Citizens' advice in general practice.
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Counseling↗
Biomedical subjects
D Bentley
Publications and source records attributed to D Bentley.
Molecular diagnosis of X-linked agammaglobulinaemia.
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Agammaglobulinemia↗
Physical mapping shows close linkage between the alpha-galactosidase A gene (GLA) and the DXS178 locus.
X-linked agammaglobulinaemia (XLA) is an inherited disorder characterised by a lack of circulating B-cells and antibodies. While the gene involved in XLA has not yet been identified, the locus for the disorder is tightly linked to the polymorphic marker DXS178, which maps to Xq22. Fabry disease is an X-linked recessive disorder caused by a deficiency in the lysosomal enzyme alpha-galactosidase A. The gene encoding this enzyme has been characterized and also maps to Xq22. Using pulsed field gel electrophoresis we have constructed a long-range restriction map that shows that the alpha-galactosidase A gene (GLA) and DXS178 lie no more than 140 kb apart on a stretch of DNA containing a number of putative CpG islands. We have also isolated yeast artificial chromosome (YAC) clones that confirm this physical linkage. The localisation of DXS178 near the alpha-galactosidase A gene will facilitate carrier detection in Fabry families using restriction fragment length polymorphism (RFLP) analysis. The identification of a number of CpG islands near DXS178 also provides candidate locations for the gene responsible for XLA.
Agammaglobulinemia↗
Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ.
Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.
Actins↗
Isolation of a putative transcriptional regulator from the region of 22q11 deleted in DiGeorge syndrome, Shprintzen syndrome and familial congenital heart disease.
A wide spectrum of birth defects are caused by deletions of the DiGeorge syndrome critical region (DGCR) at human chromosome 22q11. Over one hundred such deletions have now been examined and a minimally deleted region of 300kb defined. Within these sequences we have identified a gene expressed during human and murine embryogenesis. The gene, named TUPLE1, and its murine homologue, encodes a protein containing repeated motifs similar to the WD40 domains found in the beta-transducin/enhancer of split (TLE) family. The TUPLE1 product has several features typical of transcriptional control proteins and in particular has homology with the yeast Tup1 transcriptional regulator. We propose that haploinsufficiency for TUPLE1 is at least partly responsible for DiGeorge syndrome and related abnormalities.
Abnormalities, Multiple↗
Development and validation of laboratory procedures for preimplantation diagnosis of Duchenne muscular dystrophy.
In order to develop and validate methods for the preimplantation diagnosis of Duchenne muscular dystrophy (DMD), we have established and evaluated PCR assays for the analysis of four loci within the DMD gene and for two Y chromosome sequences in single cells. A model system using buccal cells picked from mouthwash samples has been used for an extensive evaluation of the sensitivity and specificity of the assays, and each assay has been tested in samples containing single cells, two cells, and three cells per tube. The four DMD and two Y assays have been combined in duplex and triplex reactions to enable simultaneous diagnosis of DMD and of fetal sex. One of the DMD markers is a highly polymorphic simple tandem repeat locus which produces a basic DNA profile, and provides a control for contamination by foreign DNA. Amplification of DMD or Y sequences was observed in 78 to 92% of single male cells, rising to 96% and 97% in tubes containing two or three male cells respectively. Coamplification of both a DMD and a Y sequence together occurred with a mean success of 74% in single male cells, increasing to 93% with two, and 95% with three cells per tube. With appropriate precautions, we believe that it is now possible to proceed to clinical application of these procedures.
Base Sequence↗
Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis.
Although the study of rostral-caudal segmentation of the insect body has been a rich source of information about embryonic pattern formation, relatively little is known of the process of proximal-distal segmentation of insect appendages. Here we demonstrate that during the period of limb segmentation, five segmentally iterated, sharply demarcated bands of cell surface alkaline phosphatase activity are expressed in embryonic grasshopper limbs. These bands span each intersegmental boundary in the limb as well as one boundary within the tarsus. Within appendages, expression is restricted to epithelial cells, where activity is present on both apical and basolateral surfaces. This epithelial alkaline phosphatase remains active at neutral pH, is insensitive to levamisole inhibition, and is strongly inhibited by nucleoside monophosphates. Treatment of embryos with phosphatidylinositol-specific phospholipase C releases almost all visible chromogenic activity, indicating that the epithelial alkaline phosphatase is anchored to the plasma membrane by glycosyl-phosphatidylinositol. When material released by phosphatidylinositol-specific phospholipase C is separated on native polyacrylamide gels, a single broad band of enzymatic activity is detected following incubation with substrate. A polyclonal antiserum raised against a 55 x 10(3) M(r) alkaline phosphatase from shrimp recognizes a single band of 56 x 10(3) M(r) on immunoblots of grasshopper membrane proteins. The spatially restricted expression of epithelial alkaline phosphatase suggests that it may be involved in epithelial cell rearrangements or shape changes associated with limb segmentation and morphogenesis. It also may contribute to definition of axon routes in the limb, since pioneer afferent growth cones turn at, and migrate along, the edge of one alkaline phosphatase-expressing epithelial domain.
Alkaline Phosphatase↗
Annulin, a protein expressed at limb segment boundaries in the grasshopper embryo, is homologous to protein cross-linking transglutaminases.
Annulin, a protein whose general stage- and position-specific expression pattern in the grasshopper embryo is described in the companion paper, is expressed in epithelial annuli in developing limbs. Here, we show that these annuli comprise narrow circumferential bands of epithelial cells at the boundaries of limb segments. At most boundaries, expression of annulin precedes the first morphological signs of segmentation. The most distal cells in a band underlie the boundary invagination. Bands arise in a stereotyped order and, within a band, expression occurs in an ordered circumferential progression. Annulin has a molecular weight of about 97 kDa and appears to be intracellular and peripherally associated with the inner leaflet of the cell membrane. Using the monoclonal antibody 7H7, two overlapping cDNA clones encoding this protein were isolated from an embryonic Schistocerca cDNA expression library. The nucleotide and deduced amino acid sequences indicate that the annulin protein does not contain either a signal sequence or a transmembrane domain. By sequence comparison, annulin appears to be a transglutaminase, one of a family of enzymes that have protein cross-linking activity. Its expression pattern within the limb and the embryo is associated with areas undergoing morphogenetic rearrangements, movements, or rapid cell division. It may stabilize cells under mechanical stress or participate in some other way in these morphogenetic activities.
Amino Acid Sequence↗
Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo.
Monoclonal antibody 6F8 was used to characterize and clone fasciclin IV, a new axonal glycoprotein in the grasshopper, and to study its function during growth cone guidance. Fasciclin IV is dynamically expressed on a subset of axon pathways in the developing CNS and on circumferential bands of epithelial cells in developing limb buds. One of these bands corresponds to the location where the growth cones of the Ti1 pioneer neurons make a characteristic turn while extending toward the CNS. Embryos cultured in the 6F8 antibody or Fab exhibit aberrant formation of this axon pathway. cDNA sequence analysis suggests that fasciclin IV has a signal sequence; long extracellular, transmembrane, and short cytoplasmic domains; and shows no homology with any protein in the available data bases. Thus, fasciclin IV appears to be a novel integral membrane protein that functions in growth cone guidance.
Amino Acid Sequence↗
Disruption of pioneer growth cone guidance in vivo by removal of glycosyl-phosphatidylinositol-anchored cell surface proteins.
Cell surface proteins anchored to membranes via covalently attached glycosyl-phosphatidylinositol (GPI) have been implicated in neuronal adhesion, promotion of neurite outgrowth and directed cell migration. Treatment of grasshopper embryos with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves the GPI anchor, often induced disruptions in the highly stereotyped migrations of peripheral pioneer growth cones and afferent neuron cell bodies. In distal limb regions of embryos treated with PI-PLC at early stages of pioneer axon outgrowth, growth cones lost their proximal orientation toward the central nervous system (CNS) and turned distally. Pioneer growth cones in treated limbs also failed to make a characteristic ventral turn along the trochanter-coxa (Tr-Cx) segment boundary, and instead continued to grow proximally across the boundary. Treatment at an earlier stage of development caused pre-axonogenesis Cx1 neurons to abandon their normal circumferential migration and reorient toward the CNS. None of these abnormal phenotypes were observed in limbs of untreated embryos or embryos exposed to other phospholipases that do not release GPI-anchored proteins. Incubation of embryos with PI-PLC effectively removed immunoreactivity for fasciclin I, a GPI-anchored protein expressed on a subset of neuronal surfaces. These results suggest that cell surface GPI-anchored proteins are involved in pioneer growth cone guidance and in pre-axonogenesis migration of neurons in the grasshopper limb bud in vivo.
Animals↗
Molecular heterogeneity of the fragile X syndrome.
The fragile X syndrome is an X-linked disorder which has been shown to be associated with the length variation of a DNA fragment containing a CGG trinucleotide repeat element at or close to the fragile site. Phenotypically normal carriers of the disorder generally have a smaller length variation than affected individuals. We have cloned the region in cosmids and defined the area containing the amplified sequence. We have used probes from the region to analyse the mutation in families. We show that the mutation evolves in different ways in different individuals of the same family. In addition we show that not all fragile X positive individuals show this amplification of DNA sequence even though they show expression of the fragile site at levels greater than 25%. One patient has alterations in the region adjacent to the CGG repeat elements. Three patients in fragile X families have the normal fragment with amplification in a small population of their cells. These observations indicate that there is molecular heterogeneity in the fragile X syndrome and that the DNA fragment length variation is not the only sequence responsible for the expression of the fragile site or the disease phenotype.
Base Sequence↗
Reproductive cycle and heavy metals in the organs of red mullet, Mullus barbatus (L.), from the northwestern Mediterranean.
Concentrations of the heavy metals Cd, Cu, Fe Mn, V and Zn have been measured in the principal tissues of the red mullet, Mullus barbatus (L.), during a complete sexual cycle. By comparison of metal levels during the reproductive and non-reproductive periods, it was found that concentrations as well as total amounts of Cu, Mn and Zn in liver and gonads of male and female mullets were dependent upon the stage of the sexual cycle. No relationship with the sexual cycle was noted for these metals measured in muscle, kidney, spleen or pyloric caecum. Because of the observed variation in liver and gonad metal concentrations, these tissues appear to be poor indicators of ambient levels of metals in the surrounding waters. However, muscle may prove useful as an indicator tissue owing to its apparent independence of effects of the sexual cycle.
Animals↗
Tracheal reconstruction. Success with a silicone tracheal prosthesis.
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Adult↗
Microtubule behavior during guidance of pioneer neuron growth cones in situ.
The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues. Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment. We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation. A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin. Complex microtubule loops often formed transiently within the growth cone. Branches both with and without microtubules were regularly observed. Microtubules did not extend into filopodia. During growth cone steering events in response to identified guidance cues, microtubule behaviour could be monitored. In turns towards guidepost cells, microtubules selectively invaded branches derived from filopodia that had contacted the guidepost cell. At limb segment boundaries, microtubules displayed a variety of behaviors, including selective branch invasion, and also invasion of multiple branches followed by selective retention in branches oriented in the correct direction. Microtubule invasion of multiple branches also was seen in growth cones migrating on intrasegmental epithelium. Both selective invasion and selective retention generate asymmetrical microtubule arrangements within the growth cone, and may play a key role in growth cone steering events.
Animals↗
Calcium ion distribution in nascent pioneer axons and coupled preaxonogenesis neurons in situ.
The "calcium hypothesis" of regulation of growth cone motility and neurite elongation has derived from analysis of a variety of neurons growing in vitro. It proposes that calcium ion concentration within growth cones is an important regulator of motility and growth. We now extend this analysis by investigating calcium concentrations within growth cones and nascent neurites of identified embryonic neurons growing on their normal substrate in situ. The pair of Ti1 pioneer neurons are the first to extend axons in limb buds of grasshopper embryos. Their growth cones migrate along a stereotyped pathway, where they encounter a series of guidance cues, including preaxonogenesis afferent neurons (guidepost cells). By injecting a pioneer neuron with fura-2 dye, we measured calcium concentration in the injected neuron, and in cells to which it became dye-coupled, at successive stages of pioneer outgrowth through the limb. Pioneer neurons undergoing axonogenesis had calcium concentrations in the above-100-nM range characteristic of a variety of neuron types studied in vitro. In pioneer neurons not yet in contact with guidepost cells, a shallow calcium concentration gradient, highest at the growth cone, was often observed. Guidepost cells that had not begun axonogenesis had lower calcium concentrations, in the 65 nM range. Therefore, an increase in cytosolic calcium concentration may be associated with the onset of axonogenesis. Nascent pioneer neurons were fura-2 dye-coupled to each other and established coupling with contacted guidepost cells. Calcium concentration measurements along pioneer neurites suggest that calcium ions also are transferred from pioneer neurons to these coupled guidepost cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Pioneer growth cone steering decisions mediated by single filopodial contacts in situ.
In grasshopper embryo limb buds, the sibling Ti1 pioneers are the first neurons to initiate axonogenesis. The pioneer growth cones migrate from the limb tip to the CNS along a in direction comprising discrete steering events. Filopodial exploration of the cellular terrain in the vicinity of the advancing growth cone appears to be important for steering. Some information is available on the identity of cells and cell types, on cell-surface characteristics, and on the involvement of basal lamina in these steering decisions. In the work reported here, we have used computer-enhanced fluorescence video microscopy to examine filopodial behavior and the process of growth cone migration and reorientation resulting from interactions with the normal guidance cues on the in situ substrate. We observed several different kinds of migration and steering events, which appear to be related to the absolute and relative affinities of the contacted substrates. On a relatively homogeneous substrate of intrasegmental epithelium, growth cones advance by extending veils between filopodia, as is commonly observed on uniform substrates in vitro. Where growth cones confront an orthogonal border between substrates of dissimilar affinity, they remain on the higher-affinity substrate by extending new branches along it. Subsequently, reorientation in the preferred direction on the higher-affinity substrate is accomplished by regression of branches extended in the nonselected direction. By contrast, a single filopodial contact with a very high-affinity substrate, such as a guidepost neuron, can reorient a growth cone, even when it is migrating on a favorable substrate. In this situation, the filopodium that contacts the high-affinity substrate expands in diameter until it becomes the nascent axon.
Animals↗
Is it possible to "achieve a balance" and meet the "safety net"? Paediatric Advisory Subcommittee to the North West Thames Regional Health Authority.
The government's document Hospital Medical Staffing--Achieving a Balance--Plan for Action introduced the concept of a "safety net"--that is, a minimum safe level of staffing--of junior doctors in the acute specialties. The North West Thames Paediatric Advisory Group has therefore reviewed the implications and consequences of implementing the safety net in respect of children's services. The group found that if a reasonable safety net was to be provided that enabled the services to stay within the projected junior staffing levels, maintain a balance, meet training needs, and reduce junior doctors' hours of work, then changes in the organisation of the services would be required. Examining the options available showed that to achieve a safety net within the projected numbers of junior staff at least six paediatric units in the region would have to close. It is doubtful if there is the political will to support the very radical changes that would be needed in the distribution of services if the government's recommendations in Achieving a Balance were to be implemented. The profession, the Department of Health, and the public must be made aware that the proposed changes in medical staffing will cause a fundamental change in the traditional pattern of delivery of health care.
Child↗
Transient pioneer neurons are essential for formation of an embryonic peripheral nerve.
In developing nervous systems, many peripheral and central pathways are established by early arising populations of pioneer neurons. The growth cones of these pioneer neurons can migrate while embryonic distances are short and while intervening tissue is relatively uncomplicated. Are these pioneers necessary? In grasshopper embryos, a pair of pioneer neurons arise at the tips of limb buds and extend axons through the limb to the central nervous system. Growth cones of later arising sensory neurons migrate along the pioneer axons. After ingrowth of sensory axons, the pioneer neurons die. If the pioneer neurons are prevented from differentiating by heat shock, then the sensory growth cones that would have migrated along them are blocked and fail to reach the central nervous system. Thus, the pioneer axons are necessary for successful migration of these sensory growth cones. By crossing a segment boundary early in embryogenesis, the pioneers circumvent an incompatibility between differentiated segment boundary cells and growth cone migration. Pioneer neurons may resolve similar problems in many systems.
Afferent Pathways↗