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Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.

Animals↗

Forthergillian Lecture. Imaging human brain function.

The non-invasive brain scanning techniques introduced a quarter of a century ago have become crucial for diagnosis in clinical neurology. They have also been used to investigate brain function and have provided information about normal activity and pathogenesis. They have been used to investigate functional specialization in the brain and how specialized areas communicate to generate complex integrated functions such as speech, memory, the emotions and so on. The phenomenon of brain plasticity is poorly understood and yet clinical neurologists are aware, from everyday observations, that spontaneous recovery from brain lesions is common. An improved understanding of the mechanisms of recovery may generate new therapeutic strategies and indicate ways of modulating mechanisms that promote plastic compensation for loss of function. The main methods used to investigate these issues are positron emission tomography and magnetic resonance imaging (M.R.I.). M.R.I. is also used to map brain structure. The techniques of functional brain mapping and computational morphometrics depend on high performance scanners and a validated set of analytic statistical procedures that generate reproducible data and meaningful inferences from brain scanning data. The motor system presents a good paradigm to illustrate advances made by scanning towards an understanding of plasticity at the level of brain areas. The normal motor system is organized in a nested hierarchy. Recovery from paralysis caused by internal capsule strokes involves functional reorganization manifesting itself as changed patterns of activity in the component brain areas of the normal motor system. The pattern of plastic modification depends in part on patterns of residual or disturbed connectivity after brain injury. Therapeutic manipulations in patients with Parkinson's disease using deep brain stimulation, dopaminergic agents or fetal mesencephalic transplantation provide a means to examine mechanisms underpinning plastic change. Other models of plastic change, such as normal visuospatial learning or re-establishing speech comprehension after cochlear implantation in the deaf illustrate how patterns of brain function adapt over time. Limitations of the scanning techniques and prospects for the future are discussed in relation to new developments in the neuroimaging field.

Brain↗

[Skeletal development in monozygotic and dizygotic twins].

The maturation and development of 27 monozygotic and 23 dizygotic twins were studied over a 10 year period with data collected at one year intervals from age 9 to adulthood. In this manner the ossification process was recorded based on X-ray films of the carpal bones. Further information was acquired through anthropometric and somatoscopic data reproduced with standard photography. Sex and phase specific genetic factors influencing the maturation process are presented and analysed.

Bone Development↗

Effects of localized cutaneous vascular alteration on reactive hyperemia.

BACKGROUND: Cutaneous microcirculation shows a distinctive spatiotemporal inhomogeneity. Therefore provocation tests are necessary to receive significant and reproducible data. The present study investigated the effect of localized cutaneous vascular alteration, like in psoriasis vulgaris, on different parameters of reactive hyperemia (peak capillary blood cell velocity [pCBV], postocclusive reactive hyperemia [PRH%], and time to peak capillary blood cell velocity [tpCBV]). MATERIAL AND METHODS: Psoriatic plaques and normal skin on the contralateral side of 20 patients with psoriasis vulgaris were examined by Laser Doppler Anemometry. Capillary blood cell velocity was measured before suprasystolic occlusion and during postocclusive hyperemia. RESULTS: Compared to normal skin psoriatic plaques showed a significant increase of peak capillary blood cell velocity (pCBV, normal skin: 0.89+/-0.23 mm/s, psoriatic plaque: 2.03+/-0.94 mm/s), resting capillary blood cell velocity (rCBV, normal skin: 0.43+/-0.12 mm/s psoriatic plaque: 0.72+/-0.20 mm/s) and postocclusive reactive hyperemia (PRH%, normal skin: 107%, psoriatic plaque: 180%). The time to peak capillary blood cell velocity (tpCBV) during reactive hyperemia did not change significantly. CONCLUSION: In reactive hyperemia, changing of pCBV and PRH% combined with unaltered tCBV indicate a dysfunction of cutaneous microcirculation. In contrast acute closure of upper and lower arterial extremity show a changed tCBV with stable pCBV and PRH%.

Blood Flow Velocity↗

[Acoustic rhinometry: the bat principle of the nose].

All cross-sectional areas of the upper airway can be measured by an acoustic signal using the acoustic reflection technique, or acoustic rhinometry. The plane of the cross-sectional areas measured was determined in nasal models. The isotemporal layers were found to be nearly parallel to the nasal valve. The acoustically measured cross-sectional areas correlated with the cross-sectional areas of cuts from nasal models. After digitizing these cuts, a CAD software calculates cross-sectional areas in all orientations and at all distances. The difference between the measured and calculated cross-sectional areas is up to 3% in the nasal cavity and up to 17% in the nasopharynx. The hypothesis that the cross-sectional areas measured lie nearly parallel to the nasal valve was confirmed. The normal rhinometric curve shows the minimal cross-sectional area (I-notch) to lie at the nasal isthmus. The second narrowest segment of the nasal cavity lies at the head of the inferior concha and septal concha (C-notch). Characteristic examples of patients with turbinate hypertrophy, choanal atresia, enlarged adenoids, and septal deviations are presented. Acoustic rhinometric curves can only be interpreted in combination with the rhinoscopic findings because different pathological conditions can produce similar curves. Recording of reliable and reproducible data by acoustic rhinometry demands that the connection between the rhinometer and the nose does not distort the valve area. When we used two different nose pieces (1.2 and 1.5 cm outer diameter) the cross-sectional areas in the anterior third of the nose of only 28% of the patients was measured correctly.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoids↗

[Public health importance of ischemic heart disease].

The high cardiovascular morbidity and mortality makes good quality and reproducible data essential to obtain adequate conclusion. Data collected in order to be well financed are inadequate, special conducted surveys are mandatory. Even in absence of such surveys the high prevalence and high mortality data are widely accepted. Theoretically the total elimination of risk factors and the application of the four preventive drugs would bring very effective (75-90%) risk reduction. The excellent perspective dose not support the sparing with preventive efforts.

Humans↗

[Incidence of nosocomial rotavirus infections in a pediatric hospital over a 3-year period].

AIM: To estimate the incidence rate of hospital-acquired rotavirus infections (HRI) in a paediatric hospital in a 3-year period; the risk of HRI associated to age, sex, ward, season; the cost determined by HRI for the healthcare system. METHODS: Retrospective epidemiological study, carried out using hospital datasets, with the analysis of some clinical records. RESULTS: In the 2003-2005 period the HRI incidence rate was 1.9% (mean age 15.2 months, SD 11.8, range 1.5-49), with a decreasing trend over years. Children aged less than 8 months were at higher risk of HRI than others (RR 1.83, CI 95% 1.18-2.85) as well as were children admitted to a 0-18 months pediatric medicine ward (RR=2.84, CI 95% 1.78-4.5) and to an infectious diseases ward (RR=4.9, CI 95% 3.4-7). The incidence of HRI was higher in the winter than in the rest of the year (RR 1.59, CI 95% 1.07-2.36). The hospital stay of children with HRI was prolonged by a mean of 5.2 days compared to age, sex and comorbidity-matched controls. (P=0.02). CONCLUSIONS: The method used in this study is easily reproducible. Data obtained can be used as internal quality indicators and to evaluate the performance of different hospital wards in infection control. Infection prevention activities should involve mothers and families of hospitalized children, as they perform on them a great deal of basic care and hygiene activities which are at risk for hospital cross-infections.

Child↗

Comparison of fifteen production batches of house dust mite extract (Dermatophagoides pteronyssinus).

An important measurement of quality of allergenic extracts is the batch to batch uniformity. Using different methods of protein and immunobiochemical analysis, we could demonstrate, that 15 production batches of house dust mite Dermatophagoides pteronyssinus extract prepared during the course of 4 years from different lots of the source material, (purified whole mite bodies) showed reproducible data.

Allergens↗

Normal cutaneous sensibility of the face.

Normal values for facial sensibility were determined in 36 healthy subjects. Sensation was evaluated using static and moving two-point discrimination and pressure and vibratory threshold in four major regions of the head and neck. Each region was directly related to the area innervated by a particular peripheral sensory nerve (supraorbital, infraorbital, inferior alveolar-mental, or the great auricular nerve). Cutaneous sensibility varied from region to region but was consistent from one normal individual to another and in the same subject on different days. Measurements of pressure and vibratory thresholds provided the most reliable, reproducible data. A relationship of these data to the types of sensory receptors in facial skin and vermilion is postulated.

Adult↗

Video-based image collection for quantitative histopathology.

Obtaining histologic images for computer-based morphometric analysis is associated with a number of standardization problems, which must be solved if reproducible data collection is expected. These problems include tissue processing, sectioning and staining, standardizing and calibrating the video camera and determining the appropriate sampling rate (pixels/micron). Suggested solutions for these problems are presented for a specific image analysis system, but are applicable to other systems with similar capabilities. Biologic variability is not eliminated by computer-assisted analysis, so it is important to minimize data-collection artifacts by parallel processing of experimental and control material, as in other investigative work.

Animals↗

Immunochemical determination of lonidamine in rat tissues and blood serum.

Lonidamine (LND) is an antitumor drug which interferes selectively with the energy metabolism of neoplastic cell. Because of its physico-chemical properties, LND determination with conventional methods, i.e. high performance liquid chromatography and spectrofluorimetry, gives rise to several problems: LND is a lipotropic drug which becomes intimately associated with biological membranes so that it is impossible to extract all the drug bound, thus leading to an underestimation of the LND content in cells and tissues. These difficulties can be overcome by the immunoenzymatic method described here. The assay is simple, rapid, practical, highly sensitive (2-5 ng/ml) and particularly suitable for the analysis of multiple samples (twelve samples in triplicate for each plate). There is, moreover, a great improvement in data reproducibility.

Animals↗

Dose-response relationship in the treatment of gastrointestinal disorders.

Numerous clinical studies have been performed to establish efficacy and safety of drugs in gastroenterological disorders. Only in a few if any of these studies, however, the rationale for the optimal dose and the dose regimens, respectively, have been addressed. Adequate and well-controlled dose finding studies play a key role in the clinical assessment of new drugs and in the evaluation of new indications. Hereby the range from the minimal effective dose to the maximal effective and well tolerated dose can be assessed and thus the optimal dose-range and dosage regimen be determined. Meaningful pharmacodynamic studies can be performed in the gastrointestinal tract also in healthy volunteers provided that a method with a high predictability for the desired therapeutic effect is available such as measurement of gastric acid secretion and its inhibition by a drug. Dose finding studies in gastroenterology can be carried out under two main aspects: First, to assess the pharmacodynamic and therapeutic effect of a compound on the gastrointestinal tract (e.g. anti-ulcer drug). Second, to evaluate the side effects of a drug on the gastrointestinal tract (e.g. gastric mucosal damage by non-steroidal anti-inflammatory drugs). For the evaluation of new drugs in gastrointestinal therapy a number of methods are available which yield accurate and reproducible data. While careful clinical-pharmacological dose-response studies using these methods have been carried out already more than a decade ago, it is surprising that therapeutic dose finding studies have become available only during the past few years. For scientific as well as for ethical reasons more trials which determine the optimal therapeutic dose are warranted.

Clinical Trials as Topic↗

[Antitoxoplasma serum--derivation of the national standard (ELISA)].

When assessing the content of specific antibodies in serum there is tendency to abandon the specification expressed as dilution and use more reproducible data, i.e. international units. To this end it is necessary to derive from the international standard the national standard according to which positive control sera in diagnostic sets, produced by SEVAC can be described in international units. In pooled positive human serum recommended as the national standard and in the international standard first the parallel course of the dose/response curve was tested. Then both sera were repeatedly compared: into a close dilution series of the national standard one dilution of the international standard was included and in both sera the dilution with same response was found (by optic density) and thus the activity of the suggested national standard was assessed as 2400 units/ml. The author discusses also the recommended routine procedure for expressing the activity of the examined sera in international units.

Animals↗

Evaluation of capillary gas chromatography for multicomponent drug analysis.

Capillary gas chromatography is evaluated for multicomponent drug analysis. A 0.32 mm id X 30 m fused silica column and a 0.75 mm id X 30 m borosilicate glass column (both with OV-1 bonded phase) are investigated. Retention times (relative to caffeine) are presented for 39 drug components representing a variety of chemical classes and pharmacological activities. Reproducibility data are presented for both isothermal and programmed temperature runs on selected drug mixtures. Recovery data are provided for 2 multicomponent drug mixtures prepared to approximate over-the-counter antihistaminic and antitussive preparations.

Antitussive Agents↗

[Morphometric analysis of fibrocystic breast disease].

We used the automated microscopic image analysis (system "Robotron A 6471"; software AMBA/R) to study the ductal epithelium in biopsies from 88 patients with fibrocystic breast disease, including 52 with proliferative mastopathy. Significant differences were demonstrated using reproducible data and selective caryo- and histometric parameters which allowed the separation of ductal hyperplasia and ductal cell proliferation with atypia. In addition, multiparameter analysis indicated that cases with proliferative mastopathy III can be separated into two groups which have probably a different high risk with respect to carcinoma.

Adult↗

Flow analysis with digital subtraction angiography: 1. Description of a simplified flow model.

An inexpensive, simplified flow model for use with digital subtraction angiographic (DSA) equipment is described. System tests show that the model gives reproducible data with a standard deviation of +/- 2.6%. Flow analysis also reveals an appropriate response to flow rates of 20-240 ml per min. The flow model can be used to investigate many of the variables and sources of error in DSA flow measurements.

Angiography↗

[Criteria for assessing the reliability and information value of diagnostic data in neurological studies].

Clinical and experimental studies of the central nervous system diseases with electroencephalographic recording processed using statistical techniques are described. Criteria are offered for the evaluation of the data reproducibility, specificity and sensitivity with special reference to their use in outpatient and hospital practice of the long term follow-up.

Electroencephalography↗

Automated histometry in fibrocystic breast disease.

Ductal epithelial proliferations of the mammary gland in biopsy material from 101 patients, including 52 with proliferative fibrocystic disease (mastopathy), were quantitatively analyzed by means of the Robotron A 6471 system together with AMBA/R software. Based on reproducible data obtained for distinct karyometric and histometric features, significant differences were found to exist between epithelial proliferations without atypical hyperplasia (mastopathy II) and those with atypia (mastopathy III). The multiparameter analysis also produced some hints that cases of proliferative mastopathy III can be divided into two groups having different risks of developing carcinoma.

Cell Division↗