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D L Becker

Publications and source records attributed to D L Becker.

At least 19 recordsLinked to original sources
PubMed2005

Limiting burn extension by transient inhibition of Connexin43 expression at the site of injury.

Extension of a burn wound over the first 24h following injury is recognised clinically, and leads to diagnostic and therapeutic dilemmas. In the central nervous system, a similar spread of damage, beyond the initial injury, can occur via the spread of death signals from injured cells to their healthy neighbours via Connexin43 (Cx43) gap junction channels. In the skin, Cx43 is expressed in the basal epidermis and in fibroblasts and dermal appendages. We have used Cx43 specific antisense oligodeoxynucleotide approach to transiently down-regulate Cx43 protein in the early stages of partial thickness cutaneous burn wound healing. Antisense ODNs reduce the spread of tissue damage and neutrophil infiltration around the wound following injury. Epithelial cell proliferation is increased and the rate of wound closure is accelerated, compared to controls. Resultant scarring is smaller with less granulation tissue and more dermal appendages than controls. These findings suggest that Cx43 antisense treatment speeds partial thickness burn wound healing and reduces scarring. We suggest that this approach may provide an effective adjunct to managing partial thickness burn wounds.

Animals↗
PubMed2002

Caspase activity and expression of cell death genes during development of human preimplantation embryos.

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

Apoptosis↗
PubMed2001

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin.

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.

Binding Sites↗
PubMed2001

Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face.

Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face.

Animals↗
PubMed2001

Regeneration of rabbit cornea following excimer laser photorefractive keratectomy: a study on gap junctions, epithelial junctions and epidermal growth factor receptor expression in correlation with cell proliferation.

Corneal wound repair was investigated in rabbits following excimer laser ablation of a 6 mm diameter and 90 microm deep disc. In the healing process particular attention was focused on the epithelium where gap junction expression and the rearrangement of desmosomes and hemidesmosomes were correlated with cell proliferation and epidermal growth factor receptor expression. Immunofluorescence-based confocal laser scanning microscopy, semithin resin section morphology and electron microscopy were utilized. In resting cornea two isotypes of gap junctions, confined to different regions in the same basal epithelial cells, were detected. Particulate connexin43 (alpha1) immunostaining was concentrated on the apical while the connexin26 type (beta2) in the baso-lateral cell membranes. This is the first report of connexin26 in the cornea. Connexin43 was found also in corneal keratocytes and endothelial cell. Since the two connexins do not form functioning heteromeric channels and have selective permeabilities they may serve alternative pathways for direct cell-cell communication in the basal cell layer. During regeneration both connexins were expressed throughout the corneal epithelium including the migrating cells. They also showed transient up-regulation 24 hr after wounding in the form of overlapping relocation to the upper cell layers. At this time, basal epithelial cells at the limbal region, adjacent to the wound and those migrating over the wounded area all expressed membrane bound epidermal growth factor receptor and they were highly proliferating. In conclusion, like in other stratified epithelia connexin26 is also expressed in the cornea. Transient up-regulation and relocation of connexins within the regenerating epithelium may reflect the involvement of direct cell-cell communication in corneal wound healing. Mitotic activity in the migrating corneal epithelial cells is also a novel finding which is probably the sign of the excessive demand for new epithelial cells in larger wounds not met alone by the proliferating limbal stock.

Animals↗
PubMed2001

Oestriol and oestradiol increase cell to cell communication and connexin43 protein expression in human myometrium.

Oestradiol increases the protein expression of connexin43 (Cx43) gap junctions in myometrium but the effect of oestriol on gap junction expression has not been described previously. Oestriol is the most abundant free oestrogen in pregnant women and there is a marked surge in oestriol concentrations before term and idiopathic preterm labour. In order to determine whether oestriol may have a physiological action on the myometrium, cultured human myometrial cells obtained from non-pregnant hysterectomy specimens were exposed to 10 nmol/l oestradiol or oestriol. Intercellular communication between myometrial cells was investigated by microinjection of confluent cultured cells with the gap junction-permeant tracer Cascade Blue. There was a progressive increase in coupling after exposure to oestradiol or oestriol (P < 0.0005). An increase in Cx43 protein expression was demonstrated by immunocytochemistry after 1 h (P < 0.01) and 3 days (P < 0.01) exposure, and by Western blotting after 1 h (P < 0.01) and 3 days (P < 0.05) exposure, to both oestradiol and to oestriol. We conclude that oestriol increases gap junction communication in human myometrium by increasing gap junction expression. Elevated oestriol concentrations may thus play a role in the initiation of labour in women, by increasing cell-cell communication in the myometrium.

Adult↗
PubMed2001

Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation.

Gap junction communication plays an essential role in follicle growth. Immunocytochemistry and confocal microscopy were used to examine the expression of gap junction connexins of the alpha and beta subfamilies in follicles from primordial to preovulatory stages in the ovaries of prepubertal and adult mice. Connexin-specific antibodies detected alpha(1), alpha(4), alpha(6), beta(1), beta(2) and beta(4) connexins within follicles. In adult ovaries connexin immunolabelling was stronger in larger (more mature) follicles than it was in smaller follicles, with comparatively reduced labelling detected in prepubertal ovaries. In healthy follicles, labelling for alpha subfamily connexins was detected between granulosa cells, whereas labelling for beta subfamily connexins was found in the theca. Labelling for beta subfamily connexins and alpha(4) connexin (preantral stage) was detected on the oocyte surface membrane. In atretic follicles, labelling for beta(4) connexin appeared between the granulosa cells. These results demonstrate that alpha and beta connexin subfamilies are segregated to separate cellular compartments in the mouse follicle. The results are discussed in the light of possible roles for differential gap junctional communication in the regulation of folliculogenesis, oocyte maturation and atresia.

Animals↗
PubMed2000

Increased connexin43 gap junction protein in hamster cardiomyocytes during cold acclimatization and hibernation.

OBJECTIVE: The physiology of hibernation is characterized by dramatic reductions of heart rate, respiration, metabolism, blood pressure and body temperature and by resistance to ventricular fibrillation. Gap junctions in the heart provide low resistance pathways, facilitating electrical and metabolic coupling between cardiac muscle cells for coordinated action of the heart and tissue homeostasis. The conductance of these junctions, and therefore their function, is likely to be affected by the physiological changes that take place during hibernation. Our objective was to quantitate gap junction protein levels in cold acclimatization, hibernation and arousal. METHODS: We have used specific antibodies to connexins 43 and 40, in combination with confocal microscopy, to quantitatively analyze the expression of connexin protein in hamster (Mesocricetus auratus) left ventricles in four animal groups: normal controls at euthermy, cold controls (cold-exposed animals that did not undergo hibernation), hibernating animals and animals aroused from hibernation for 2 h. RESULTS: Connexin40 immunostaining was not detected in ventricular cardiomyocytes in any animal group but connexin43 was found in all groups. Connexin43 expression was significantly enhanced in hibernation and cold control ventricular cardiomyocytes. Total plaque area, numerical density and plaque size were higher in the cold controls and hibernating hamsters compared to normal controls and animals aroused from hibernation. CONCLUSION: It is possible that the increased size and number of connexin43 gap junction plaques in the cold controls may represent a compensatory response in order to maintain sufficient gap junction communication during physiological conditions that would reduce conductance. These changes may represent a mechanism by which the hamster avoids ventricular fibrillation during hibernation and arousal.

Adaptation, Physiological↗
PubMed2000

Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development.

Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death. Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earle's balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4'-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy. IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% (+IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 +/- 2.9% (-IGF-I) to 8.7 +/- 1.4% (+IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 +/- 4.6 with IGF-I; 54.5 +/- 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.

Apoptosis↗
PubMed1999

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II.

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.

Anticoagulants↗
PubMed1999

Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach.

Gap junctional communication has been implicated in embryonic development and pattern formation. The gap junction protein, alpha 1 connexin (Cx43) is expressed in dynamic and spatially restricted patterns in the developing chick embryo and its expression correlates with many specific developmental events. High levels of expression are found in regions of budding, which leads to shaping and appears to be a necessary prelude for tissue fusions. In order to investigate the role of alpha 1 connexin in these morphogenetic events, we developed a novel method of applying unmodified antisense deoxyoligonucleotides (ODNs) to chick embryos. The use of pluronic gel to deliver antisense ODNs has allowed us to regulate the expression of alpha 1 connexin protein, both spatially and temporally. This "knockdown" results in some striking developmental defects that mimic some common congenital abnormalities, such as spina bifida, anencephaly, myeloschisis, limb malformation, cleft palate, failure of hematopoiesis, and cardiovascular deformity. The results imply a major role for alpha 1 connexin communication in the integration of signaling required for pattern formation during embryonic development. This novel antisense technique may also be widely applicable.

Animals↗
PubMed1999

Connexin alpha1 and cell proliferation in the developing chick retina.

During the formation of the eye, high levels of connexin alpha1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin alpha1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2-4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin alpha1, to one eye of early chick embryos results in a rapid and significant reduction of alpha1 protein which lasts for 24-48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin alpha1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 +/- 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin alpha1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.

Animals↗
PubMed1998

Basic FGF increases communication between cells of the developing neocortex.

We have found that basic fibroblast growth factor (bFGF), applied to cortical progenitor cells in vitro, produces an increase in the expression of the gap junction protein connexin (Cx) 43 and in the mRNA encoding Cx 43. This effect was evident in both proliferating and nonproliferating cells. The elevated levels of mRNA suggest that bFGF is likely to exert its effect by upregulating the rate of transcription of the Cx 43 gene. We have further shown that the increase in Cx 43 expression is mediated through the receptor tyrosine kinase pathway and is associated with enhanced intercellular dye-coupling mediated by gap junctions. These results suggest that gap junction channels provide a direct conduit for mitogens released in response to bFGF to effectively regulate proliferation during corticogenesis.

Animals↗
PubMed1998

Co-capping studies reveal CD8/TCR interactions after capping CD8 beta polypeptides and intracellular associations of CD8 with p56(lck).

CD8 is a T cell surface glycoprotein that participates in recognition of peptide/MHC class I molecules by binding to their alpha 3 domains. In addition, the cytoplasmic domain of CD8 associates with the intracellular tyrosine kinase p56(lck) (lck) promoting recruitment of lck to the TCR signaling complex. Recent data have suggested also that CD8 may interact with the TCR to promote energetically favorable conformations which increase its ligand binding. We have used the techniques of co-capping and confocal microscopy to ask whether we can detect an association between CD8 and the TCR independently of their binding to MHC class I molecules. We show that capping CD8 heterodimers with antibodies to the CD8 beta polypeptide is significantly more efficient than antibodies to the CD8 alpha polypeptide at inducing co-localization of TCR molecules with CD8, suggesting that there may be preferred conformations of CD8 which stabilize interactions with the TCR. In addition, we show by microscopy that intracellular lck redistributes very efficiently to the area of a CD8 cap, suggesting that there is a stronger association between lck and CD8 than has been proposed from immunoprecipitation analyses.

Animals↗
PubMed1998

Expression of major gap junction connexin types in the working myocardium of eight chordates.

The alpha1 connexin (connexin43) is regarded as the major gap junction protein of the myocardium because it predominates there in mammals. Here, we show that it is not the major connexin of the working myocardium in non-mammalian vertebrates, which instead express beta1-like connexins homologous to mammalian connexin32. A phylogenetic series of hearts was immunostained with seven antibodies raised against peptide sequences specific for three distinct members of the gap junction connexin family: alpha1, beta1 and alpha5 (mammalian connexin40/avian connexin42). Working myocardium from two ascidian chordates (Ciona and Mogula), a teleost (Carassius), a frog (Xenopus) and two reptiles (Anolis and Alligator) was found to express a beta1-like connexin, rather than an alpha1-like connexin. An alpha1-like connexin was nevertheless often detected in other cardiac tissues. In the chicken (by ancestry a reptile), the developing myocardium expressed a beta1-like connexin strongly on embryonic day 6 but less strongly at hatching, and minimally in the adult. Myocardial expression of alpha5 connexin increased during development, but remained strongest in the coronary vascular endothelial and cardiac conduction tissues. The arteriolar smooth muscle of the chicken expressed alpha1 connexin throughout development, but its myocardium did not. In contrast, the working myocardium of a marsupial mammal (the opossum Trichosurus) strongly expressed an alpha1 connexin just like placental mammals. These results imply that a shift from beta1 to alpha1 connexin expression in the heart occurred prior to the evolution of the opossums. The beta and alpha connexin subfamilies have different permeabilities and gating properties, and we discuss factors that might have made this shift beneficial.

Animals↗
PubMed1997

Fibroblast growth factor 4 directs gap junction expression in the mesenchyme of the vertebrate limb Bud.

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.

Animals↗