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Biomedical subjects

C W Lo

Publications and source records attributed to C W Lo.

At least 19 recordsLinked to original sources
PubMed1995

Nonoverlapping expression of Cx43 and Cx26 in the mouse placenta and decidua: a pattern of gap junction gene expression differing from that in the rat.

We characterized the expression of two gap junction genes (Cx26 and Cx43) in the mouse decidua and placenta. In the decidua, in situ hybridization analysis and immunostaining studies revealed a high level of Cx43 expression. In contrast, Cx26 expression was not detected. Analysis of the placenta revealed that both Cx43 and Cx26 transcripts are expressed, but in nonoverlapping cell populations. Cx26 transcripts were observed only in the labyrinthine trophoblast layer of the placenta, a tissue of ectoplacental cone derivation. In contrast, no Cx43 transcripts were found in the placenta proper, but only in the maternally derived decidual cap covering the placenta. These results, in conjunction with previous observations in the mouse and rat, indicate that there may be species-specific differences in the pattern of Cx43/Cx26 expression in the placenta and decidua.

Animals↗
PubMed1995

Lethality of Rw/Rw mouse embryos during early postimplantation development.

Three mutations in the mouse, white spotting (W), rump white (Rw), and patch (Ph), are described as a "gene triplet" on the basis of their close genetic linkage and similar mutant phenotypes. The finding that the W phenotype results from mutations altering the c-kit protooncogene, and that Ph is associated with the deletion of Pdgfra, suggested specific molecular reagents which could be used for the analysis of the chromosomal structure of the third mutation, rump white. Such studies indicated that Rw is associated with a large chromosomal inversion. In this study, we showed that it is possible to generate molecular markers specific for the Rw chromosome, as recombination is suppressed between the inverted portion of the Rw chromosome and the wild-type homologue. Using one such marker, we were able to genotype the offspring of Rw/+ intercrosses. This enabled us to show that Rw homozygote embryos die around 9.5 days of gestation. Histological analysis revealed that the embryos undergo gastrulation, forming three germ layers, and in some cases, exhibit a defined axial midline with an apparent notochordal plate. However, mutant embryos are significantly smaller than the wild-type, with the size difference evident from Day 7.5 and becoming more disparate as development progresses. These morphological data further support the genetic evidence indicating that the developmental lethality of the Rw mutation is not caused by the disruption of a gene within the cluster of RTKs in the central portion of mouse chromosome 5. Furthermore, we present evidence that both Kit and Pdgfra are expressed from the Rw chromosome in several adult tissues. The results of these studies suggest that the identification of the sequence(s) disrupted by the Rw mutation will provide further insight into the regulation of early postimplantation development.

Animals↗
PubMed1995

Identification and characterization of a fibroblast marker: FSP1.

We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast-like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.

3T3 Cells↗
PubMed1995

Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells.

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.

3T3 Cells↗
PubMed1992

Restrictions in gap junctional communication in the Drosophila larval epidermis.

We characterized gap junctional communication in the Drosophila larval epidermis by monitoring the pattern of dye spread following the intracellular injection of the fluorescent dye, Lucifer yellow. We found that dye injected into the epidermis spread extensively from cell to cell, but at segment borders and also at boundaries positioned at the lateral aspects of each body segment, dye spread was restricted. The precise position of these boundaries of restricted gap junctional communication was determined by examining the distribution of the fluorescent tracer in thick sections of each dye-injected specimen. These results show that each of the thoracic and abdominal segments is segregated into four domains or communication compartments. We also observed the presence of dye in the procuticle and epicuticle, and examined the possible basis for this dye localization.

Animals↗
PubMed1992

Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains.

The expression of the gap junction gene, Cx43, during mouse embryogenesis was characterized by an in situ hybridization analysis of mouse embryos from gestation days 4.5 to 12.5. This analysis revealed that Cx43 transcripts are differentially expressed as a function of development beginning at the blastocyst stage. In many regions of the embryo, Cx43 transcripts were found in discrete spatially restricted domains. This was observed in conjunction with the development of the brain, neural tube, prevertebra, limb, and various aspects of organogenesis. In some cases, the differential localization of Cx43 transcripts is associated with developmental processes mediated by inductive interactions, such as that of the eye, otic vesicle, kidney, and the branchial arches. In addition, in the 10.5 day embryo, Cx43 transcripts appear to be distributed as a gradient in regions spanning the midbrain/hindbrain junction, in the telencephalon, and in the limb mesenchyme. Surprisingly, our results also suggest that neural crest and sclerotomal cells, i.e., cells that are presumably migratory, express high levels of Cx43 transcripts. Overall, these results suggest that gap junctions encoded by Cx43 may play a role in various aspects of mouse development, possibly including relaying second messengers emanating from signal transduction pathways that mediate inductive interactions.

Animals↗
PubMed1990

Successful resuscitation and survival following massive overdose of metoprolol.

A 23-year-old female was found unconscious and deteriorated rapidly to cardiac asystole. Prolonged resuscitation was required, and she remained in severe cardiogenic shock, despite high doses of positive inotropic agents. Massive beta-adrenoceptor blocker overdose was suspected (and subsequently confirmed). IV administration of glucagon was followed by prompt haemodynamic improvement and recovery, illustrating the beneficial role of glucagon in overwhelming beta-adrenoceptor blockade.

Adult↗
PubMed1989

Chromosomal recombination and breakage associated with instability in mouse centrometric satellite DNA.

A mouse L cell line containing the centromeric insertion of herpes thymidine kinase genes (tk) was previously shown to undergo a high frequency of DNA rearrangement at the site of tk insertion. Analysis of TK- revertants had demonstrated that DNA rearrangements were usually associated with DNA deletion and were always mediated by intrachromosomal recombinations. In this study, we further analyzed several TK+ subclones to examine the mode of DNA rearrangements in the absence of negative selection pressure. In two clones, LC2-3F and LC2-3E17, rearrangements were accompanied by DNA amplification and were mediated by intrachromosomal recombination. In subclone LC2-3E17-19, we further detected perturbations in the pattern of centromeric heterochromatization. This was associated with chromosome instability, as evidenced by chromosome breakage at the centromere. The analysis of three other sibling clones, LC2-3, LC2-6 and LC2-15, further suggests that reciprocal recombination events may play a role in such centromeric rearrangements. These results suggest that DNA rearrangements in the centromere may be mediated by a number of different mechanisms, and generally do not affect chromosome stability except when accompanied by changes in the pattern of heterochromatization.

Animals↗
PubMed1989

Introduction of human DNA into mouse eggs by injection of dissected chromosome fragments.

A procedure has been developed for introducing exogenous DNA into mouse eggs by injection of chromosome fragments. Chromosome fragments were dissected from human metaphase spreads and microinjected into the pronuclei of fertilized mouse eggs. Many of the injected eggs subsequently exhibited normal pre- and postimplantation development. Embryos obtained from eggs injected with centromeric fragments retained human centromeric DNA as demonstrated by in situ hybridization analysis. From eggs injected with noncentromeric fragments, a mouse was obtained whose tail tissue exhibited the presence of human DNA. This procedure should facilitate incorporation of very large (more than 10 megabases) DNA fragments into cells and embryos without the need for cloned sequences.

Animals↗
PubMed1989

Gap junctional communication in the extraembryonic tissues of the gastrulating mouse embryo.

We characterized gap junctional communication in the extraembryonic tissues of the 7.5-d gastrulating mouse embryo. At this stage of development, the extraembryonic tissues form a large part of the conceptus, and link the embryo proper to the maternal tissue. Using Lucifer yellow injections, cells in most extraembryonic tissues were observed to be very well dye coupled, the only exception being the peripheral regions of the ectoplacental cone. Of particular interest was the fact that no dye coupling was detected between the three major extraembryonic tissues. Thus, the extraembryonic ectoderm (EEC), the extraembryonic endoderm (EEN), and the ectoplacental cone (EPC) corresponded to separate communication compartments, with the EPC being further subdivided into three compartments. Interestingly, the EEN was observed to exhibit a very low level of dye coupling with the adjacent visceral embryonic endoderm (EN), and consistent with the latter dye coupling results was the finding that the EEN was ionically coupled to the EN, but not with any other extraembryonic tissues. However, in the EPC, ionic coupling studies show that the central region was well coupled ionically to the EEC, but only weakly coupled to the peripheral EPC. These findings, in conjunction with our previous study (1988. J. Cell Biol. 107:241-255), demonstrate that the 7.5-d mouse conceptus is subdivided into at least nine major Lucifer yellow-delineated communication compartments, with ionic coupling across some of these compartments effectively unifying the embryo into two large domains corresponding to the embryo proper and the major extraembryonic tissues.

Animals↗
PubMed1989

A comparative evaluation of a minute ventilation sensing and activity sensing adaptive-rate pacemakers during daily activities.

Most studies evaluating the rate response of adaptive-rate pacemakers have been based on treadmill or bicycle exercise. These studies disregard the fact that few pacemaker recipients voluntarily undertake such activities. The rate responses of nine patients (mean age 62 years, range 33-79 years) with implanted minute ventilation sensing (Meta) pacemakers were studied. The indications for pacing were complete heart block (seven patients), sick sinus syndrome (one patient), and five nodal disease (one patient). Significant improvement in maximum distance covered during a 12-minute walking test was observed in the rate adaptive compared to the VVI pacing mode (989 +/- 104 vs 921 +/- 90 m, P less than 0.02). The rate responses of this pacemaker during daily activities were recorded with telemetry during a variety of structured daily activities. The rate responses were also compared to those of an externally attached Activitrax pacemaker in each patient and to a group of ten age and sex matched volunteers. For less strenuous activities such as walking, descending stairs, washing, and bed making, both pacemakers achieved adequate rate responses compared to normal subjects. For more strenuous activities, the Activitrax pacemaker failed to achieve an adequate rate response. For example, the pacing rate achieved on ascending stairs was lower than that achieved on descending stairs (92 +/- 3 vs 102 +/- 3 bpm, P less than 0.02). The direction of rate responses was more appropriate for the Meta pacemaker. Similar to the normal subjects, the maximum rate was reached before the end of an activity with the Activitrax pacemaker.(ABSTRACT TRUNCATED AT 250 WORDS)

Activities of Daily Living↗
PubMed1989

Changes in the gastrointestinal tract during enteral or parenteral feeding.

Intestinal adaptation, in terms of increasing intestinal length and weight, usually occurs rapidly after small-bowel resection. However, this response depends on provision of enteral nutrients. If total parenteral nutrition without enteral feeding is prolonged, hypoplasia of the intestinal mucosa results. Adaptation is probably mediated through the presence of luminal nutrients, particularly glutamine, which is preferentially used by the intestine. However, systemic hormonal factors, possibly gastrin, cholecystokinin, and glucagon, also influence intestinal adaptation. Thus, in the management of short-bowel syndromes, enteral nutrition should be added to total parenteral nutrition as soon as possible.

Adaptation, Physiological↗
PubMed1988

Communication compartments in the gastrulating mouse embryo.

We characterized the pattern of gap junctional communication in the 7.5-d mouse embryo (at the primitive streak or gastrulation stage). First we examined the pattern of dye coupling by injecting the fluorescent tracers, Lucifer Yellow or carboxyfluorescein, and monitoring the extent of dye spread. These studies revealed that cells within all three germ layers are well coupled, as the injected dye usually spread rapidly from the site of impalement into the neighboring cells. The dye spread, however, appeared to be restricted at specific regions of the embryo. Further thick section histological analysis revealed little or no dye transfer between germ layers, indicating that each is a separate communication compartment. The pattern of dye movement within the embryonic ectoderm and mesoderm further suggested that cells in each of these germ layers may be subdivided into smaller communication compartments, the most striking of which are a number of "box-like" domains. Such compartments, unlike the restrictions observed between germ layers, are consistently only partially restrictive. In light of these results, we further monitored ionic coupling to determine if some coupling might nevertheless persist between germ layers. For these studies, Lucifer Yellow was coinjected while ionic coupling was monitored. The injected Lucifer Yellow facilitated the identification of the impalement sites, both in the live specimen and in thick sections in the subsequent histological analysis. By using this approach, all three germ layers were shown to be ionically coupled, indicating that gap junctional communication is maintained across the otherwise dye-uncoupled "germ layer compartments." Thus our results demonstrate that partially restrictive communication compartments are associated with the delamination of germ layers in the gastrulating mouse embryo. The spatial distribution of these compartments are consistent with a possible role in the underlying development.

Animals↗
PubMed1988

Organization of chromosomes in the mouse nucleus: analysis by in situ hybridization.

We used in situ hybridization with various biotinylated DNA probes to characterize the distribution of mouse chromosomes in the nucleus. This entailed an examination of plasmid-marked chromosomes from two strains of transgenic mice and also an examination of the distribution of centromeres and total mouse genomic DNA. Our results show that, regardless of the hybridization probes used or the tissue types examined, with sections of mouse tissues embedded in paraffin all chromosomal DNA appeared to be localized to the nuclear periphery. This was observed in tissues fixed with either crosslinking or non-crosslinking fixatives, and submitted to a variety of prehybridization treatments. Further experiments with non-embedded nuclei fractionated from mouse liver revealed a similar distribution of DNA at the telomeric end of chromosome 3, but for centromeric DNA and total mouse genomic DNA, hybridization signals were observed both in the interior and at the periphery of the nucleus. These observations preclude the exclusive localization of chromosomes to the nuclear periphery. However, they indicate that a subset of centromeres are likely to be associated with the nuclear membrane, and that there is such an association at the telomeric end of chromosome 3. Overall, these results are compatible with previous observations of other investigators showing regions of contact between chromosomes and the nuclear membrane.

Animals↗