Biomedical subjects
Publications and source records attributed to P Kucera.
This study is based on the in-vitro analysis of the normal and pathological colon smooth muscle contraction. Two different methods, supported by computer analysis, are presented. This approach will probably help the physician and the surgeon to better understand the physiopathology of some characteristical diseases, such as Hirschsprung's disease.
The penetration of the CVS strain of rabies virus and its avirulent derivative AvO1 into peripheral neurons was investigated after intramuscular inoculation into the forelimbs of adult mice. It was found that CVS directly penetrates both the sensitive and motor routes with equal efficiency, without prior multiplication in muscle cells. Infected neurons became detectable 18 h after infection. The second cycle of infection occurred within 2 days, and at day 3 there was a massive invasion of the spinal cord and sensory ganglia. In sensory ganglia, where it was possible to identify cell outlines, it was evident that the infection did not proceed directly from cell body to cell body. The avirulent strain AvO1 penetrated motor and sensory neurons with the same efficiency as CVS. Restriction of viral propagation was observed from the second and third cycles onwards. No further development of the infection could be seen after day 3, and by that time the lysis of primarily infected neurons seemed to occur.
Teratogenic and toxic effects of DXM and DPH were tested using a standardized chick embryo culture. Survival, growth and malformations were scored with respect to the drug concentrations used. DXM (greater than 10(-8) mol/l) inhibited the differentiation of the extraembryonic blood circulation and induced craniofacial anomalies. DPH (greater than 1.5 10(-5) mol/l) induced cardiomegaly, craniofacial and somitic anomalies. Both drugs were lethal at 10(-3) mol/l. Comparison of results obtained with 8 drugs shows that the method has a good discriminative power and specificity and that it can be used as a simple, reliable and economical primary screening test, making it possible to reduce the use of animals in toxicological studies.
Distribution of myosin, tubulin and laminin immunoreactive cells in the area opaca of the young chick embryo (Stages 4-8 HH) was studied using immunofluorescence technique. For the three markers, the number of stained cells increased with the age of the blastoderm. Cells stained for tubulin and laminin, were distributed throughout the area opaca, showing no supracellular organization. On the contrary, the cells stained for myosin became organized in a ring surrounding the area pellucida. This pattern appeared at the stage 6. Such an heterogenous distribution of the markers suggests a functional diversification of the ectodermal cell monolayer forming at these early developmental stages the area opaca. This idea is also supported by the results of autoradiography for tritiated thymidin which showed that the edge cells did not synthetize DNA and consequently did not divide.
The carbon dioxide production of the chick embryo cultured in vitro has been determined during the first 24 h of post-laying development using a non-invasive conductometric microtechnique. The mean CO2 production of the whole blastoderm (1) increased from 16 nmol/h at laying to 231 nmol/h at early neurulation, (2) became dependent on exogenous glucose and (3) was closely linked to mechanical tension generated in the blastoderm (loosening from vitelline membrane resulted in a decrease of 56%). In our experimental conditions, no significant influence of carbonic anhydrase on the CO2 production has been detected. The value of the respiratory exchange ratio varied from about 3 at pregastrular stages to 1 at neurula stage and CO2 was produced transiently in presence of antimycin A. Such results indicate that the source of CO2 is not exclusively mitochondrial and that the relative proportions of mitochondrial and non-mitochondrial CO2 productions might vary significantly throughout the early development. Our findings confirm that the metabolism of the chick embryo becomes more and more oxidative from laying onwards and suggest that the modifications of metabolism observed during the studied period of development could be associated with functional differentiation.
This multi-center trial compared two doses of parenterally administered interferon alpha-n1 (Wellferon) in men and women with recurrent/resistant genital warts. Patients received either 1 or 3 MU/m2 daily for 14 days, then 3 times weekly for 4 weeks; non-responders could receive an additional four weeks of treatment. A total of 107 patients were enrolled, and 102 were evaluable after six weeks of study. The principal dose comparison was in 57 women assigned alternately to the two doses. Median lesion measurements were reduced significantly from baseline at weeks 2, 4 and 6 in both groups. Statistical analysis showed no difference in response to 1 versus 3 MU/m2. The overall complete response (CR) plus partial response (PR) rate at week 6 was 69% for the two doses. Two additional groups of 21 women and 24 men were treated at the higher dose with CR plus PR rates of 75 and 50%, respectively. Week 10 disease evaluations for all groups showed 19 of 77 patients to be completely cleared. Of these 19, only one had recurrent disease at the end of the 6-month study period. Analysis of the incidence of symptomatic side effects showed a significantly higher frequency among women treated with 3 MU/m2 than among women treated with 1 MU/m2. Five dose reductions and two withdrawals for toxicity occurred, all in the high dose group. This study demonstrates that parenterally administered Wellferon produces clearance of resistant genital warts in many patients, and that rates of clearance do not appear to vary between groups receiving moderate or low dose therapy.
An in vitro culture of chicken embryo is described: the embryos at the stage of gastrulation are explanted from eggs into transparent silicone chambers where they continue to develop normally under controlled conditions for additional 3 d. This period corresponds to 2-5 wk of postconceptional age in the human embryo. In both the chick and man, this period is very sensitive to physicochemical perturbations which can lead to surviving malformations. Six chemical agents were tested with this culture: methotrexate, cadmium chloride, caffeine, phenobarbital, aspirin, and saccharin. Survival scores, growth perturbations, and early signs of anomalies of the nervous, skeletomotor, and cardiovascular systems were analyzed with respect to the used concentrations. The dose-response curves were obtained with good precision and allowed a discrimination between the teratogenetic and unspecific toxic effects and a comparison of the toxic potency of the six drugs. The evaluation of one drug took, roughly, 3 wk, one technician, and about 150 eggs. The advantages (simplicity, rapidity, reproducibility, specificity, economy, no suffering, and no use of mature animals) and disadvantages (nonmammalian species, absence of detoxicating organs) of the method are discussed. The method is proposed as a routine teratogenicity and embryotoxicity test which allows primary screening of many compounds and which can thus substantially reduce the ultimate experiments that use pregnant mammalian females.
A conductometric micromethod combined with image analysis system has been developed allowing to determine the CO2 production within 'two-dimensional' tissues, i.e., flat and thin cell layers or epithelial sheets. The preparation was mounted into an airtight chamber separated in two compartments by a thin silicone membrane permeable to gases. The lower compartment contained the nutritive medium and the preparation. The upper compartment and a conductivity measuring capillary connected in series were perfused with a solution of Ba(OH)2. The CO2 produced by the tissue precipitated as BaCO3 and the resulting decrease of electrical conductivity was linearly related to the total CO2 production. In addition, the pattern of CO2 production was directly observable as the BaCO3 crystals formed upon the silicone membrane over the regions which produced CO2. The spatial distribution of the crystals was quantified by video image processing and the regional CO2 production evaluated with a spatial resolution of 100 microns. This new microtechnique was originally developed to study the CO2 production in the early chick blastoderm which is a disc 1-5 cells thick. At the stage of young neurula the CO2 production was found to be 235 +/- 37 nmol.h-1 (mean +/- SD; n = 10) per blastoderm and large variations of local CO2 production were detected from one region to another (from 0.6 to 6.5 nmol.h-1.mm-2). These results indicate a high metabolic and functional differentiation of cells within the blastoderm. The possible applications and improvements of such a microtechnique are discussed.
The mechanical behaviour of ectodermal cells in the area opaca and the supracellular organization of fibronectin in the adjacent extracellular matrix were studied in whole chick blastoderms developing in vitro. The pattern of spontaneous mechanical activity and its modification by immunoglobulins against fibronectin were determined using a real-time image-analysis system. The pattern of fibronectin was studied using immunocytochemical techniques. It was found that the ectodermal cells in the area opaca actively develop a radially oriented contraction, which leads to a distension of the area pellucida from which the embryo develops. Abnormally increased tension resulted in perturbations of gastrulation and neurulation. An optimized mechanical equilibrium within the blastoderm seems to be necessary for normal development. Anti-fibronectin antibodies applied to the basal side of the blastoderm led rapidly and reversibly to an increase of tension in the contracted cells. This observation indicates that modifications of the extracellular matrix can be transmitted to cytoskeletal elements within adjacent cells. The extracellular matrix of the area opaca contains fibronectin arranged in radially oriented fibrils. This orientation corresponds to the direction of migration of the mesodermal cells. Interestingly, the radial pattern of fibronectin is found in the regions where the ectodermal cells are contracted and develop radially oriented forces. This observation suggests that the supracellular assembly of the extracellular materials could be influenced by the mechanical activity of adjacent cells. Possible modulations of the supracellular organization of extracellular matrix by other factors, e.g. diffusible metabolites, is also discussed. The presence of characteristically organized extracellular matrix components, of spatially differentiated cell activities and of reciprocal interactions between them makes the young chick blastoderm an excellent system for physiological studies of the coordinated cellular activities that lead to changes in form, complexity and function.
Oxygen uptake was studied during the establishment of cephalocaudal polarity in the very early chick embryo, i.e., 10 hr before (stage VI) and at laying (stage X). Oxygen fluxes in minute regions of the intact blastoderms were measured in vitro by scanning microspectrophotometry in the presence or absence of glucose. The oxygen consumption of the whole blastoderm remained constant (6 nmol O2 X hr-1) throughout the period studied, although the number of cells increased more than twofold. The regional oxygen fluxes varied from 0.41 to 1.13 nmol O2 X hr-1 X mm-2 at stage VI and from 0.42 to 0.70 nmol O2 X hr-1 X mm-2 at stage X. At stage VI, the oxygen flux in the center of the blastoderm was significantly higher than that in its periphery. This pattern remained evident when the values were corrected for cell number or for cytoplasmic volume. At stage X, there was a tendency for the oxygen fluxes to decrease from the posterior to the anterior regions of the area pellucida. Thus the pattern of oxidative metabolism in the late uterine embryos seems to change from radial to bilateral. This change of symmetry probably reflects the process of formation of the embryonic axis. In addition, the fact that the oxygen uptake was similar in the presence or absence of glucose suggests that early chick embryos metabolize essentially intracellular stores.
The mechanical activity of embryonic cells was studied in vitro in whole gastrulating chick blastoderms by means of video-image analysis. Displacements of identified points were recorded and interpreted in terms of deformation and active movement. From the deformations of the vitelline membrane to which blastoderms attach by the edge cells, it was deduced that the blastoderms developed and maintained a tension of 1-1.2 X 10(4) dyn/cm2. This tension was generated by a radial contraction of cells situated in the area opaca. This contraction stretched the area pellucida where the embryogenesis took place. Additional experimentally induced distension of the vitelline membrane affected the migration of the edge cells and the formation of the primitive streak. Decrease in temperature, anoxia, colchicine, and cytochalasin B relaxed the tension in the membrane but resulted in different deformation patterns in the blastoderm. A focalized electrical stimulation led to local contractions of cells in all regions of the blastoderm except the edge cells. The tension within the contracted cells increased up to 4 X 10(4) dyn/cm2. In the area pellucida, movements of migrating mesoblastic cells showed periodic saccades lasting about 5 min. Repetitive electrical stimuli applied at the same period induced ectopic migration of cells in the area pellucida but not in the area opaca. These results are discussed with respect to factors possibly modulating the activity of embryonic cells during early morphogenesis.
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Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.
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Explore the source record for details and available documents.
In monkeys with a unilateral subtotal lesion of the bulbar pyramid, a partial recovery of manual dexterity occurred within 4-6 weeks. This recovery may have been due to sprouting of fibers from the intact pyramid into the partly denervated areas of the spinal gray matter. In order to test this hypothesis, small injections of radioamino acids were made in the precentral hand area of two experimental animals (3 and 18 months after pyramidotomy) and one normal control animal. The pattern of spinal labeling was analyzed in autoradiographs from the cervicothoracic segments. No statistically significant additional labeling was found in laminae VI-VIII where the majority of the ipsilateral corticospinal fibers terminate. However, in the monkey with a long survival time, many large perikarya, situated exclusively within lamina IX in segments C8 and T1, were labeled with aggregations of silver grains. On the basis of the position and size of these neurons we conclude that a postsynaptic labeling occurred selectively in those motoneurons which control hand movements and which were deprived from their contralateral pyramidal control. We tentatively suggest that such a labeling may reflect an influence of the (injected) ipsilateral cortex on these distal motoneurons via direct connections which are normally not observed in the intact monkey.
14C-2-deoxyglucose (DG), currently employed in in vivo studies of brain glucose metabolism, has been used for determination of glucose consumption in the in vitro developing chick embryo. DG, presented in traces, accumulates in the embryo in proportion with incubation time. Analysis of tissue homogenates shows that the accumulated radioactivity is due to both phosphorylated (DGP) and nonphosphorylated DG. As it is only the radioactivity originating from the DGP that is proportional to glucose utilization, the nonphosphorylated DG must be washed out. The washout shows two distinct kinetics: a fast one corresponding to DG that has entered the cells but has not yet been phosphorylated and a slow one that is probably due to a dephosphorylated DGP coming from a different cellular compartment. On the basis of these results the optimal experimental conditions have been defined, allowing quantitative studies of glucose metabolism during the first day of development of the chicken embryo. From 18 to 24 hr of incubation (end of gastrulation), total glucose consumption increases from 50 nmol X h-1 at stage 3-4 to 90 nmol X h-1 at stage 6-7. This increase mainly reflects the growth of the blastodisc. Comparison with the values of O2 uptake measured at the same period of development suggests that only a fraction of the glucose consumed is oxidized, the major part being converted aerobically to lactate.