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Biomedical subjects

S E Webb

Publications and source records attributed to S E Webb.

At least 19 recordsLinked to original sources
PubMed2006

Nuclear translocation of the calcium-binding protein ALG-2 induced by the RNA-binding protein RBM22.

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.

Active Transport, Cell Nucleus↗
PubMed2002

Energy and fat intake in obese and lean children at varying risk of obesity.

OBJECTIVE: This study compared lean children at high risk (HR) and low risk (LR) of obesity and obese children (OB) to assess the relationship between their energy (EI) and fat intake and adiposity. DESIGN: Cross-sectional study of energy and fat intake in children, using 7-day weighed intakes validated by doubly labelled water (DLW) energy expenditure. SUBJECTS: A total of 114 pre-pubertal children, 50 HR (mean+/-s.d., 6.7+/-0.6 y, 25.7+/-4.8 kg, 21.3+/-6.6% body fat), 50 LR (mean+/-s.d., 6.6+/-0.8 y, 23.6+/-3.7 kg, 18.9+/-5.7% body fat) and 14 OB (mean+/-s.d., 6.8+/-1.0 y, 37.7+/-5.3 kg, 34.8+/-5.6% body fat). MEASUREMENTS: Body fatness was measured using deuterium dilution, total energy expenditure (TEE) by DLW and dietary intake using 7-day weighed records. RESULTS: EI was 98% of TEE in LR children, 95% in HR children and 86% in OB children. Although EI was similar in each group (LR, 7.03+/-1.26 MJ/day; HR, 7.30+/-1.46 MJ/day; OB, 7.55+/-1.67 MJ/day), obese +/-4.6%; P<0.05). There was a significant linear trend towards increasing fat intake (percentage energy) with increasing risk of obesity (P<0.05). While HR children were heavier and fatter than LR children (P<0.05), their EI and fat intake were not significantly greater (HR, 73+/-17 g, 37.3+/-4.4%). Dietary fat intake (percentage energy) was weakly but significantly related to body fatness (r(2)=0.05, P=0.02) by step-wise regression. Since energy from fat was the only macronutrient that was a significant predictor of body fatness, results were therefore analysed using quartiles of fat intake (percentage energy) as cut-offs. When grouped in this way children with the lowest intakes were leaner than those with the highest intakes (19.5+/-7.5 vs 24.9+/-9.4% body fatness; P<0.05). There was a significant trend for increasing fatness as fat intake increased (P<0.05). CONCLUSION: Fat intake is related to body fatness in childhood.

Anthropometry↗
PubMed2001

Application of the stretched exponential function to fluorescence lifetime imaging.

Conventional analyses of fluorescence lifetime measurements resolve the fluorescence decay profile in terms of discrete exponential components with distinct lifetimes. In complex, heterogeneous biological samples such as tissue, multi-exponential decay functions can appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, but the assumption of multiple discrete components is essentially arbitrary and is often erroneous. Moreover, interactions, both between fluorophores and with their environment, can result in complex fluorescence decay profiles that represent a continuous distribution of lifetimes. Such continuous distributions have been reported for tryptophan, which is one of the main fluorophores in tissue. This situation is better represented by the stretched-exponential function (StrEF). In this work, we have applied, for the first time to our knowledge, the StrEF to time-domain whole-field fluorescence lifetime imaging (FLIM), yielding both excellent tissue contrast and goodness of fit using data from rat tissue. We note that for many biological samples for which there is no a priori knowledge of multiple discrete exponential fluorescence decay profiles, the StrEF is likely to provide a truer representation of the underlying fluorescence dynamics. Furthermore, fitting to a StrEF significantly decreases the required processing time, compared with a multi-exponential component fit and typically provides improved contrast and signal/noise in the resulting FLIM images. In addition, the stretched-exponential decay model can provide a direct measure of the heterogeneity of the sample, and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.

Animals↗
PubMed2001

Time-domain whole-field fluorescence lifetime imaging with optical sectioning.

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.

Animals↗
PubMed2000

Calcium signalling during zebrafish embryonic development.

Calcium signals appear throughout the first 24 hours of zebrafish development. These begin at egg activation, then continue to be generated throughout the subsequent zygote, cleavage, blastula, gastrula, and segmentation periods. They are thus associated with the major phases of pattern formation: cell proliferation, cell differentiation, axis determination, the generation of primary germ layers, the emergence of rudimentary organ systems, and therefore the establishment of the basic vertebrate body plan. When signals need to be transmitted across significant distances they take the form of waves, either intracellular waves when the cell size is large, or later in development when the cell size is reduced, intercellular waves. We will consider both types of calcium signals and their integration into signalling networks, and discuss their possible functions and developmental significance with regard to pattern formation. BioEssays 22:113-123, 2000.

Animals↗
PubMed2000

On the mechanism of ooplasmic segregation in single-cell zebrafish embryos.

It has been previously shown that localized elevations of free cytosolic calcium are associated with a morphological contraction in the forming blastodisc and animal hemisphere cortex during ooplasmic segregation in zebrafish zygotes. It was subsequently proposed, in a hypothetical model, that these calcium transients might be linked to the contraction of a cortically located actin microfilament network as a potential driving force for segregation. Here, by labeling single-cell embryos during the major phase of segregation with rhodamine-phalloidin, direct evidence is presented to indicate that the surface contraction was generated by an actin-based cortical network. Furthermore, while zygotes incubated with colchicine underwent normal ooplasmic segregation, those incubated with cytochalasin B did not generate a constriction band or segregate to form a blastodisc. During segregation at the single-cell stage, ooplasm simultaneously moved in two directions: toward the blastodisc within the so-called axial streamers, and toward the vegetal pole in the peripheral ooplasm. The velocities of both axial and peripheral streaming movements are reported. By injection of a fluorescein isothiocyanate (FITC)-labeled 2000 kDa dextran into the peripheral ooplasm it was demonstrated that a portion of it feeds into the bases of the extending streamers, which helps to explain the lack of accumulation of ooplasm at the vegetal pole. These new data were incorporated into the original model to link the bipolar ooplasmic movements with the calcium-modulated, actin-mediated contraction of the animal hemisphere cortex as a means of establishing and driving ooplasmic segregation in zebrafish.

Actins↗
PubMed2000

Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos.

Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.

Aequorin↗
PubMed1999

A wave of free cytosolic calcium traverses zebrafish eggs on activation.

The activation process in a variety of deuterostome and protostome eggs is accompanied by cytosolic calcium transients that usually take the form of either a single or multiple propagating waves. Here we report that the eggs of zebrafish (Danio rerio) are no exception in that they generate a single activation wave that traverses the egg at a velocity of around 9 microm/s. There appears, however, to be no difference between the calcium-mediated activation response of eggs with regard to the presence or absence of sperm in the spawning medium. This leads us to suggest that these eggs are normally activated when they come in contact with their spawning medium and are then subsequently fertilized. The aspermic wave is initiated at the animal pole in the region of the micropyle, appears to propagate mainly through the yolk-free egg cortex, and then terminates at the vegetal pole. As neither sperm nor external calcium is required for the initiation (or propagation) of the activation wave, this suggests that an alternative wave trigger must be involved.

Animals↗
PubMed1999

Hepatocyte growth factor stimulates chemotactic response in mouse embryonic limb myogenic cells in vitro.

In this study we investigate the influence of Hepatocyte Growth Factor (HGF) on the motility of embryonic forelimb myoblasts. Using Blindwell chemotactic chambers, it was found that HGF at concentrations of 1-50 ng/ml dramatically enhanced the ability of myogenic cells to migrate. This stimulatory effect was elicited in a dose-dependent fashion and the effect was reversed with the addition of HGF neutralizing antibodies. A checkerboard analysis was performed and it revealed that HGF's effect on limb myoblast motility was through both chemokinesis and chemotaxis. HGF was also examined for its ability to stimulate myogenic cell proliferation, using MF20 antibody as the myogenic marker. At all concentrations tested, HGF did not stimulate an overall increase in the numbers of MF20-positive myoblasts in culture. To examine the chemokinetic effect of HGF on cell migration in the limb, cells were isolated from the proximal regions of the limb (areas rich in myogenic cells), exposed to HGF, labeled with DiI and transplanted into 11.5 day mouse forelimbs. After 36 h of culture, it was found that DiI-labeled limb cells, pretreated with HGF, migrated significantly further in the limb than labeled cells that have not been exposed to HGF. The chemotactic effect of HGF was also investigated by implanting beads loaded with and without HGF into the 11.5 day limb. Proximal to the beads, DiI-labeled limb cells were also transplanted. It was found that HGF was able to chemotactically attract and direct the migration of DiI-labeled limb cells. Immunohistological staining was performed with HGF antibodies to determine the distribution of HGF in the 11.5 day mouse forelimb. It was found that HGF was strongly expressed by the apical ectodermal ridge (AER), the ectoderm and the mesenchyme directly beneath the AER. Positive staining was also obtained for the myogenic regions. However, the pattern was heterogeneous--punctuated with myogenic cells expressing and not expressing HGF.

Animals↗
PubMed1999

Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.

Aequorin↗
PubMed1999

Neurotransmitters, peptides, and neural cell adhesion molecules in the cortices of normal elderly humans and Alzheimer patients: a comparison.

Immunocytochemical techniques was used to compare the proportion of neurons expressing various neurotransmitters (tyrosine hydroxylase, choline acetyltransferase and gamma-aminobutyric acid), neuropeptides (Leu-enkephalin and substance P) and neural cell adhesion molecules (NCAM) in the hippocampus, frontal (area 10) and occipital (area 17) cortices of neurologically normal elderly humans to that of age-matched Alzheimer disease (AD) patients. There was no difference in the proportion of GABAergic and cholinergic cells between the normal and AD groups in all three brain regions studied. However, the catecholaminergic cells in the frontal cortex of the AD patients revealed a significant decrease. The catecholaminergic cells present in the cortex were both neurons and astrocytes, as revealed by a double immunostaining of tyrosine hydroxylase and glial fibrillary acid protein (GFAP). Furthermore, the difference in the proportion of cells expressing Substance P and Leu-enkephalin was minimal between the two groups studied. Although there was little difference in the levels of NCAM in the occipital cortex and hippocampus of the two groups, there were significantly fewer positive NCAM neurons in the frontal cortex of AD than normal aging individuals.

Aged↗
PubMed1998

Calcium transients accompany ooplasmic segregation in zebrafish embryos.

Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (KD = 1.5 micromol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: > or = 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (KD = 0.15 micromol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.

Aequorin↗
PubMed1997

Localized calcium transients accompany furrow positioning, propagation, and deepening during the early cleavage period of zebrafish embryos.

Through the injection of f-aequorin (a calcium-specific luminescent reporter) and the use of an imaging photon detector, we see a distinct localized elevation of intracellular calcium that accompanies the appearance of the first furrow arc at the blastodisc surface: the furrow positioning signal. As the leading edges of the arc progress outward toward the margins of the blastodisc, they are accompanied by two subsurface slow calcium waves moving at about 0.2 micron/s: the furrow propagation signal. As these wave fronts approach the edge of the blastodisc, another calcium signal arises in the central region where the positioning signal originally appeared. Like the propagation signal, it extends outward to the margins of the blastodisc, but in this case it also moves downward, accompanying the deepening process that separates the daughter cells: the furrow deepening signal. Both of these furrow deepening progressions move at around 0.1 to 0.2 micron/s. The deepening signal begins to diminish from the center outward, returning to precleavage resting levels on completion of cytokinesis. The signaling sequence is repeated during the second cell division cycle. These localized transients do not require external calcium and they can be dissipated after they have begun by introducing calcium shuttle buffers, resulting in furrow delocalization and regression. They also occur in parthenogenetically activated eggs in which, in an attenuated form, they accompany abortive cleavages.

Aequorin↗
PubMed1997

Fibroblast growth factors 2 and 4 stimulate migration of mouse embryonic limb myogenic cells.

Fibroblast growth factors (FGFs) are believed to be vital for limb outgrowth and patterning during embryonic development. Although the effect of FGFs on the formation of the skeletal elements has been studied in detail, their effect on the development of the limb musculature is still uncertain. In this study, we used Blindwell chemotactic chambers to examine the effect of FGF-2 and FGF-4 on the motility of myogenic cells obtained from the proximal region of the day 11.5 mouse forelimbs. The limb myogenic cells were found to be chemotactically attracted to FGF-2 and FGF-4 at 10-50 ng/ml. Both FGFs increased myogenic cell migration in a dose-dependent manner, with maximal responses attained at 1-50 ng/ml for FGF-2 and at 10 ng/ml for FGF-4; however, FGF-2 was found to be a more potent chemoattractant than FGF-4. It was possible to inhibit the myogenic cells' response to FGF-2 and FGF-4 by the addition of the appropriate neutralizing antibody. The effects of FGF-2 on cell migration were further investigated by loading this cytokine into Affi-Gel blue beads and transplanting them into day 11.5 forelimb buds. The results showed that FGF-2 attracted DiI-labelled proximal cells to migrate toward the implanted beads and that the migration was more extensive than that observed in the absence of FGF-2. A checkerboard assay was performed in which various concentrations of FGF-2 and FGF-4 were introduced to both the upper and lower wells of the Blindwell chambers. The results indicated that both FGF isoforms can stimulate chemokinesis as well as chemotaxis in myogenic cells. In addition, the effect of FGF-2 at 0.1-10 ng/ml stimulated a significant increase in the number of myocytes expressing sarcomeric myosin on examination after 48 hr in culture, but the effect of FGF-4 was negligible at all concentrations analyzed; however, both FGF-2 and FGF-4 inhibited myocyte fusion compared with the spontaneous fusion observed in control cultures. Finally, we used in situ hybridization and immunohistochemical techniques to determine the distribution of myogenic cells and FGF-2 protein in the day 11.5 mouse forelimbs.

Animals↗
PubMed1997

A role for microtubules in Plasmodium falciparum merozoite invasion.

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 microM-1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

Animals↗
PubMed1997

Effect of platelet-derived growth factor isoforms on the migration of mouse embryo limb myogenic cells.

The effect of platelet-derived growth factor (PDGF) isoforms on limb myoblast migration was examined in vitro. Using Blindwell Chemotaxis chambers, the ability of PDGF-AA, -AB and -BB to stimulate the migration of myoblasts, obtained from the proximal region of 11.5 day mouse forelimb buds, was examined. Immunocytochemistry, with the anti-sarcomeric myosin antibody MF-20, was used to identify the myogenic cells in the heterogeneous cell population. Myoblasts, suspended in PDGF-free medium in the upper chamber, migrated across the polycarbonate filter of the Blindwell chamber to 1-10 ng/ml PDGF-AB and 1-100 ng/ml PDGF-BB situated in the lower well. At 1-10 ng/ml of either PDGF-AB or -BB migration increased in a dose-dependent manner. PDGF-AA, however, was unable to elicit a significant locomotory response in forelimb myoblasts. A Checkerboard assay, with various concentrations of PDGF-AA, -AB or -BB in the upper and lower wells of the chamber, indicated that -AB and -BB but not -AA stimulated the random migration of limb myoblasts. The differential effect of PDGF isoforms on myoblast migration was compared with other aspects of skeletal muscle development. At 0.1-10 ng/ml all three isoforms were able to stimulate an increase in the number of differentiated myoblasts, indicated by the expression of sarcomeric myosin, on examination after 48 h when cultured at low density. In high density cultures, however, these isoforms inhibited myoblast fusion when compared to the spontaneous fusion observed in untreated cultures. Immunohistochemical studies of both cultured limb cells and cryosections of 11.5 day whole limbs revealed that myoblasts expressed both PDGF alpha- and beta-receptors which suggests that the action of PDGF isoforms on limb myoblasts is receptor-mediated. Finally, having demonstrated that the PDGF-B monomer stimulates migration in limb myoblasts, by immunohistochemistry, the presence of PDGF-B was confirmed and its distribution examined in the 11.5 day forelimb.

Animals↗
PubMed1996

Neurotransmitters and peptides in the developing human facial nucleus.

The human facial nucleus can be sub-divided into five structurally discrete regions. Immunohistochemistry was used to locate various neurotransmitters and neuropeptides in the neurons and nerve fibres of the human facial nucleus at 14 and 27 weeks of gestation and in the neonate. Whilst choline acetyltransferase-positive neurons were observed in the facial nucleus at all stages of development, dopamine beta-hydroxylase-positive neurons were only found in the neonate. In addition, afferent nerve fibres positive for choline acetyl-transferase, enkephalin and substance P were observed at all stages of development. In the younger specimens these fibres were evenly distributed; however, in the neonates the fibres were asymmetrically distributed as the different types became concentrated in the various structurally distinct regions of the facial nucleus.

Choline O-Acetyltransferase↗