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Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells.

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non-cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum-deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.

3T3 Cells↗

Understanding the uneven spread of HIV within Africa: comparative study of biologic, behavioral, and contextual factors in rural populations in Tanzania and Zimbabwe.

BACKGROUND: Large differences in the spread of HIV have been observed within sub-Saharan Africa. GOAL: The goal was to identify factors that could explain differences in the spread of HIV within sub-Saharan African populations. STUDY DESIGN: Ecologic comparison of data from population-based surveys in high and relatively low HIV prevalence rural areas in Zimbabwe, Manicaland, and Tanzania, Kisesa. RESULTS: HIV prevalence in Manicaland and Kisesa was 15.4% and 5.3% in men aged 17-44 years and 21.1% and 8.0% in women aged 15-44 years (odds ratios, 3.3 and 3.1, respectively). Marriage is later, spatial mobility more common, cohabitation with marital partners less frequent, education levels are higher, and male circumcision is less common in Manicaland. However, adjustment for differences in these factors increased the odds ratios for HIV infection in Manicaland versus Kisesa to 6.9 and 4.8 for men and women, respectively. Sexually transmitted infection levels were similar, but syphilis was only common in Kisesa. Respondents in Kisesa started sex earlier and reported more sexual partners. Age differences between partners were similar in the 2 locations. CONCLUSION: Substantial differences exist between the contemporary sociodemographic profiles of rural Manicaland and Kisesa. However, these differences did not translate into measurable differences in the biologic or behavioral factors for which data were available and did not explain the much higher HIV prevalence found in Manicaland. These findings might reflect more extensive AIDS-selective mortality and behavior change or greater bias in reporting of sexual behavior in Zimbabwe.

Adolescent↗

Nonparametric 1-D temperature restoration in lossy media using Tikhonov regularization on sparse radiometry data.

Microwave thermometry has the potential to characterize thermal gradients in lossy materials down to a few centimeters depth. The problem of retrieving temperature profiles from sets of brightness temperatures is studied using Galerkin expansion of one-dimensional (1-D) temperature profiles combined with Tikhonov regularization and predefined boundary conditions. From a priori knowledge of the temperature field shape, smooth Chebyshev polynomials are used as basis functions in the series expansion. The proposed estimator does not require iterative calculations that are normally performed using conventional numerical methods for signal parameter estimation and is, thus, very fast. Noise effects versus bandwidth limitations (smoothness of solutions) are studied in terms of four performance indexes defined in the text. In general, statistical spread of the temperature estimator increases with increasing number of Chebyshev polynomials. Systematic deviation from true values (bias) decreases as the number of Chebyshev polynomials increases. Results show that smooth temperature profiles can be reproduced using 6-7 Chebyshev polynomials. With additional constraints such as boundary conditions and maxima localization, a three-frequency-band radiometric scan is sufficient to produce acceptable results in regions with low thermal gradients. As the spatial variability of the 1-D temperature profile increases, more radiometric bands (5-6) are required to provide nonbiased estimates.

Algorithms↗

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis.

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.

Animals↗

Continuity-based and discontinuity-based segmentation in transparent and spatially segregated global motion.

The mechanisms underlying the parsing of a spatial distribution of velocity vectors into two adjacent (spatially segregated) or overlapping (transparent) motion surfaces were examined using random dot kinematograms. Parsing might occur using either of two principles. Surfaces might be defined on the basis of similarity of motion vectors and then sharp perceptual boundaries drawn between different surfaces (continuity-based segmentation). Alternatively, detection of a high gradient of direction or speed separating the motion surfaces might drive the process (discontinuity-based segmentation). To establish which method is used, we examined the effect of blurring the motion direction gradient. In the case of a sharp direction gradient, each dot had one of two directions differing by 135 degrees. With a shallow gradient, most dots had one of two directions but the directions of the remainder spanned the range between one motion-defined surface and the other. In the spatial segregation case the gradient defined a central boundary separating two regions. In the transparent version the dots were randomly positioned. In both cases all dots moved with the same speed and existed for only two frames before being randomly replaced. The ability of observers to parse the motion distribution was measured in terms of their ability to discriminate the direction of one of the two surfaces. Performance was hardly affected by spreading the gradient over at least 25% of the dots (corresponding to a 1 degrees strip in the segregation case). We conclude that detection of sharp velocity gradients is not necessary for distinguishing different motion surfaces.

Discrimination, Psychological↗

Cellular epigenetics: topochronology of progressive "spontaneous" transformation of cells under growth constraint.

Early passages of NIH 3T3 cells yield about 10 transformed foci for every 10(5) cells seeded after the cells multiply to confluence in a standardized 2-week assay. The question arose whether more cells would give rise to foci if given more time for their development. This question could not be answered simply by extending the incubation period, since the original foci spread to cover much of the area of the culture dish. Transformed cells can also detach into the medium from the original foci to initiate new foci by reattaching at a distance. These problems were averted by growing cells in multiwell plates which in effect simulated partitioned culture dishes. All the wells in a given plate were assayed for focus formation at successive intervals up to 14 weeks. The results indicated the spatial pattern and sequence of transformation on different parts of the "partitioned" dish. The number of multiwells containing focus-forming cells increased steadily with time, indicating that all parts of a dish eventually undergo transformation. Also, most of the transformations were recorded long after confluence in the multiwells was reached. Hence such a transformation is much more likely to occur in the nondividing state rather than in the dividing state of the cells and is thus inconsistent with a mutational basis. The results suggest that "spontaneous" transformation is a population-wide, epigenetic phenomenon. This agrees with the results from clonal analysis and other studies and is well described by the concept of progressive state selection, in which "spontaneous" transformation represents a heterogeneous, adaptive response of competent cells to moderate constraints on cell growth.

3T3 Cells↗

Meningococcal disease and influenza-like syndrome: a new approach to an old question.

The temporal and spatial association of meningococcal disease and influenza-like syndrome (ILS) was assessed from surveillance data on these diseases in France for a 6-year period (1985-1990). Using time series methods to account for the usual seasonal pattern meningococcal disease, the incidence of meningococcal disease in a given week was found to be linked to incidence of ILS in the 5 previous weeks but not to that in subsequent weeks. Geographic spread of meningococcal disease correlated with spread of ILS. This study also suggests that meningococcal disease is more severe for a 2-month period during and after an ILS epidemic: The proportion of cases with purpura fulminans increased by 24% and those resulting in death by 26% during this period. No shift in the age distribution was observed. When an ILS epidemic is identified, medical practitioners should be informed of the likelihood of an increased incidence and severity of meningococcal disease.

Adolescent↗

Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro.

Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.

Adolescent↗

Spatial distribution of the DF508 mutation in cystic fibrosis: a review.

The recent finding of the most common mutation (DF508) in cystic fibrosis in white populations has led to the publication of numerous data regarding its distribution and frequency. A review of the available data shows that there is a gradient in both the incidence of cystic fibrosis and the frequency of the DF508 mutation in Europe, the highest values being found in western Europe. It is postulated that the DF508 mutation was present in a western European population before the arrival of the Indo-Europeans from the Middle East; the mutation then spread into these migrating populations.

Cystic Fibrosis↗

Intratumoral DNA stem-line heterogeneity in superficial spreading melanoma.

BACKGROUND: In primary melanomas, data on the degree of intratumoral heterogeneity to date have been lacking. OBJECTIVE: Our purpose was to investigate intratumoral DNA stem-line heterogeneity in superficial spreading melanoma (SSM). METHODS: Multiple measuring fields of 54 SSMs (tumor thickness median 1.60 mm) were studied by DNA image cytometry to obtain data on the number of DNA stem lines per tumor, their ploidy characteristics, and intratumoral distribution. Results were compared with standard histopathological criteria. RESULTS: Twenty-three of 54 SSMs were found to have two or three distinct proliferating tumor cell stem lines (1.46 +/- 0.57 per tumor). Stem lines appeared spatially separated in 22 of 23 SMMs. At least 3 measuring fields per tumor were necessary to identify all stem lines with a likelihood of 95%. DNA heterogeneity correlated with tumor thickness, but occurred in 5 of 19 cases of pT1 melanoma. CONCLUSIONS: Primary SSMs can be regarded as potentially clonally unstable with a tendency for spatial separation of tumor cell stem lines.

Aged↗

Habitat-related prevalence of macroscopic Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula), Hohonu Range, Westland, New Zealand.

AIM: To identify broadscale habitat factors influencing the prevalence of macroscopic Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) at a site in Westland, New Zealand. METHODS: During 1973/74, 1989/90 and 1997, we undertook repeated cross-sectional surveys of M. bovis infection in a possum population on the Hohonu Range , Westland. Data were analysed to determine the influence of site-specific habitat characteristics (land form, aspect, slope, altitude), distance from forest-pasture margin and time since infection on the spatial and temporal prevalence of macroscopic M. bovis infection. RESULTS: The prevalence of M. bovis infection was highest in 1973/74 (13.4%), compared with 1989/90 (3.1%) and 1997 (9.4%). The prevalence of macroscopic M. bovis infection was significantly influenced by habitat, as indexed by altitude and slope in this study site. Every 100 m increase in elevation was associated with a 29% decrease in the odds of infection, and every 10 degrees increase in slope was associated with a 20% decrease in the odds of infection. For possums caught in the lowland podocarp forest (altitude 100-200 m, average slope=5.7 degrees ), the odds of infection were nearly 30-fold higher than those for possums caught in high-altitude hardwood forest near the tree line (altitude 900-1000 m, average slope=28 degrees ). Whilst the prevalence of disease fluctuated markedly between surveys, its broadscale spatial distribution changed little over time. Proximity to the forest-pasture margin had no significant influence on the prevalence of disease, once the effect of habitat was taken into account. CONCLUSION: The prevalence of macroscopic M. bovis infection in possums was strongly influenced by habitat type, being highest in habitats that supported the highest density of possums, and lowest in habitats where population density was low. There was no evidence of progressive spread of M. bovis infection in possums into forest away from pasture-forest margins over the 24-year period of these surveys.

Journal Article↗

Three-dimensional point spread function measurement of cone-beam computed tomography system by iterative edge-blurring algorithm.

With separability assumed, we decompose a three-dimensional point spread function (3D PSF) into two-dimensional (2D) PSFs and further into one-dimensional (ID) PSFs. Based on the observation of the location invariance of a step edge under convolution, we propose a rectification procedure to automatically establish the step-edge function from a blurred edge profile. The ID PSF is modelled as a single-parameter Gaussian function, which is determined by iteratively blurring a step-edge function into a spread edge profile. A plastic solid ball (diameter approximately 6 mm) is used to provide double-edged rectangular functions along scanlines passing through the ball centre, and correspondingly, the reconstructed digital volume provides the blurred rectangular profiles. Experimenting with a cone-beam computed tomography system, we demonstrate the iterative edge-blurring algorithm for PSF measurement. By repositioning the ball phantom in the object support space, we measure the system's spatial variance in terms of full-width-at-half-maximum (FWHM) of the local PSFs. Specifically, we obtained the FWHMs for three specific locations at (0, 0, -40 mm), (0, 0, 0) and (0, 0, 40 mm), which are given by 0.92 +/- 0.10 mm, 0.65 +/- 0.08 mm and 0.93 +/- 0.10 mm, respectively.

Algorithms↗

The progression and topographic distribution of interleukin-1beta expression after permanent middle cerebral artery occlusion in the rat.

The cytokine interleukin-1 (IL-1) has been implicated in the exacerbation of ischemic damage in the brains of rodents. This study has ascertained the cellular localization and chronologic and topographic distribution of pro/mature interleukin-1beta (IL-1beta) protein 0.5, 1, 2, 6, 24, and 48 hours after ischemia by subjecting rats to permanent unilateral occlusion of the middle cerebral artery. Interleukin-1beta was localized immunocytochemically in vibratome sections of perfusion-fixed brains. The cells that expressed IL-1beta had the morphologic features of microglia and macrophages. Interleukin-1beta was first detected 1 hour after occlusion in ipsilateral meningeal macrophage-like cells. By 6 hours, pro/mature IL-1beta-immunoreactive (IL-1(beta)ir) putative microglia were present in the ischemic cerebral cortex, corpus callosum, caudoputamen, and surrounding tissue. By 24 and 48 hours after ischemia, the number and spread of IL-1(beta)ir cells increased greatly, including those resembling activated microglia and macrophages, as the core of the infarct became infiltrated. Interleukin-1(beta)ir cells also were present in apparently undamaged tissue, adjacent to the lesion ipsilaterally, and contralaterally in the cerebral cortex, dorsal corpus callosum, dorsal caudoputamen, and hippocampus. These results support the functional role of IL-1 in ischemic brain damage and reveal a distinct temporal and spatial expression of IL-1beta protein in cells believed to be microglia and macrophages.

Animals↗

X-ray computed tomography in the presence of arbitrary symmetrical focal spot intensity distributions.

A loss of resolution in tomographic images is due to the well-known bimodal spatial variations in photon emission across the x-ray focal spot combined with the finite width of the x-ray detector. Such departures from the ideal infinitely narrow beams which are usually considered can be taken into account by replacing the customary line integrals by suitably chosen integrals over strips. The image degradation resulting from using those strip integrals as ingredients in conventional reconstruction formulas is expressible as a complicated point spread function, which can always be computed in any given situation. Restoring the reconstructed image to provide higher resolution is a nontrivial operation, which however greatly benefits from experience already gained in dealing with optical imagery degraded by the aberrations of astigmatism and curvature of field. Typical point spread functions are shown for a simple bimodal source intensity distribution of primordial importance.

Technology, Radiologic↗

[Architecture and epidemiology in gynaecology and obstetrics (author's transl)].

The authors, who are doctors, architects and hospital engineers, made every effort to work out the architectural rules that were imposed on them at one and the same time by the epidemiology, the humanization of the work and spread in a specialty that is becoming more and more specialised and most attractive; having consultations between the disciplines take place, as they have done when a new isolated building for the department of gynaecology and obstetrics was being planned. An original study was carried out using information about the flow rates of people and the coefficients of interest in the siting of the various departments on small models, and this allowed a spatial tridimensional programme to be worked out so that the structure could become something like an ideal building in which the different needs of the functional element, the human element, the preventive epidemiological element and also the economic and budgetary possibilities could come together. The thoughts and suggestions of the authors concerned with this building should help to develop new concepts in hospital architecture which are of interest for gynaecologists and obstetricians.

Architecture↗

Ranging patterns of a troop of baboons (Papio anubis) in Kenya.

A troop of olive baboons, Papio anubis, was studied for 11 months in an open-country habitat near Gilgil, Kenya. Detailed mapping of troop movements was undertaken and provided a precise picture of range usage, showing that a small part of the range was used intensively. Gradations of home range use are described quantitatively. Tendencies to spread out and coalesce in different parts of the range are investigated. Shifting ranging patterns are examined together with meteorological factors, seasonal food supplies and water availability.

Age Factors↗

Serial magnetic resonance diffusion and hemodynamic imaging in a neonatal rabbit model of hypoxic-ischemic encephalopathy.

Dynamic changes in relative cerebral blood volume (rCBV) and apparent diffusion coefficient (ADC) were investigated, using high speed magnetic resonance imaging (MRI) in an acute neonatal rabbit model of hypoxic-ischemic encephalopathy (HIE). Serial rCBV imaging used a magnetic susceptibility blood pool contrast agent. Interleaved ADC and rCBV images were acquired with 9 s temporal resolution. Rabbits received unilateral common carotid artery (CCA) ligation followed by hypoxia. rCBV increased bilaterally within 1-2 min after the onset of hypoxia. A biphasic ADC decline was observed: a slowly declining phase (84 +/- 18% of baseline) followed by a rapid, focal drop to 55 +/- 8% of baseline in the ipsilateral cortex, which was paralleled by a rapid focal rCBV drop to 70 +/- 17% of baseline. ADC decline generally began in a small region of ipsilateral cortex and spread over the ipsilateral cortex, ipsilateral subcortical tissue and contralateral cortex. The initial ADC drop usually preceded the initial rCBV drop by approximately 60 s, however at later timepoints rCBV decline sometimes preceded ADC decline. Upon normoxia, rCBV recovered to about baseline values while ADC recovered to baseline or above. This method provides a sensitive means of non-invasively visualizing acute hemodynamic- and metabolic-related changes in HIE with good temporal and spatial resolution.

Animals↗

Propagation of excitation in idealized anisotropic two-dimensional tissue.

This paper reports on a simulation of propagation for anisotropic two-dimensional cardiac tissue. The tissue structure assumed was that of a Hodgin-Huxley membrane separating inside and outside anisotropic media, obeying Ohm's law in each case. Membrane current was found by an integral expression involving partial spatial derivatives of Vm weighted by a function of distance. Numerical solutions for transmembrane voltage as a function of time following excitation at a single central site were computed using an algorithm that examined only the portion of the tissue undergoing excitation at each moment; thereby, the number of calculations required was reduced to a large but achievable number. Results are shown for several combinations of the four conductivity values: With isotropic tissue, excitation spread in circles, as expected. With tissue having nominally normal ventricular conductivities, excitation spread in patterns close to ellipses. With reciprocal conductivities, isochrones approximated a diamond shape, and were in conflict with the theoretical predictions of Muler and Markin; the time constant of the foot of the action potentials, as computed, varied between sites along axes as compared with sites along the diagonals, even though membrane properties were identical everywhere. Velocity of propagation changed for several milliseconds following the stimulus. Patterns that would have been expected from well-known studies in one dimension did not always occur in two dimensions, with the magnitude of the difference varying from nil for isotropic conductivities to quite large for reciprocal conductivities.

Animals↗