection. Recovery of 6-MP after protein precipitation was consistently > 85% and the drug-mBrB adduct was found to be stable for at least 2 weeks at room temperature. With plasma samples containing 30 nM 6-MP, the assay displayed within-run (n = 6) and between-day (n = 6) coefficients of variation of 2.2 and 10.6%, respectively. The limit of detection for 6-MP in plasma was 3 nM (500 pg/ml) and the standard curve was linear up to 3 microM. Using this method, we have observed that 6-MP is stable in heparinized whole blood for at least 24 h provided samples are maintained on ice. Since this method requires few manipulations during sample preparation and is readily adaptable to automated techniques, it may prove useful in the routine clinical laboratory setting.

Bridged Bicyclo Compounds↗
PubMed2006

Color demosaicing using variance of color differences.

This paper presents an adaptive demosaicing algorithm. Missing green samples are first estimated based on the variances of the color differences along different edge directions. The missing red and blue components are then estimated based on the interpolated green plane. This algorithm can effectively preserve the details in texture regions and, at the same time, it can significantly reduce the color artifacts. As compared with the latest demosaicing algorithms, the proposed algorithm produces the best average demosaicing performance both objectively and subjectively.

Algorithms↗
PubMed1986

A sensitive and specific assay to detect Ro(SS-A) and La(SS-B) antibodies.

This paper describes a sensitive and specific immunoblotting procedure to detect Ro(SS-A) and La(SS-B) autoantibodies in the serum of patients with lupus erythematosus. In order to perform this procedure, we have partially purified the Ro(SS-A) and La(SS-B) antigens from human spleen extracts by DEAE-cellulose and Sephacryl S-300 chromatography. The Ro(SS-A) and La(SS-B) antigens were immobilized on nitrocellulose paper after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested against a panel of sera at different dilutions: normal human sera (n = 14), Ro(SS-A) antisera (n = 6), La(SS-B) (n = 7), Ro(SS-A)/La(SS-B) (n = 8), Sm-nRNP-La(SS-B) (n = 2). We found that Ro(SS-A) antisera react with a protein of an approximate Mr of 58K, whereas the La(SS-B) antisera reacted with two bands of Mr 42 and 40K, respectively. Antisera with both autoantibodies [Ro(SS-A) and La(SS-B)] reacted with both antigens, whereas the control NHS, anti-nRNP, and anti-Sm did not stain the Ro(SS-A) and La(SS-B) protein bands. In addition, some of the positive sera continued reacting with the respective antigens at extremely high dilutions. This procedure can be easily adapted to test many serum samples and produce data which include Mr of the antigen and titer of autoantibodies in the patient's serum.

Antibodies, Antinuclear↗
PubMed2004

Effect of vitamin E supplementation on various functional properties of macrophages and neutrophils obtained from weaned piglets.

Sixteen piglets were used to determine the effect of vitamin E supplementation on several functional properties of macrophages and neutrophils obtained from weaned piglets. Piglets, immediately following weaning, were assigned to one of three experimental groups: control (no vitamin E supplementation), low level of vitamin E supplementation (100 mg DL-alpha-tocopheryl acetate/kg diet) and high level of vitamin E supplementation (300 mg DL-alpha-tocopheryl acetate/kg diet). Supplementation of vitamin E lasted for a period of 36 days, following a 3-day adaptation period after weaning. Blood samples were collected on days 0, 12, 24 and 36 of the experimental period, monocytes/macrophages and neutrophils were isolated and the following parameters were determined in macrophages and neutrophils activated by phorbol myristate acetate: total cell-associated and membrane-bound urokinase plasminogen activator (u-PA) activity and superoxide anion production. Results showed that macrophages and neutrophils isolated from piglets that received supplemental vitamin E had higher (P < 0.05) total and membrane-bound u-PA activities as well as higher (P < 0.05) superoxide anion production compared with the values of the corresponding cells obtained from control piglets on day 12 of the experimental period. Both levels of vitamin E supplementation (low and high) were equally effective. In contrast, vitamin E supplementation had no effect (P > 0.05) on total and membrane-bound u-PA activities and superoxide anion production by porcine macrophages and neutrophils on days 24 and 36 of the experimental period. In conclusion, the low level of vitamin E supplementation is recommended for piglets for the first 2 weeks after weaning.

Animals↗
PubMed1990

Detection and identification of E. coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment.

The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI). Amplification primers were designed to detect E. coli strains of human as well as porcine origin. This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI). In addition, three clinical E. coli isolates of human and one of porcine origin were tested. All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method. All strains yielded the expected 275 bp DNA fragment after enzymatic amplification. This fragment was further identified by allele specific oligonucleotide hybridization. Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E. coli isolated from humans and pigs. The detection limit determined in water with the ATCC 37218 strain was 20 bacteria. The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI. All of the strains tested showed this polymorphism as expected. Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively. This method may be adapted to environmental and food samples.

Animals↗
PubMed1998

siaD PCR ELISA for confirmation and identification of serogroup Y and W135 meningococcal infections.

Non-culture diagnosis and serogroup determination of meningococcal infection is important in contact management where vaccination may be possible. A serogroup B and C PCR ELISA assay for the non-culture diagnosis and serogroup determination has proved invaluable for enhanced epidemiological surveillance and contact management. A polymerase chain reaction assay, based on a restriction fragment length polymorphism in the meningococcal serogroup Y and W135 sialyltransferase (siaD) gene, was developed to enhance the range of non-culture diagnosis of meningococcal infection from clinical samples. The PCR assay was adapted to an ELISA format incorporating hybridisation with serogroup-specific Y and W135 oligonucleotide probes. The serogroup-specific W135 and Y PCR ELISA is a useful addition to currently available serogroup B and C assay for non-culture diagnosis of meningococcal infection and outbreak investigation.

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Serum 5'nucleotidase activity in rats: a method for automated analysis and criteria for interpretation.

A manual kit for determining serum 5'nucleotidase (5'NT, EC 3.1.3.5) activity was adapted for use with rat samples on a large discrete clinical chemistry analyzer. The precision of the method was good (within-run C.V. = 2.14%; between-run C.V. = 5.5%). A comparison of the new automated method with a manual and semi-automated method gave regression statistics of y = 1.18X -3.66 (Sy. x = 4.54), and y = 0.733X + 1.97 (Sy. x = 1.69), respectively. Temperature conversion factors provided by the kit manufacturer for human samples were determined to be inaccurate for converting results from rat samples. Analysis of components contributing to normal variation in rat serum 5'NT activity showed age and sex to be major factors. Increased serum 5'NT activity was observed in female rats when compared to male rats beginning at about 5 to 6 weeks of age. An analysis of variance of serum 5'NT, alkaline phosphatase, and GGT activities observed over a 9-week period in normal rats suggests several advantages for 5'NT as a predictor of biliary lesions in rats.

Journal Article↗
PubMed2002

Distribution and behaviour of glabrous cutaneous receptors in the human foot sole.

To document the activity of cutaneous mechanoreceptors in the glabrous skin of the foot sole, tungsten microelectrodes were inserted through the popliteal fossa and into the tibial nerve of thirteen healthy human subjects. A total of 104 cutaneous mechanoreceptors were identified in the glabrous skin of the foot. This sample consisted of 15 slow adapting type I (14 %), 16 slow adapting type II (15 %), 59 fast adapting type I (57 %), and 14 fast adapting type II units (14 %). The location of the receptors and the outline of the receptive fields were determined by using nylon monofilaments perpendicularly applied against the surface of the skin. This revealed that the receptors were widely distributed without an accumulation of receptors in the toes. There were also larger receptive fields predominantly isolated on the plantar surface of the metatarsal-tarsal region of the foot sole. Furthermore, with the foot in an unloaded position, there was no background discharge activity in any of the cutaneous receptors in the absence of intentionally applied stimulation. These findings suggest that skin receptors in the foot sole behave differently from those receptors found on the glabrous skin of the hand. This may reflect the role of foot sole skin receptors in standing balance and movement control.

Adult↗
PubMed1994

Dose-volume distributions: a new approach to dose-volume histograms in three-dimensional treatment planning.

A new approach to calculating and displaying dose-volume relationships in 3D radiation therapy is presented. We have developed a concept of a dose-volume distribution (DVD) and its corresponding differential dose-volume distribution (DDVD), based on organization of the data in the volume rather than in the dose domain. The new concepts make full use of the information that can be obtained from the dose calculation points and the sampling pattern and are designed to overcome shortcomings of the classical concepts of dose-volume histograms (DVH) and differential dose-volume histograms (DDVH). The new concepts can be applied to any number of dose calculation points, but they are especially advantageous when a small number of points is used. DVDs are particularly well suited to pseudo- and quasi-random sampling of dose distributions. We have developed an error analysis for DVDs and DDVDs in the case of pseudorandom sampling. We also describe an adaptive technique for minimizing the amount of data needed for purposes of display.

Biophysical Phenomena↗
PubMed2002

In-gel kinase assay as a method to identify kinase substrates.

Phosphorylation of proteins by kinases is central to many cellular processes, including signal transduction. Thus, assays to identify or characterize kinases are a key tool for research in this area. Kinase substrates can be incorporated into polyacrylamide gels and used to characterize kinase activity in mixed samples. This methodology can be adapted for the identification of novel kinase-substrates or kinase-kinases that participate in the regulation of cell signaling. Here, I review the rationale and principles of an in-gel kinase assay. This strategy relies on co-polymerization of a substrate within the gel matrix, followed by detection of enzymatic activity in situ. The following Protocol provides a detailed method for performing the in-gel kinase assay and discusses the uses of the assay to evaluate kinase activity in the context of proliferation, differentiation, and survival pathways.

Autoradiography↗
PubMed2005

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique.

AIMS: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. METHODS/RESULTS: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlater. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. CONCLUSIONS: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.

Biomedical Research↗
PubMed1995

Molecular techniques and their potential application in monitoring the microbiological quality of indoor air.

Health effects associated with poor indoor air quality have created a need for accurate, reproducible methods of monitoring the microbiological content of indoor air. Improved methods of detection may allow researchers to clarify the effect of individual species present in the indoor environment on human health. This review discusses the shortcomings of current methods of identification and detection and focuses on the potential for molecular techniques in this emerging field. Probe techniques, restriction endonuclease analysis, karyotyping, and DNA and polymerase chain reaction fingerprinting methods available to detect and identify bacteria and fungi significant in the indoor air environment are discussed. Problems that may be encountered using these techniques are also considered. The authors have included a brief discussion on current air sampling techniques as well as adapting these techniques for use with molecular detection methods.

Air Microbiology↗
PubMed2004

Phase I and pharmacologic study of intermittently administered 9-nitrocamptothecin in patients with advanced solid tumors.

PURPOSE: 9-Nitrocamptothecin (9NC) is an oral camptothecin analogue currently administered at 1.5 mg/m(2)/day x 5 days/week in Phase III studies for pancreatic carcinoma. In an effort to increase the dose administered per day and determine whether the daily dose or number of days of treatment influence toxicity, we performed a Phase I study of 9NC using intermittent schedules of administration. EXPERIMENTAL DESIGN: On schedule A, 9NC was administered orally daily x 5 days for 2 weeks every 4 weeks (one cycle). On schedule B, 9NC was administered orally daily x 14 days every 4 weeks (one cycle). Dose levels were determined by adaptive dose finding. Serial blood samples were obtained on day 1 of each schedule for pharmacokinetic studies of 9NC and its 9-aminocamptothecin (9AC) metabolite, and lactone forms were measured by high-performance liquid chromatography. RESULTS: The recommended Phase II doses for schedules A and B were 2.43 and 1.70 mg/m(2)/day, respectively, each providing the same dose intensity (i.e., 24 mg/m(2)/cycle). The primary toxicities on schedules A and B were neutropenia, thrombocytopenia, and diarrhea. On schedule A, two patients with gastric cancer and two patients with pancreatic cancer had stable disease for more than six cycles. On schedule B, one patient with pancreatic cancer had stable disease for more than six cycles, and a patient with pancreatic cancer had a partial response. There was significant interpatient variability in the disposition of 9NC and 9AC. Most of the drug remained in the 9NC form with a ratio of 9NC to 9AC of approximately 4 to 1. CONCLUSIONS: These studies suggest that 9NC administered on an intermittent schedule is tolerable and may be an active regimen in patients with gastric or pancreatic cancers. Dosing 9NC on a mg/m(2) basis does not reduce pharmacokinetic variability.

Administration, Oral↗
PubMed1994

Evaluation of a kinetic enzyme-linked immunosorbent assay for detection of caprine arthritis-encephalitis virus-specific antibodies.

A kinetic indirect enzyme-linked immunosorbent assay (k-ELISA) was evaluated for detection of antibody to caprine arthritis-encephalitis virus (CAEV), using sodium dodecyl sulfate-treated CAEV-63 as antigen. Two hundred fifteen caprine sera submitted to the diagnostic laboratory were tested for CAEV antibody by the k-ELISA and by immunoprecipitation of [35S]-methionine-labeled CAEV. A k-ELISA positive cutoff point of 80 yielded a sensitivity of 94.4% and a specificity of 100%, as compared with immunoprecipitation. A k-ELISA cutoff point of 50 resulted in a sensitivity of 100%, with 95.6% specificity. When sera with k-ELISA scores between 50 and 80 were considered suspect, testing of 1,001 diagnostic sera resulted in < 1.5% suspect reactions. Using the 80 cutoff point, the CAEV k-ELISA had good sensitivity and specificity, with the added advantages of quick turn-around time, few suspect reactions, and adaptability to large numbers of samples

Animals↗
PubMed2006

Using aptamers as capture reagents in bead-based assay systems for diagnostics and hit identification.

Most applications of xMAP (Luminex) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules--so-called aptamers--are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human alpha-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.

Antithrombins↗