Biomedical subjects
Publications and source records attributed to C Brownlee.
Recent studies indicate that sphingosine-1-phosphate - known to be an important signalling molecule in animal cells - is involved in Ca(2+)-dependent signalling in yeast and higher plants, raising the likelihood that it is a universal signalling molecule with a diverse range of functions in eukaryotes.
The acquisition and expression of polarity during early embryogenesis underlies developmental pattern. In many multicellular organisms an initial asymmetric division of the zygote is critical to the determination of different cell fates of the early embryonic cells. Zygotes of the marine fucoid algae are initially apolar and become polarized in response to external cues. This results in an initial asymmetric division of the zygote. Subsequent divisions occur in a highly ordered spatial and temporal pattern. A combination of cell biological and biochemical studies is providing new details, and some controversies concerning the mechanisms by which zygotic polarity is acquired and amplified. Here, we discuss some of the more recent studies that are allowing improved understanding of polarization in this system.
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How do plant cells accomplish a net uptake of Ca(2+) but keep the membrane potential under control? Consideration of the voltage dependence of the depolarization-activated calcium channel and hyperpolarization-activated calcium channel types, and two other major transporters in the plasma membrane, the H(+)-ATPase and I(K,out), suggests that one channel is well suited for both nutritive and signalling Ca(2+) uptake whereas the other could be limited to a signalling function.
In eukaryotic cells, the basic machinery of cell cycle control is highly conserved. In particular, many cellular events during cell cycle progression are controlled by cyclin-dependent kinases (CDKs). The cell cycle in animal early embryos, however, differs substantially from that of somatic cells or yeasts. For example, cell cycle checkpoints that ensure that the sequence of cell cycle events is correct have been described in somatic cells and yeasts but are largely absent in embryonic cells. Furthermore, the regulation of CDKs is substantially different in the embryonic and somatic cells. In this study, we address the nature of the first cell cycle in the brown alga Fucus, which is evolutionarily distant from the model systems classically used for cell cycle studies in embryos. This cycle consists of well-defined G1, S, G2, and M phases. The purine derivative olomoucine inhibited CDKs activity in vivo and in vitro and induced different cell cycle arrests, including at the G1/S transition, suggesting that, as in somatic cells, CDKs tightly control cell cycle progression. The cell cycle of Fucus zygotes presented the other main features of a somatic cell cycle, such as a functional spindle assembly checkpoint that targets CDKs and the regulation of the early synthesis of two PSTAIRE CDKs, p32 and p34, and the associated histone H1 kinase activity as well as the regulation of CDKs by tyrosine phosphorylation. Surprisingly, the synthesis after fertilization of p32 and p34 was translationally regulated, a regulation not described previously for CDKs. Finally, our results suggest that the activation of mitotic CDKs relies on an autocatalytic amplification mechanism.
Although iterative development can be uncoupled from morphogenesis in plant organs, the relationship between the cell cycle and developmental events is not well established in embryos. Zygotes of fucoid algae, including Fucus and Pelvetia are particularly well suited for studying the interaction(s) between cell cycle progression and the early morphogenetic events, as the establishment of polarity and its morphogenetic expression, i.e. germination, and the first cell cycle are concomitant. We have previously demonstrated that, in Fucus zygotes, various aspects of cell cycle progression are tightly controlled by cyclin-dependent kinase (CDK)-like proteins, including two PSTAIRE CDK-like proteins, p34 and p32, which are synthesised after fertilisation. We show that specific inhibition of CDK-like proteins, either with purine derivatives such as olomoucine and amino-purvalanol or by microinjection of the CDK inhibitor p21(cip1), prevents germination and cell division. Whereas direct inhibition of DNA replication by aphidicolin did not affect polarised development, olomoucine, which has previously been shown to prevent entry in S phase, and other purine derivatives also inhibited photopolarisation. Early microinjection of a monoclonal anti-PSTAIRE antibody also prevented germination and cell division. Only p34 had affinity for amino-purvalanol, suggesting that among PSTAIRE CDKs, this protein is the main target of purine derivatives. Models to account for the simultaneous control of early cell cycle progression and polarisation are proposed.
Fucoid algae, including the genus Fucus and Pelvetia, are recognized as model systems to study early embryogenesis in plants. In particular the zygotes of these fucoid algae are highly suitable experimental systems for investigating the establishment of polarity and its requirement for later embryogenesis. However, the transduction pathways involved in the initiation of polarization are still poorly understood, and the link between the early polarization processes and embryo long-term patterning has never been experimentally demonstrated. We, therefore, have investigated the putative role of protein phosphorylation in the regulation of early embryogenesis, using a combined pharmacological and biochemical approach. Among the various protein kinase inhibitors tested, a subset of well-known PTK inhibitors, including genistein, prevented germination but had no effect on growth of germinated zygotes and embryos. Inhibition of germination appeared to be a direct consequence of prevention of polarization since genistein and other PTK inhibitors specifically inhibited axis formation in a light-independent manner. Genistein inhibited cellular events associated with polarization such as polarized secretion of cell wall sulfated compounds. Anchorage of F-actin at the rhizoid pole was also inhibited and F-actin redistributed in response to a new light vector. Zygotes inhibited in the polarization process over the period of axis formation recovered from the treatment and displayed differentiated cellular structures after a few days. However, they exhibited a deeply disorganized pattern, suggesting that the early polarization process is essential for normal patterning of the embryo. Western blot analysis of protein phosphorylation showed that the patterns of protein phosphorylation changed during development and were disturbed by treatments with genistein. This drug also inhibited in vitro autophosphorylation. The nature of the genistein-sensitive kinases required for polarization and long-term patterning is discussed in light of these data.
Plant cells can respond qualitatively and quantitatively to a wide range of environmental signals. Ca(2+) is used as an intracellular signal for volume regulation in response to external osmotic changes. We show here that the spatiotemporal patterns of hypo-osmotically induced Ca(2+) signals vary dramatically with stimulus strength in embryonic cells of the marine alga Fucus. Biphasic or multiphasic Ca(2+) signals reflect Ca(2+) elevations in distinct cellular domains. These propagate via elemental Ca(2+) release in nuclear or peripheral regions that are rich in endoplasmic reticulum. Cell volume regulation specifically requires Ca(2+) elevation in apical peripheral regions, whereas an altered cell division rate occurs only in response to stimuli that cause Ca(2+) elevation in nuclear regions.
The ability to uncover the fine details of intracellular Ca(2+) signals has improved remarkably in recent years, largely as a result of developments in methods for reporting Ca(2+), coupled with great improvements in measurement instrumentation. The cell biologist wishing to image intracellular Ca(2+) has a range of options to consider. These include the use of photoproteins, commercially available fluorescent indicators or the new generation of fluorescent protein Ca(2+) probes. Molecular biology and biophysics are now joining forces to bring major advances in the art of deciphering the complexity of spatiotemporal Ca(2+) signals.
This study investigated inorganic carbon accumulation in relation to photosynthesis in the marine dinoflagellate Prorocentrum micans. Measurement of the internal inorganic carbon pool showed a 10-fold accumulation in relation to external dissolved inorganic carbon (DIC). Dextran-bound sulfonamide (DBS), which inhibited extracellular carbonic anhydrase, caused more than 95% inhibition of DIC accumulation and photosynthesis. We used real-time imaging of living cells with confocal laser scanning microscopy and a fluorescent pH indicator dye to measure transient pH changes in relation to inorganic carbon availability. When steady-state photosynthesizing cells were DIC limited, the chloroplast pH decreased from 8.3 to 6.9 and cytosolic pH decreased from 7.7 to 7.1. Re-addition of HCO3- led to a rapid re-establishment of the steady-state pH values abolished by DBS. The addition of DBS to photosynthesizing cells under steady-state conditions resulted in a transient increase in intracellular pH, with photosynthesis maintained for 6 s, the amount of time needed for depletion of the intracellular inorganic carbon pool. These results demonstrate the key role of extracellular carbonic anhydrase in facilitating the availability of CO2 at the exofacial surface of the plasma membrane necessary to maintain the photosynthetic rate. The need for a CO2-concentrating mechanism at ambient CO2 concentrations may reflect the difference in the specificity factor of ribulose-1,5 bisphosphate carboxylase/oxygenase in dinoflagellates compared with other algal phyla.
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We describe a simple isocratic HPLC method for the accurate and precise measurement of retinol, alpha-tocopherol and the major carotenoids in plasma using UV detection. Reference ranges for retinol, alpha-tocopherol and five carotenoids are determined in a healthy population group. The most abundant carotenoids found in plasma were beta-carotene, lycopene, lutein and cryptoxanthin. Retinol, alpha-tocopherol and carotenoids were determined simultaneously using two internal standards, retinol acetate for retinol and tocopherol acetate for alpha-tocopherol and carotenoids. The use of echinenone as an internal standard for carotenoids was investigated. The protective effect of an antioxidant (ascorbic acid) on the stability of samples and extracted material is documented. The method is useful for the routine measurement of plasma retinol, alpha-tocopherol and carotenoids and could also be used in large scale epidemiological studies.
OBJECTIVE: To describe pediatric housestaff resuscitation experience and their ability to perform key resuscitation skills. DESIGN: Cohort study of 63 pediatric residents in a university-based training program. PARTICIPANTS AND METHODS: Investigators observed, scored, and timed resident performance on 4 key resuscitation skills. Cognitive ability was tested with 4 written scenarios. Housestaff provided self-reports of the number of months since their last American Heart Association Pediatric Advanced Life Support course, number of mock and actual codes attended, number of times skills were performed, and self-confidence with respect to resuscitation. RESULTS: A total of 45 pediatric residents (71%) participated. Median cognitive score was 5 (range, 1-5). Of all residents, 44 (97%) successfully bag mask-ventilated the mannequin; 24 (53%) and 36 (80%) used the correct bag and mask size, respectively. Thirty-nine residents (87%) placed a tube in the mannequin trachea, 12 (27%) checked that suction was working prior to intubation, and 30 (67%) chose the correct endotracheal tube size. Forty residents (89%) discharged the defibrillator, and 25 (56%) and 32 (71%) correctly chose asynchronous mode and infant paddles, respectively. Thirty-eight residents (84%) inserted an intraosseous line; 35 (78%) had correct placement. Median times for successful skill completion were 83 seconds for bag mask ventilation, 136 seconds for intubation, 149 seconds for defibrillation, and 68 seconds for intraosseous line placement. CONCLUSION: Pediatric housestaff previously trained in pediatric advanced life support were generally able to reach the end point of 4 key resuscitation skills but less frequently performed the specific subcomponents of each skill. This poor performance and the prolonged time to skill completion suggest the need for greater attention to detail during training.
Zygotes of fucoid algae acquire polarity de novo. The initial polarity establishes the apical-basal polarity of the multicellular embryo and the adult plant. Acquisition of polarity involves the translation of external vectorial signals into spatial information within the cell via spatial photoreceptor activation at the level of the plasma membrane. Fixation of the polar axis involves interactions between the cytoskeleton, plasma membrane and the cell wall. Recently a central role for targeted secretion in polar axis fixation has been identified. In the multicellular embryo, evidence is accumulating for roles of cell wall and intercellular communication via diffusible signals in pattern formation and control of cell fate.
Protoplasts isolated from root cap cells of maize were shown to secrete fucose-rich polysaccharides and were used in a patch-clamp study to monitor changes in whole-cell capacitance. Ca2+ was required for exocytosis, which was measured as an increase in cell capacitance during intracellular dialysis with Ca2+ buffers via the patch pipette. Exocytosis was stimulated significantly by small increases above normal resting [Ca2+]. In the absence of Ca2+, protoplasts decreased in size. In situ hybridization showed significant expression of the maize annexin p35 in root cap cells, differ-entiating vascular tissue, and elongating cells. Dialysis of protoplasts with maize annexins stimulated exocytosis at physiological [Ca2+], and this could be blocked by dialysis with antibodies specific to maize annexins. Dialysis with milli-molar concentrations of GTP strongly inhibited exocytosis, causing protoplasts to decrease in size. GTPgammaS and GDPbetaS both caused only a slight inhibition of exocytosis at physiological Ca2+. Protoplasts were shown to internalize plasma membrane actively. The results are discussed in relation to the regulation of exocytosis in what is usually considered to be a constitutively secreting system; they provide direct evidence for a role of annexins in exocytosis in plant cells.
The early embryo of the brown alga Fucus comprises two cell types, i. e. rhizoid and thallus which are morphogically and cytologically distinguishable. Previous work has pointed to the cell wall as a source of position-dependent information required for polarisation and fate determination in the zygote and 2-celled embryo. In this study we have analysed the mechanism(s) of cell fate control and pattern formation at later embryonic stages using a combination of laser microsurgery and microinjection. The results indicate that the cell wall is required for maintenance of pre-existing polarity in isolated intact cells. However, all cell types ultimately have the capacity to re-differentiate or regenerate rhizoid cells in response to ablation of neighbouring cells. This regeneration is regulated in a position-dependent manner and is strongly influenced by intercellular communication, probably involving transport or diffusion of inhibitory signals which appear to be essential for regulation of cell fate decisions. This type of cell-to-cell communication does not involve symplastic transport or direct cell-cell contact inhibition. Apoplastic diffusible gradients appear to be involved in pattern formation in the multicellular embryo.
The plant extracellular matrix has multiple roles in determining pattern during plant development. These include provision of anchorage sites for focal adhesion-like structures which may play a direct signalling role and provide a reference for cytoskeletal elements involved in nuclear rotation and orientation of the cell division plane. The activity of mechanosensitive ion channels in the plasma membrane can also be regulated by the mechanical properties of the cell wall. Moreover, there is increasing evidence from a variety of systems suggesting that the cell wall may be a direct source of factors which specify cell fate in response to position. These may be inserted into the wall by differentiating cells and may act by providing signals to adjacent cells or by providing positive feedback to the protoplast contained therein, maintaining its fate according to its position.