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Biomedical subjects

M Ringuette

Publications and source records attributed to M Ringuette.

13 recordsLinked to original sources
PubMed2000

Association of SPARC (osteonectin, BM-40) with extracellular and intracellular components of the ciliated surface ectoderm of Xenopus embryos.

SPARC (Secreted Protein, Acidic, Rich in Cysteine) was detected by immunohistochemistry in the sensorial layer of the bilayered embryonic epidermis of Xenopus laevis during neurulation, when a subset of the sensorial cells are selected to differentiate into ciliated cell precursors. After the ciliated cells had intercalated into the outer layer and had undergone ciliogenesis, intense SPARC immunostaining was associated with the cilia and remained associated with the cilia throughout their persistence on the epidermis. Circumferential SPARC immunostaining was also detected at the interface between surface epithelial cells. Animal cap explants indicated that the embryonic activation of SPARC expression in the dorsal ectoderm does not require signaling from factors secreted by the underlying mesoderm. Immunoelectron microscopy revealed that SPARC is intimately associated with the 9 + 2 microtubule arrays of cilia. Our data indicate that SPARC plays a role in the development and function of the surface ciliated epidermis of Xenopus embryos. We propose that the counter-adhesive activity of SPARC facilitates the intercalation of ciliary cell precursors to the surface epithelial layer, where its Ca(2+)-binding abilities promote cell-cell adhesion. Based on its association with ciliary microtubule arrays, we also propose that intracellular SPARC may play a role in regulating ciliary beat frequency and polarity.

Animals↗
PubMed1999

A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm cell migration during Xenopus laevis gastrulation: evidence of counter-adhesive activity in vivo.

Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+-binding, counter-adhesive, extracellular glycoprotein associated with major morphogenic events and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPARC is expressed first by dorsal mesoderm cells at the end of gastrulation and undergoes complex, rapid changes in its pattern of expression during early organogenesis. Another study has reported that precocious expression of SPARC by injection of native protein into the blastocoele cavity of pregastrula embryos leads to a concentration-dependent reduction in anterior development. Thus, normal development requires that the timing, spatial distribution, and/or levels of SPARC be regulated precisely. In a previous study, we demonstrated that injection of a synthetic peptide corresponding to the C-terminal, Ca2+-binding, EF-hand domain of SPARC (peptide 4.2) mimicked the effects of native SPARC. In the present investigation, peptide 4.2 was used to examine the cellular and molecular bases of the phenotypes generated by the aberrant presence of SPARC. Exposure of late blastula embryos to LiCl also generated a concentration-dependent reduction in anterior development; therefore, injections of LiCl were carried out in parallel to highlight the unique effects of peptide 4.2 on early development. At concentrations that caused a similar loss in anterior development (60-100 ng peptide 4.2 or 0.25-0.4 microg LiCl), LiCl had a greater inhibitory effect on the initial rate of chordomesoderm cell involution, in comparison with peptide 4.2. However, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of LiCl. Moreover, peptide 4.2 and LiCl had distinct influences on the expression pattern of dorso-anterior markers at the neural and tail-bud stages of development. Scanning electron microscopy showed that peptide 4.2 inhibited spreading of migrating cells at the leading edge of the involuting chordomesoderm. While still in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in the direction of migration. In contrast, LiCl had no effect on the spreading or shape of involuting cells. These data are the first evidence of a counter-adhesive activity for peptide 4.2 in vivo, an activity demonstrated for both native SPARC and peptide 4.2 in vitro.

Animals↗
PubMed1998

Regulation of SPARC expression during early Xenopus development: evolutionary divergence and conservation of DNA regulatory elements between amphibians and mammals.

SPARC (Secreted Protein, Acidic, Rich in Cysteine/osteonectin/BM-40) is a highly conserved metal-binding extracellular matrix (ECM) glycoprotein which is first expressed by Xenopus embryos during late gastrulation/early neurulation (stage 12/13), by presumptive notochord and somitic cells. When animal cap explants of stage 9 embryos were cultured in vitro, SPARC expression was not detected until sibling embryos reached late neurula stage (stage 19). Addition of activin, a potent dorsal mesoderm inducer, to animal caps resulted in SPARC being expressed by the time sibling embryos reached stage 16. While basic fibroblast growth factor (bFGF), a ventral mesoderm inducer, had modest effects on SPARC mRNA expression, the combination of both activin and bFGF was synergistic. The appearance, however, of SPARC transcripts 11 h after the addition of activin and bFGF, indicates that unknown intermediates were likely to be involved in activating SPARC expression. In order to identify the potential intermediate regulatory factors which may activate and control SPARC expression, we examined the genomic organization of the 5' end of the Xenopus SPARC gene. No significant homology to the equivalent region that is highly conserved in the mouse, bovine and human SPARC genes was observed. Thus, while mammalian SPARC promoters lack TATA or CAAT boxes, the Xenopus gene contains a consensus TATA box. Moreover, promoter-proximal GGA-box repeats necessary for high level expression of mammalian SPARC are absent in Xenopus. When reporter constructs containing the 5' flanking region of the Xenopus gene were microinjected into two-cell embryos, 868 bp of 5' flanking DNA was sufficient to mimic the temporal and tissue-specific pattern of SPARC expression observed in whole embryos. While a bovine SPARC promoter reporter construct containing 740 bp of the 5' flanking DNA was expressed at a significant level in Xenopus embryos, significant differences in the cell-type expression of the reporter genes were obtained between the bovine and Xenopus constructs. The data indicate that zygotic activation of SPARC mRNA is mediated by regulatory factors acting downstream of major mesoderm induction events. The high DNA sequence conservation at the 5' end of mammalian SPARC genes is not conserved in Xenopus. These differences led to differences in their ability to direct tissue-specific gene expression in early Xenopus embryos.

Activins↗
PubMed1998

Expression of SC1 is associated with the migration of myotomes along the dermomyotome during somitogenesis in early mouse embryos.

SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts were first detected in mouse embryos after day 8.5 post coitus (p.c.) in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continued as they began involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration was completed, SC1 mRNA expression was downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1997

Spatiotemporal distribution of SPARC/osteonectin in developing and mature chicken retina.

Expression of SPARC (Secreted Protein, Acidic, Rich in Cysteine), a counteradhesive, calcium-binding extracellular matrix (ECM) glycoprotein, is associated with several morphogenetic events during early development. In this study, changes in the spatiotemporal distribution of SPARC transcripts and the protein during chicken retinal development were documented by in situ hybridization and indirect immunofluorescence microscopy. SPARC transcripts were first detected within the proliferating neural ectoderm at embryonic day 4. 5 (E4.5), followed short thereafter (E5) by appearance of SPARC. SPARC was enriched within the inner plexiform layer (IPL) by E10 and within the outer plexiform layer (OPL) by E14, several days after these layers became morphologically distinct. Significant levels of SPARC transcripts were first observed within the ganglion cell layer (GCL) at E17 prior to accumulation of SPARC within the nerve fiber layer, seen first at E20. SPARC protein was first detected within the developing retinal pigment epithelium (RPE) at E10 and increased significantly at RPE cells ceased to proliferate and continued differentiating. Of special note was the restriction of SPARC to the basal-half of the RPE cells. SPARC transcripts were similarly distributed in the adult retina, but at lower levels than in the period just prior to hatching. In the adult retina SPARC was retained in the nerve fiber layer and present in the inner nuclear layer (INL) and outer nuclear layer (ONL), but lost from the IPL and OPL. These changes in expression pattern with time indicate that SPARC is developmentally regulated and therefore may have important function(s) in both morphological development of the retina and functioning of the mature eye.

Amino Acid Sequence↗
PubMed1993

Developmental anomalies of Xenopus embryos following microinjection of SPARC antibodies.

The function of SPARC (Secreted Protein, Acidic, Rich in Cysteine) in early embryonic development was assayed by microinjecting affinity-purified antibodies directed against SPARC into the blastocoel cavity of Xenopus embryos. Microinjection of SPARC antibodies did not appear to interfere with development until late neurulation. By hatching, a broad spectrum of external developmental anomalies were observable, including bent embryonic axes, accentuated ventral masses, shortened embryonic axes, and lack of visible eye pigment. Histological sections of injected embryos demonstrated that lack of visible eye pigmentation was often associated with deformities in eye development. Bending and shortening of the embryonic axis was associated with highly disorganized myotome patterns and loss of segmental boundaries. The results indicate a requirement for SPARC in the early morphological development of several tissues in Xenopus.

Animals↗
PubMed1992

Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine). A highly conserved acidic calcium-binding extracellular-matrix protein.

SPARC (Secreted Protein, Acidic, Rich in Cysteine) is expressed as a 1.6 kb mRNA in Xenopus laevis. On the basis of cDNA sequence analysis, Xenopus SPARC has a core Mr of 32643, with one potential N-glycosylation site. Western analysis of SPARC isolated from Xenopus long bone indicates that the mature protein has an Mr of 43,000. At the amino acid level, Xenopus SPARC has 78-79% sequence similarity to mouse, bovine and human SPARC. The least-conserved region is found within the N-terminal glutamic acid-rich domain, with the C-terminal Ca(2+)-binding domain being the most conserved. Adult Xenopus tissues show the same pattern of tissue-specific distribution of SPARC mRNAs as adult mouse.

Amino Acid Sequence↗
PubMed1992

Cytoplasmic localization of the DNA virus frog erythrocytic virus.

In situ hybridization, using a biotinylated clone of frog erythrocytic virus (FEV), was conducted to determine the location of viral sequences in bullfrog erythrocytes. FEV-specific hybridization signals were found to correspond to mature cytoplasmic viral particles and assembly sites. These data are consistent with electron microscopic observations of viral assembly in the erythrocyte cytoplasm. Although FEV has morphological and biochemical properties similar to frog virus 3, our data suggest that the site of DNA replication and assembly of FEV is more similar to that of the poxviruses.

Amino Acid Sequence↗
PubMed1991

Expression of SPARC/osteonectin in tissues of bony and cartilaginous vertebrates.

To explore the biological functions of SPARC (secreted protein, acidic, rich in cysteine), a Ca(2+)-binding extracellular glycoprotein, we have examined its expression in an evolutionary diverse group of organisms. Similar patterns of SPARC mRNA expression were observed in adult mouse and rat tissues. SPARC transcripts represented 0.0002-0.0025% of the total RNA found in calvarium, lung, brain, and heart, whereas relatively low levels of SPARC RNA were detected in liver and kidney. Within nonmuscular tissues, a statistically significant correlation was observed between the tissue distribution of SPARC and cytoskeletal actin transcripts. Southern blot analysis revealed SPARC as a low or single-copy gene in an evolutionary diverse group of vertebrates. No hybridization signal was observed with the invertebrates examined. The tissue distribution of SPARC transcripts in the vertebrates examined was similar, except for sea lamprey and sea skate, two vertebrates that do not form mineralized bone. These data suggest that SPARC has multiple functions in mineralized and nonmineralized tissues of vertebrates.

Actins↗
PubMed1987

Localization of Escherichia coli RNA polymerase-binding sites on bacteriophage S13 replicative form I DNA by protection of restriction enzyme cleavage sites.

Protection of restriction endonuclease cleavage sites by Escherichia coli RNA polymerase bound to the replicative form I of bacteriophage S13 DNA has been used to identify a number of regions of RNA polymerase binding. Digestion with HincII, AluI, HinfI, or HaeIII, under conditions optimized for "open" complex formation, revealed 12 regions of RNA polymerase binding. Based on differential salt sensitivities, five of the regions were classified as strong or tight binding sites. These were located before genes A (two sites), B, and D and at the 5' end of gene F. The seven regions which exhibited weaker binding were located at the 5' end of gene C (two sites), in the middle of gene D, just before and at the 3' end of gene F, at the 5' end of gene G, and in the middle of gene H. The sites before genes B and D coincide with sites previously identified as promoters in bacteriophage phi X174. One of the sites before gene A, that at nucleotides 5175-5211, represents a new putative promoter site in bacteriophage S13 and phi X174 located before the previously identified A gene promoter at nucleotides 10-45.

Base Sequence↗
PubMed1980

Stimulation of transcription of chromatin by specific small nuclear RNAs.

Small molecular weight nuclear RNAs (SnRNA) purified from the chromatin of SV40-transformed W138 human fibroblasts have been found to stimulate transcription of chromatin in homologous isolated nuclei as well as in nuclei of untransformed human and monkey cells. Stimulation in normal cell nuclei involves an increase in both initiation sites and rate of elongation of RNA chains. Fractionation of the SnRNA in polyacrylamide gradient slab gels revealed that the "active" RNA was 160-175 nucleotides in length.

Cell Line↗