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At least 217 records · Page 12Linked to original sources
PubMed1994

Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.

The practical application of highly efficient fluorescence-based methods for the semi-automated genotyping of polymerase chain reaction-based microsatellite markers will depend on the development of robust protocols that provide accurate and reproducible data. In the present report we compare the accuracy of a fluorescence-based protocol with a benchmark radiolabeling method that depends on a known sequence ladder or amplified DNA from reference individuals for sizing by autoradiography. Three microsatellite markers, IGF1 (mfd 1), D4S174 (mfd 59), and D5S211 (mfd 154), with products overlapping in size were each labeled with a different fluorophore and run simultaneously with an internal size standard in a single electrophoretic lane. The size of each allele was compared for these markers by using both techniques for five larger CEPH families (884, 1331, 1332, 1333, and 1362). Of 462 possible alleles, four discrepancies (0.8%) were identified when the two approaches were compared. We conclude that the fluorescence-based protocol is at least as accurate as the standard radiolabeling technique since none of the sizing errors arose as a result of the fluorescence-based technique. We describe the adaptation of this fluorescence-based protocol to the simultaneous analysis of up to 24 microsatellite loci per electrophoretic lane. These highly accurate and efficient semi-automated techniques will be useful in high-resolution genomic analyses.

Alleles↗
PubMed1991

Morphometry of the corpus callosum in normal aging and Alzheimer's disease.

Changes of the human corpus callosum in normal aging and Alzheimer's disease were analysed by means of morphometry. A standardized computerized evaluation program was implemented allowing objective, quantitative and reproducible data. The various parts of the corpus callosum showed a different pattern of changes in normal aging as compared to Alzheimer's disease. In conclusion, in normal aging affects mostly the front-temporal interhemispheric fiber systems, whereas in Alzheimer's disease the parietotemporal commissural fibers are altered.

Aging↗
PubMed1981

Analysis of 99mTc-pyridoxylidene-glutamate complex formation and its preparation with tin-adsorbed-resin: Sn-Resin kit method.

99mTc-Pyridoxylidene-glutamate (Tc-PG) was introduced in 1975 by Baker et al. as a cholescintigraphic agent. Nevertheless its routine use has been limited due to the autoclaving process involved. To shorten the labeling procedure, an analysis of Tc-PG complex formation using the stannous chloride method was carried out. Sn-Resin (stannous ion adsorbed onto cation exchange resin) was used for labeling a stable Tc-PG with high efficiency. A preautoclaved complex of pyridoxal and glutamate was required but the labeling procedure took only 10-15 min after the elution of 99m-TcO4- from the generator. Formation of a complex other than a 99mTc-complex of the Schiff -base ligand pyridoxylidene-glutamate is discussed. The new formulation of a Tc-PG kit (Sn-Resin) simplified the labeling method and reproducible data to that already reported by Baker was obtained in chromatographic studies. Its suitability in hospital departments has already been shown in clinical studies.

Bile↗
PubMed1983

Neurobehavioral tests in single- and repeated-dose toxicity studies in small rodents.

The discovery of neurobehavioral hazards is an important goal of the majority of the toxicological studies. Quite often it is possible to detect signs of neurobehavioral toxicity through non-instrumental observations and from the response of the animals to simple manipulations. Psychopharmacologists have long used these techniques to identify new drugs with potential usefulness as psychotropic agents. Their approach may also be applicable for the detection of neurotoxic chemicals. Based on a review of the literature and personal experience a neurobehavioral check-list is proposed which can easily be incorporated into the animal care and treatment routine used in single- and repeated-dose toxicity studies with small rodents. The instrumental tests which measure motor activity, neuromuscular functions, muscle strength, emotionality and social behavior are also discussed. The usefulness of these procedures in toxicology is not yet established. Their introduction into the testing routine creates considerable logistic difficulties and might not be compatible with good laboratory practices. Therefore, it is suggested to explore the procedures developed by psychopharmalogists and behavioral scientists and to validate them with a variety of neurobehavioral toxins. Tests which produce relevant and reproducible data may then be added to the toxicological protocols, preferably within the framework of safety pharmacology.

Animals↗
PubMed1979

Application of linear system analysis to the horizontal vestibulo-ocular reflex of the alert rhesus monkey using pseudorandom binary sequence and single frequency sinusoidal stimulation.

Horizontal eye movements of the alert rhesus monkey resulting from both pseudorandom binary sequence (PRBS) and single frequency sinusoidal rotational stimulation were analyzed using a PDP 11/40 computer in order to generate gain, phase, and coherence estimates at discrete frequencies between 0.008 and 1.28 Hz. A computer simulation of vestibular induced eye movements was used to validate our analysis procedures and to determine the effects of digital noise. Frequency domain transfer functions derived from gain and phase estimates revealed that the responses to PRBS stimulation and to single frequency sinusoids were not appreciably different. PRBS testing was accomplished in approximately one third the time required for sinusoidal testing and yielded highly reproducible data. We conclude that PRBS stimulation is a reliable and efficient method for assessing linear system parameters of the horizontal vestibulo-ocular reflex. PRBS testing may be particularly advantageous in studies of vestibulo-oculomotor plasticity in which rapid assessment of alterations in system dynamics is essential.

Animals↗
PubMed1989

Classification of Y chromosome polymorphisms by DNA content and C-banding.

A system has been developed using a microscope photometer to measure the DNA content of individual chromosomes. After correcting for the systematic and statistical errors the measured values are highly reproducible. Data are presented of measurements of human Y chromosomes from a cytogenetically normal population. When compared with the C-banding pattern from the same specimens the DNA content is found to change in discrete steps of 1.36 x 10(-14)g or 13.26 x 10(6) bp for each of the Y chromosome polymorphic types.

Base Composition↗
PubMed1984

Investigation of proteases with chromogenic substrates after isoelectric focusing (IEF).

A technique is described for the detection of protease isoenzymes which is more sensitive than disc electrophoresis. Supernatants of crude rat and human organ homogenates are subjected to analytical isoelectric focusing (IEF) and the gel strips are finally incubated in histochemical media containing 4-methoxy-2-naphthylamine amino acids or peptides and diazonium salts for simultaneous or post-coupling. The incubation media are identical with those used for section histochemistry of proteases. This combination of IEF and proteases histochemistry yields excellent and reproducible data which cannot be obtained by protease histochemistry alone. Post-coupling delivers less and more diffuse bands than simultaneous coupling. For simultaneous coupling, Fast Blue B and Fast Black K are the most suitable diazonium salts. More bands are found in agarose gels compared with polyacrylamide. Sex-differences exist for endopeptidases in the submandibular gland, but are absent in other rat organs. Despite their uniform membrane localization in tissue sections, aminopeptidase (AP) A and M and dipeptidylpeptidase (DPP) IV and gamma-glutamyltranspeptidase (GGT) show striking heterogeneous band patterns depending on the investigated organ. The similar band patterns of APA and APM can be specified by the use of activators or inhibitors. In rat kidney, up to 26 bands are obtained with DPP II and IV substrates, 3 for APA and APM and up to 12 for GGT. DPP IV of human liver is different from that in rat liver.

Animals↗
PubMed1989

[Postoperative, intra-abdominal adhesions--a new standardized and objective animal model and testing of substances for the prevention of adhesions].

80 male Sprague Dawley rats were divided in 8 groups of equal size. After median laparotomy defined abrasions of the serosa of abdominal wall, cecum and ileum were performed (400p, abrasive paper 280 grains/cm2, 8 cm2). The control group (I) received no medication. In the treatment groups 5 ml of the following agents were instilled before closure of the abdominal wall: normal saline (II), 16,250 IE Neomycin with 1250 IE Bacitracin (III), 30% dextrose (MW 70,000) (IV), 50,000 IE Streptokinase with 12,500 IE Streptodornase (V), or TCDO in concentrations of 10% (VI), 50% (VII), and 100% (VIII). All animals were relaparotomized on the 7th postoperative day, the adhesions were dissected and their extent was calculated by computer aid. Furthermore specimens were obtained for microscopic studies. Compared to the controls no reduction of adhesions could be achieved by dextrose. Significantly more adhesions were observed after treatment with Neomycin/Bacitracin. A reduction of about 23% was registered with normal saline and TCDO 10%. The greatest reduction of adhesions was seen after application of Streptokinase/Streptodornase as well as TCDO in concentrations of 50% and 100% (63%). The results were significant after evaluation with the t-test. Histologically there was a correlation between the extent of adhesions and the fibrin film. The new animal model has proven to result in reproducible data if used to evaluate substances for prevention of adhesions. Clinical studies with the best of these agents could serve as an approach to solve the problem of adhesion ileus.

Abdomen↗
PubMed1984

Intermittent ambulatory blood pressure recordings in children. Methodological aspects and influence of family history on hypertension.

In 100 children of different school classes (10-12 years, participation rate ca. 50%) ambulatory blood pressure (BP) was recorded by a semi-automatic non-invasive device (Remler system). BP was measured every 1/2 h between 2 and 7.30 p.m. During each measurement the children protocolled their activity. These BP recordings were repeated five times at monthly intervals in 86 children. In the same children casual and near basal BP was determined. The mean ambulatory BP was 103/64 +/- 7/6 mm Hg. The average retest reliability of the monthly mean values was r = 0.57 for systolic and r = 0.45 for diastolic BP. The variability of BP (standard deviation) was not reproducible. Data recording failed in 32% of all single systolic and in 42% of all single diastolic BP measurements. These drop-outs were caused by inappropriate use rather than technical problems. There was a relationship between procolled activities and systolic BP. On average, ambulatory BP was lower than BP under standardized conditions. Under all conditions, children with hypertensive parents exhibited a 6 mm Hg higher systolic BP than children without a similar family history. No sex differences were found.

Ambulatory Care↗
PubMed1987

New microbiological techniques for hospital epidemiology.

Infection control teams rely on microbiology laboratories for accurate, reproducible data to trace the spread of microorganisms in the hospital. Since few problems are recognized immediately, laboratories should save important nosocomial pathogens so that outbreak strains will be available for analysis by more sophisticated techniques, particularly since automation has reduced the amount of epidemiologically useful data generated by routine procedures. Traditional supplemental reference procedures include phage typing, biotyping, serotyping and bacteriocin typing. Recently, analysis of antibiotic resistance determinants has provided the hospital epidemiologist with additional tools. Resistant strains have been characterized by their production of specific antibiotic inactivating enzymes. Agarose gel electrophoresis and restriction endonuclease analysis of plasmid DNA have permitted precise characterization of nosocomial strains, and the spread of antibiotic resistance genes can now be followed by sensitive and specific DNA probes. DNA probes also show promise for distinguishing nosocomial strains bearing known virulence factors from strains with less pathogenic potential. Molecular genetics techniques have found an additional role in elucidating the epidemiology of nosocomial viruses, especially the herpesviruses.

Bacteriological Techniques↗
PubMed2004

A pre-clinical study showing how dopaminergic drugs administered during pre-exposure can impair or facilitate latent inhibition.

RATIONALE: It has been suggested that, in classical conditioning, dopamine (DA) codes for (a) attention to the conditioned stimulus (CS) or (b) the intensity of the unconditioned stimulus. OBJECTIVES: To clarify the role of DA in pre-clinical classical conditioning studies. METHODS: An existing model of classical conditioning presented by Schmajuk, Lam, and Gray (J Exp Psychol Anim Behav Process 22:321-349, 1996) suggests that DA cells in the ventral midbrain area code for the attentionally modulated internal representation of the CS. It is assumed that this representation is increased by dopaminergic agonists and decreased by dopaminergic antagonists. Computer simulations with the model describe the effect of nicotine and haloperidol on latent inhibition. RESULTS: Simulations replicate experimental results demonstrating that both nicotine and haloperidol affect latent inhibition when administered during the pre-exposure phase. In addition, the model reproduces data showing that administration of nicotine or haloperidol results in the impairment or facilitation of latent inhibition depending on the duration of CS or the number of CSs. CONCLUSIONS: The model demonstrates that pre-clinical experimental results, including cell activity and pharmacological data, are consistent with an attentional role for DA in classical conditioning.

Computer Simulation↗
PubMed2005

Multiscale modeling of lipids and lipid bilayers.

A multiscale modeling approach is applied for simulations of lipids and lipid assemblies on mesoscale. First, molecular dynamics simulation of initially disordered system of lipid molecules in water within all-atomic model was carried out. On the next stage, structural data obtained from the molecular dynamics (MD) simulation were used to build a coarse-grained (ten sites) lipid model, with effective interaction potentials computed by the inverse Monte Carlo method. Finally, several simulations of the coarse-grained model on longer length- and time-scale were performed, both within Monte Carlo and molecular dynamics simulations: a periodical sample of lipid molecules ordered in bilayer, a free sheet of such bilayer without periodic boundary conditions, formation of vesicle from a plain membrane, process of self-assembly of lipids randomly dispersed in volume. It was shown that the coarse-grained model, developed exclusively from all-atomic simulation data, reproduces well all the basic features of lipids in water solution.

Computer Simulation↗
PubMed1997

Continuous intratumoral microdialysis during high-dose methotrexate therapy in a patient with malignant fibrous histiocytoma of the femur: a case report.

We used a microdialysis technique to assay intratumoral methotrexate (MTX) levels during high-dose (12 g/m2 given as a 4-h infusion) therapy in a 43-year-old man with a malignant fibrous histiocytoma in the medial femoral condyle. Additional microdialysis probes were implanted in muscle tissue contralateral to the tumor and in an antecubital vein. Microdialysis was attempted during the initial two high-dose courses, but the two latter probes were removed at the start of the second treatment cycle due to leakage. No attempt to correct for microdialysis recovery was made. The intratumorally localized probe gave reproducible data on tumor MTX exposure of 9.3-14% of unbound systemic MTX. There was a close correlation between tumor and systemic levels for both MTX and its major extracellular metabolite 7-hydroxymethotrexate. Although limited to the study of MTX pharmacokinetics in a single subject, the experiment demonstrates that intratumoral microdialysis may provide data on tumor drug exposure, although of an indirect nature and dependent on the probe characteristics, the flow rate, and, possibly, the time after probe implantation. We propose that the application of microdialysis may prove useful for elucidation of the relationship between local drug exposure and the therapeutic response in normally inaccessible compartments during cancer pharmacotherapy.

Adult↗
PubMed2005

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer-Villiger monooxygenase catalysed lactone synthesis.

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L(-1) glycerol). Following induction of CHMO activity biomass concentrations of up to 6 gDCW L(-1) were obtained. Cells from each fermentation were subsequently used for the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L(-1)) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 microL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.

Acinetobacter calcoaceticus↗
PubMed2005

Prostate brachytherapy for localized prostate cancer.

After skin cancer, prostate cancer (CaP) is the most common cancer diagnosed in men. As a result of screening with prostate-specific antigen, CaP is being caught earlier than it was in the past. This has led to an increase in cure rates across all treatment modalities. There are no firm, reproducible data that demonstrate the superiority of one modality over another. The expectations for cure should be approximately 90% for low-risk patients and approximately 80% for intermediate-risk patients, regardless of treatment modality. The toxicity of available treatment modalities discriminates among them. All modalities have acute toxicity of similar severity; however, prostate brachytherapy (PI) has the least amount of long-term toxicity when compared with external beam radiotherapy and radical prostatectomy. Therefore, a patient who is confronted with the diagnosis of CaP is counseled to choose among the modalities based on the toxicity rather than the efficacy of the treatment options available. Adopting this evidence-based algorithm has led to the increased application of PI.

Antineoplastic Protocols↗
PubMed1989

Spectrophotometric determination of tryptophan in intact proteins by the acid ninhydrin method.

The interaction of tryptophan, lysozyme and tyrosine with ninhydrin in strong acid media has been investigated at 20, 25, 30, and 35 degrees C by spectrophotometry. Second-order rate constants and molar absorptivity values have been evaluated from an analytical point of view. Optimum conditions for the selective estimation of tryptophan, tryptophan residues in intact proteins, and indoles--without the disturbing effect of tyrosine--have been given. Under optimum conditions, in the concentration range from 2.5 X 10(-8) to 3.0 X 10(-7)M, molar absorptivity values and reproducibility data for various reactants have been reported. Molar absorptivity values (Am X 10(-3)/M X cm) of tryptophan (21.35), lysozyme (19.33), bovine serum albumin (21.05), human serum albumin (21.00), casein (17.85), alpha-chymotrypsin (18.28), trypsin (14.43), indole (5.03), and indole-3-acetic acid (13.75) have been measured with a standard error of 2.3% or less for any particular reactant.

Hydrogen-Ion Concentration↗
PubMed1990

Milacemide stimulates deficient glial Na+, K(+)-ATPase in freezing-induced epileptogenic cortex of cats.

We investigated the influence of milacemide, a glycinamide derivative with putative antiepileptic activity, on the K(+)-activation of Na+,K(+)-ATPase in bulk isolated glial cells and synaptosomes of control and epileptogenic cortex of cats with a chronic freeze lesion. In the primary and secondary epileptic foci of non-treated animals, glial Na+,K(+)-ATPase lost its physiological K(+)-activation, while the synaptosomal enzyme was unchanged. These data reproduced previous work done on the kinetic measurement of the enzymic activities. In treated animals (500 mg/kg milacemide given orally for 2 weeks after the freeze lesion), the glial enzyme showed a normal K(+)-activation in the epileptic foci. These results confirm the existence of an abnormal glial Na+,K(+)-ATPase in cold-induced focal epilepsy and suggest that the antiepileptic activity of milacemide might be secondary to an activation of glial Na+,K(+)-ATPase, contributing to antagonize ictal transformation and seizure spread.

Acetamides↗
PubMed1993

Effect of cervical vagotomy on catecholaminergic neurons in the cranial division of the parasympathetic nervous system.

This study provides evidence of catecholaminergic neurons in the cranial division of the parasympathetic nervous system. Presumptive catecholaminergic preganglionic neurons in the dorsal motor nucleus of the vagus (DMX) were revealed by a clearcut depletion of intracellular catecholamine-synthesizing enzyme immunoreactivity induced by unilateral cervical vagotomy and identified on tissues immunocytochemically processed for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H) or phenylethanolamine N-methyltransferase (PNMT). This experimental design was essential because of the recent failure in two species to reproduce data previously obtained in double-label (combined immunocytochemical-retrograde transport) studies. Vagotomy data confirmed three spatially-segregated populations of catecholaminergic visceromotor neurons in the DMX. These cell bodies were morphologically identical to preganglionic neurons observed on alternate tissues stained for Nissl substance or immunostained for choline acetyltransferase (ChAT), the enzyme biosynthesizing acetylcholine. Neurons in the central and medial DMX demonstrated fall-off of TH-like immunoreactivity (LI) ipsilateral to the vagotomy at levels caudal to the obex. This cell group is assumed to be predominantly dopaminergic since relatively few neurons at this level of the DMX expressed D beta H-LI and none were immunostained for PNMT. A second population of immunoreactive neurons, concentrated in the rostral-lateral region of the DMX, was depleted of D beta H-LI on the ipsilateral side but did not express PNMT. These visceromotor neurons may, therefore, biosynthesize noradrenaline and belong to the rostral pole of the A2 area. A third population of presumptive adrenergic vagal dorsomotor neurons in the rostral-medial DMX was depleted of TH-, D beta H- and PNMT-LI at levels of the ipsilateral nucleus anterior to obex. Patterns of depletion of cytoplasmic enzyme-immunoreaction product were identical in all cases irrespective of the site of the transection or the postoperative survival period. Quantitative analysis demonstrated statistically significant loss of immunolabeled neurons in rostral and caudal subgroups of the DMX on the side ipsilateral to the vagotomy. It is concluded that catecholaminergic processes in the vagus nerve, as previously identified by the aldehyde-induced histofluorescence method, may partly arise from the lower brainstem.

Animals↗