Search PubMed⌕ Search

Biomedical subjects

C W Lo

Publications and source records attributed to C W Lo.

At least 19 recordsLinked to original sources
PubMed2005

Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects.

As part of a large-scale noninvasive fetal ultrasound screen to recover ethylnitrosourea (ENU)-induced mutations causing congenital heart defects in mice, we established a high-throughput ultrasound scanning strategy for interrogating fetal mice in utero utilizing three orthogonal imaging planes defined by the fetus' vertebral column and body axes, structures readily seen by ultrasound. This contrasts with the difficulty of acquiring clinical ultrasound imaging planes which are defined by the fetal heart. By use of the three orthogonal imaging planes for two-dimensional (2D) imaging together with color flow, spectral Doppler, and M-mode imaging, all of the major elements of the heart can be evaluated. In this manner, 10,091 ENU-mutagenized mouse fetuses were ultrasound scanned between embryonic days 12.5 and 19.5, with 324 fetuses found to die prenatally and 425 exhibiting cardiovascular defects. Further analysis by necropsy and histology showed heart defects that included conotruncal anomalies, obstructive lesions, and shunt lesions as well as other complex heart diseases. Ultrasound imaging also identified craniofacial/head defects and body wall closure defects, which necropsy revealed as encephalocele, holoprosencephaly, omphalocele, or gastroschisis. Genome scanning mapped one ENU-induced mutation associated with persistence truncus arteriosus and holoprosencephaly to mouse chromosome 2, while another mutation associated with cardiac defects and omphalocele was mapped to mouse chromosome 17. These studies show the efficacy of this novel ultrasound scanning strategy for noninvasive ultrasound phenotyping to facilitate the recovery of ENU-induced mutations causing congenital heart defects and other extracardiac anomalies.

Abnormalities, Multiple↗
PubMed2004

Tbx2 represses expression of Connexin43 in osteoblastic-like cells.

Tbx2 belongs to a family of developmental transcription regulatory factors. We evaluated whether the gap junction protein Connexin43 (Cx43), an important regulator of osteoblast function and bone development, may be a downstream target gene regulated by Tbx2. The Cx43 promoter contains direct repeats of the consensus T-box binding motif, TCACAC, and moreover, Tbx2 and Cx43 show overlapping expression domains in precursors to bone and in osteoblasts. In vitro analysis showed that the Cx43 promoter contains two Tbx2 binding sites, and this binding was dependent on the TCACAC consensus sequence. Transient transfection analysis with a Cx43 promoter-driven lacZ reporter construct revealed negative regulation mediated by these two Tbx2 binding sites in osteoblast-like cells. Thus, downregulation of Tbx2 led to de-repression of wild-type Cx43 promoter activity, whereas a promoter construct with mutated binding sites showed no de-repression. In stably transfected osteosarcoma cells in which expression of the endogenous Tbx2 gene was downregulated with a Tbx2 antisense construct, a marked de-repression of the endogenous Cx43 gene was observed. This was accompanied by a marked increase in the abundance of Cx43 gap junctions and increased functional gap junction-mediated cell-cell communication. Analysis of lacZ expression in transgenic mice containing the mutated Cx43 promoter-driven lacZ construct further suggested de-repression of the Cx43 promoter in limb buds, a region destined to give rise to long bones of the limbs. Taken together, these findings indicate that the promoter of Cx43 is repressible by Tbx2, both in cultured osteoblast-like cells in vitro and likely in the developing embryo.

Animals↗
PubMed2003

Divergent effects of two sequence variants of GJB3 (G12D and R32W) on the function of connexin 31 in vitro.

Recently, we identified several missense mutations of the connexin gene GJB3 encoding connexin 31 (Cx31) in erythrokeratodermia variabilis (EKV), an autosomal dominant skin disorder. These mutations include G12D, which replaces a conserved glycine residue in the amino-terminus of Cx31 and is associated with a severe EKV phenotype. In contrast, the biologic relevance of the GJB3 sequence variant R32W located in the first transmembrane domain of Cx31 is disputed. To examine the effects of these sequence variants on Cx31 biogenesis and gap junction activity we expressed wild type and mutant Cx31-Flag constructs in HeLa cells. Using immunostaining, all expression variants were detected in the cytoplasm and in a punctate pattern at the cell surface, indicating that G12D and R32W did not interfere with either protein synthesis or transport to the cell membrane. Similarly, oligomerization into hemichannels appeared not impaired when expressing either Cx31 mutant as assessed by size exclusion chromatography, immunoblotting and immunostaining. However, dye transfer experiments and monitoring of intracellular calcium levels in response to serum stimulation revealed that G12D-Cx31 did not form functional gap junction channels, probably due to incorrect assembly or altered properties of Cx31 channels. In contrast, intercellular coupling between cells expressing R32W-Cx31 was comparable to that of wtCx31, suggesting that R32W is a functionally inconsequential polymorphism of Cx31.

Base Sequence↗
PubMed2002

Use of reconstruction nails to manage ipsilateral displaced femoral neck-shaft fractures: assessment of a new approach.

PURPOSE: Owing to unpredictable femoral neck reduction, reconstruction nails are not suitable for fixation of group 3 ipsilateral femoral neck-shaft fractures. We developed a new one-step fixation technique to overcome this problem. This study aims to assess this new technique at the Orthopaedic Department, Chi-Mei Foundation Medical Center, Tainan. METHODS: Of 31 consecutive patients with femoral fractures treated by reconstruction nails, five patients had group 3 ipsilateral femoral neck-shaft fractures, 4 of whom were treated by a new surgical technique. Two 5.0-mm drills were firstly inserted to tether the trochanter fragment, and distal locking screws were secondly applied to immobilise the shaft fracture. The neck-shaft angle was then restored in a closed fashion and proximal cephalomedullary screws were attached. Patients were followed up by post-operative radiography. RESULTS: All 5 cases of group 3 ipsilateral femoral neck-shaft fracture obtained radiographic union without significant surgical sequelae. Three of the patients had implants removed. No patients presented with osteonecrosis at the 3-year follow-up. CONCLUSION: The new approach to manage ipsilateral femoral neck-shaft fractures by using reconstruction nails obtains relatively good clinical results.

Adult↗
PubMed2002

An essential role for connexin43 gap junctions in mouse coronary artery development.

Connexin43 knockout mice die neonatally from conotruncal heart malformation and outflow obstruction. Previous studies have indicated the involvement of neural crest perturbations in these cardiac anomalies. We provide evidence for the involvement of another extracardiac cell population, the proepicardial cells. These cells give rise to the vascular smooth muscle cells of the coronary arteries and cardiac fibroblasts in the heart. We have observed the abnormal presence of fibroblast and vascular smooth muscle cells in the infundibular pouches of the connexin43 knockout mouse heart. In addition, the connexin43 knockout mice exhibit a variety of coronary artery patterning defects previously described for neural crest-ablated chick embryos, such as anomalous origin of the coronary arteries, absent left or right coronary artery, and accessory coronary arteries. However, we show that proepicardial cells also express connexin43 gap junctions abundantly. The proepicardial cells are functionally well coupled, and this coupling is significantly reduced with the loss of connexin43 function. Further analysis revealed an elevation in the speed of cell locomotion and cell proliferation rate in the connexin43-deficient proepicardial cells. A parallel analysis of proepicardial cells in transgenic mice with dominant negative inhibition of connexin43 targeted only to neural crest cells showed none of these coupling, proliferation or migration changes. These mice exhibit outflow obstruction, but no infundibular pouches. Together these findings indicate an important role for connexin43 in coronary artery patterning, a role that probably involves the proepicardial and cardiac neural crest cells. We discuss the potential involvement of connexin43 in human cardiovascular anomalies involving the coronary arteries.

Animals↗
PubMed2001

Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.

Connexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.

Animals↗
PubMed2001

Null mutation of connexin43 causes slow propagation of ventricular activation in the late stages of mouse embryonic development.

Connexin43 (Cx43) is the principal connexin isoform in the mouse ventricle, where it is thought to provide electrical coupling between cells. Knocking out this gene results in anatomic malformations that nevertheless allow for survival through early neonatal life. We examined electrical wave propagation in the left (LV) and right (RV) ventricles of isolated Cx43 null mutated (Cx43(-/-)), heterozygous (Cx43(+/)(-)), and wild-type (WT) embryos using high-resolution mapping of voltage-sensitive dye fluorescence. Consistent with the compensating presence of the other connexins, no reduction in propagation velocity was seen in Cx43(-/-) ventricles at postcoital day (dpc) 12.5 compared with WT or Cx43(+/)(-) ventricles. A gross reduction in conduction velocity was seen in the RV at 15.5 dpc (in cm/second, mean [1 SE confidence interval], WT 9.9 [8.7 to 11.2], Cx43(+/)(-) 9.9 [9.0 to 10.9], and Cx43(-/-) 2.2 [1.8 to 2.7; P<0.005]) and in both ventricles at 17.5 dpc (in RV, WT 8.4 [7.6 to 9.3], Cx43(+/)(-) 8.7 [8.1 to 9.3], and Cx43(-/-) 1.1 [0.1 to 1.3; P<0.005]; in LV, WT 10.1 [9.4 to 10.7], Cx43(+/)(-) 8.3 [7.8 to 8.9], and Cx43(-/-) 1.7 [1.3 to 2.1; P<0.005]) corresponding with the downregulation of Cx40. Cx40 and Cx45 mRNAs were detectable in ventricular homogenates even at 17.5 dpc, probably accounting for the residual conduction function. Neonatal knockout hearts were arrhythmic in vivo as well as ex vivo. This study demonstrates the contribution of Cx43 to the electrical function of the developing mouse heart and the essential role of this gene in maintaining heart rhythm in postnatal life.

Animals↗
PubMed2001

Multimeric connexin interactions prior to the trans-Golgi network.

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial beta-galactosidase fused to the C terminus of connexin43 (Cx43/beta-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/beta-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a subhexameric complex. Cx43/beta-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/beta-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/beta-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

3T3 Cells↗
PubMed2001

Cx43/beta-gal inhibits Cx43 transport in the Golgi apparatus.

A connexin construct consisting of bacterial beta-galactosidase fused to the C-terminus of connexin43 (Cx43/beta-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/beta-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a sub-hexameric complex, and that Cx43/beta-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/beta-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.

3T3 Cells↗
PubMed2001

Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.

The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.

Animals↗
PubMed2001

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways.

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.

Animals↗
PubMed2000

Conotruncal anomalies in the trisomy 16 mouse: an immunohistochemical analysis with emphasis on the involvement of the neural crest.

The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse.

Animals↗
PubMed2000

Migration of cardiac neural crest cells in Splotch embryos.

Pax3 encodes a transcription factor expressed during mid-gestation in the region of the dorsal neural tube that gives rise to migrating neural crest populations. In the absence of Pax3, both humans and mice develop with neural crest defects. Homozygous Splotch embryos that lack Pax3 die by embryonic day 13.5 with cardiac defects that resemble those induced by neural crest ablation in chick models. This has led to the hypothesis that Pax3 is required for cardiac neural crest migration. However, cardiac derivatives of Pax3-expressing precursor cells have not been previously defined, and Pax3-expressing cells within the heart have not been well demonstrated. Hence, the precise role of Pax3 during cardiac development remains unclear. Here, we use a Cre-lox method to fate map Pax3-expressing neural crest precursors to the cardiac outflow tract. We show that although Pax3 itself is extinguished prior to neural crest populating the heart, derivatives of these precursors contribute to the aorticopulmonary septum. We further show that neural crest cells are found in the outflow tract of Splotch embryos, albeit in reduced numbers. This indicates that contrary to prior reports, Pax3 is not required for cardiac neural crest migration. Using a neural tube explant culture assay, we demonstrate that neural crest cells from Splotch embryos show normal rates of proliferation but altered migratory characteristics. These studies suggest that Pax3 is required for fine tuning the migratory behavior of the cardiac neural crest cells while it is not essential for neural crest migration.

Animals↗
PubMed1999

Connexin 43 expression reflects neural crest patterns during cardiovascular development.

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick.

Animals↗
PubMed1999

Genes, gene knockouts, and mutations in the analysis of gap junctions.

Gop junctions are cell junctions found between most cells and tissues. They contain membrane channels that mediate the cell-to-cell diffusion of ions, metabolites, and small cell signaling molecules. Cell-cell communication mediated by gap junctions has been proposed to have a variety of functions, including roles in regulating events in development, cell differentiation, and cell growth and proliferation. The analysis of these possibilities has been confounded by the fact that there are over a dozen connexin genes encoding polypeptides that make up vertebrate gap junctions. This complexity, coupled with the fact that most cells express multiple connexin isotypes, likely explains why recent studies using reverse genetic and genetic approaches to disrupt connexin gene function have yielded only limited insights into the physiological roles of gap junctions. Nevertheless, studies in vivo and in vitro together have provided evidence for gap junctions being involved in the regulation of cell metabolism, growth, and differentiation in restricted cell and tissue types. Surprisingly, studies in invertebrates suggest that their gap junctions are encoded not by connexins, but by a family of proteins referred to as innexins. Analysis of various Drosophila and C. elegans mutants suggest that innexins may be functional homologs to the connexins. However, whether innexins are the elusive invertebrate gap junction proteins or, rather, accessory proteins that facilitate gap junction formation remains an open question. Given the rapid progress being made in the cloning and functional analysis of gap junctions in many diverse species, confusion and difficulties with nomenclature are coming to a head in this rapidly expanding field. It may be timely to form a Nomenclature Committee to establish a uniform classification scheme for naming gap junction proteins.

Animals↗
PubMed1999

Total parenteral nutrition and home parenteral nutrition: an effective combination to sustain malnourished children with cancer.

A patient with Wilms' tumor and severe failure to thrive required total parenteral nutrition (TPN) for "catch-up" growth. This case underscores how TPN might be useful in the management of a child with cancer. Cancer cachexia, chemotherapy, radiation, and infections caused by immune suppression can lead to potentially serious macro- and micronutrient deficiencies.

Failure to Thrive↗