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Biomedical subjects

Y Chai

Publications and source records attributed to Y Chai.

At least 37 records · Page 2Linked to original sources
PubMed1999

Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants.

Posterior midbrain and anterior hindbrain neuroectoderm trans-differentiate into cranial neural crest cells (CNCC), emigrate from the neural folds, and become crest-derived ectomesenchyme within the mandibular and maxillary processes. To investigate the growth factor requirement specific for the initiation of tooth morphogenesis, we designed studies to test whether nerve growth factor (NGF) can support odontogenesis in a first branchial arch (FBA) explant culture system. FBA explants containing neural-fold tissues before CNCC emigration and the anlagen of the FBA were microdissected from embryonic day 8 (E8) mouse embryos, and cultured for 8 days in medium supplemented with 10% fetal calf serum only, or serum-containing medium further supplemented with either NGF or epidermal growth factor (EGF) at three different concentrations: 50, 100, or 200 ng/ml. Morphological, morphometric, and total protein analyses indicated that growth and development in all groups were comparable. Meckel's cartilage and tongue formation were also observed in all groups. However, odontogenesis was only detected in explants cultured in the presence of exogenous NGF. NGF-supplemented cultures were permissive for bud stage (50 ng/ml) as well as cap stage of tooth morphogenesis (100 and 200 ng/ml). Morphometric analyses of the volume of tooth organs showed a significant dose-dependent increase in tooth volume as the concentration of NGF increased. Whole-mount in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction for Pax9, a molecular marker of dental mesenchyme, further supported and confirmed the morphological data of the specificity and dose dependency of NGF on odontogenesis. We conclude that (1) E8 FBA explants contain premigratory CNCC that are capable of emigration, proliferation, and differentiation in vitro; (2) serum-supplemented medium is permissive for CNCC differentiation into tongue myoblasts and chondrocytes in FBA explants; and (3) NGF controls CNCC cell fate specification and differentiation into tooth organs.

Animals↗
PubMed1999

Inhibition of transforming growth factor-beta type II receptor signaling accelerates tooth formation in mouse first branchial arch explants.

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through their cognate receptors to determine cell phenotypes during embryogenesis. Our previous studies on the regulation of first branchial arch morphogenesis have identified critical components of a hierarchy of different TGF-beta isoforms and their possible functions in regulating tooth and cartilage formation during mandibular morphogenesis. Here we tested the hypothesis that TGF-beta type II receptor (TGF-beta IIR) is a critical component in the TGF-beta signaling pathway regulating tooth formation. To establish the precise location of TGF-beta ligand and its cognate receptor, we first performed detailed analyses of the localization of both TGF-beta2 and TGF-beta IIR during initiation and subsequent morphogenesis of developing embryonic mouse tooth organs. A possible autocrine functional role for TGF-beta and its cognate receptor (TGF-beta IIR) was inferred due to the temporal and spatial localization patterns during the early inductive stages of tooth morphogenesis. Second, loss of function of TGF-beta IIR in a mandibular explant culture model resulted in the acceleration of tooth formation to the cap stage while the mandibular explants in the control group only showed bud stage tooth formation. In addition, there was a significant increase in odontogenic epithelial cell proliferation following TGF-beta IIR abrogation. These results demonstrate, for the first time, that abrogation of the TGF-beta IIR stimulates embryonic tooth morphogenesis in culture and reverses the negative regulation of endogenous TGF-beta signaling upon enamel organ epithelial cell proliferation.

Adenoviridae↗
PubMed1999

Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70.

At least eight inherited human neurodegenerative diseases are caused by expansion of a polyglutamine domain within the respective proteins. This confers dominant toxicity on the proteins, leading to dysfunction and loss of neurons. Expanded polyglutamine proteins form aggregates, including nuclear inclusions (NI), within neurons, possibly due to misfolding of the proteins. NI are ubiquitinated and sequester molecular chaperone proteins and proteasome components, suggesting that disease pathogenesis includes activation of cellular stress pathways to help refold, disaggregate or degrade the mutant disease proteins. Overexpression of specific chaperone proteins reduces polyglutamine aggregation in transfected cells, but whether this alters toxicity is unknown. Using a Drosophila melanogaster model of polyglutamine disease, we show that directed expression of the molecular chaperone HSP70 suppresses polyglutamine-induced neurodegeneration in vivo. Suppression by HSP70 occurred without a visible effect on NI formation, indicating that polyglutamine toxicity can be dissociated from formation of large aggregates. Our studies indicate that HSP70 or related molecular chaperones may provide a means of treating these and other neurodegenerative diseases associated with abnormal protein conformation and toxicity.

Animals↗
PubMed1999

Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro.

Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is one of at least eight inherited neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein. Here we present two lines of evidence implicating the ubiquitin-proteasome pathway in SCA3/MJD pathogenesis. First, studies of both human disease tissue and in vitro models showed redistribution of the 26S proteasome complex into polyglutamine aggregates. In neurons from SCA3/MJD brain, the proteasome localized to intranuclear inclusions containing the mutant protein, ataxin-3. In transfected cells, the proteasome redistributed into inclusions formed by three expanded polyglutamine proteins: a pathologic ataxin-3 fragment, full-length mutant ataxin-3 and an unrelated GFP-polyglutamine fusion protein. Inclusion formation by the full-length mutant ataxin-3 required nuclear localization of the protein and occurred within specific subnuclear structures recently implicated in the regulation of cell death, promyelocytic leukemia antigen oncogenic domains. In a second set of experiments, inhibitors of the proteasome caused a repeat length-dependent increase in aggregate formation, implying that the proteasome plays a direct role in suppressing polyglutamine aggregation in disease. These results support a central role for protein misfolding in the pathogenesis of SCA3/MJD and suggest that modulating proteasome activity is a potential approach to altering the progression of this and other polyglutamine diseases.

Acetylcysteine↗
PubMed1999

[Coverage of the lower leg defects using the reversed island skin flap based on the nutrient vessels of the saphenous nerve].

OBJECTIVE: To report the application of the distally based neurocutaneous saphenous island flap for the coverage of soft tissue defects in the lower limb. METHODS: According to the size and site of the defect and the rotation point, the flap was designed, and it was based on the arterial axis associated with the saphenous nerve and the greater saphenous vein. RESULTS: Six neurocutaneous saphenous island flaps were used and all survived. Flap dimensions were as large as 6 cm x 8 cm. The results were satisfactory after 6 to 20 months' follow-up. CONCLUSION: The vascularity of the saphenous nerve is closely connected with the vascularity of the skin. The flap supplied by the accompanying vessels of the cutaneous nerve can be utilized with good results.

Adolescent↗
PubMed1999

Cloning and sequencing of the fragment of angiostatin K (1-3) gene.

OBJECTIVE: To secure anti-endotheliocyte hyperplasia functional fragment of human angiostatin K (1-3) gene. METHODS: As the template of leukocytotic cDNA, functional fragment of human angiostatin K (1-3) cDNA was amplified by PCR, cloned into the vector pGEM-3Zf, and sequenced according to Dye primer sequencing kit. RESULTS: The functional fragment of angiostatin K (1-3) cDNA (859 bp) was obtained by PCR and determined by sequencing. CONCLUSION: The angiostatin K (1-3) gene has been cloned. The gene is of great significance in tumor-antiangiogenesis therapy.

Angiogenesis Inhibitors↗
PubMed1999

[Improved method of obtaining medial plantar cutaneous graft and its clinical application].

OBJECTIVE: To introduce a skin flap containing the middle cutaneous branch of the medial plantar artery. METHODS: Microanatomic study was performed on 8 fresh cadaveric feet, the arteries were dissected and infused with methylene blue to observe their vascular distribution and the skin area supplied by the middle cutaneous branch. Furthermore, the clinical application was reported. A local pedicled flap containing the middle cutaneous branch was used to repair the soft tissue defects of the foot in 7 patients, and free cutaneous graft was used to repair the skin defects of the fingers in 6 patients. RESULTS: The results showed that the medical plantar artery gave off 3 cutaneous branches to supply the medial aspect of the foot, among which the middle branch was the largest one and anastomosed with the other two branches. The skin flaps used clinically were all survived completely. CONCLUSION: Medial plantar cutaneous graft had a reliable blood supply, and it's one of the best choice in repairing small to middle sized skin defects of the foot and the fingers.

Adolescent↗
PubMed1999

[Expression of genes aroG and pheA in phenylalanine biosynthesis].

aroG and pheA genes, encoding 3-Deoxy-D-arabinoheptulonate-7-phosphate synthase(DS) and Chorismate mutase (CM)-prephenate dehydratase(PD) in the pathway of phenylalanine biosynthesis respectively, were amplified by polymerase chain reaction(PCR). The genes were assembled on the multicopy vectors and expressed in both Escherichia coli and Brevibacterium. The products of two gene were detected by SDS-PAGE. The activities of relevant enzymes were measured in the crude extract of the host strain. When aroG-pheA genes were introduced into E. coli p2392, the activities of DS, CM and PD were increased by 4.3-fold, 4.4-fold and 2.2-fold respectively. Whereas in the case of Brevibacterium flavum 2732, the activities of DS, CM and PD were increased by 12.3-fold, 2.3-fold and 5.6-fold, respectively. As the results, the overproduction of phenylalanine was brought about by using the genetic engineering strain of B. flavum.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1999

[Toxin-binding activity of cyanogen bromide cleaved peptide fragments of the Chinese-cobra serum antitoxic protein].

OBJECTIVE: For further study of the structure-function relationship of the Chinese cobra serum antitoxic protein. METHODS: Cyanogen bromide was used to cleave the peptide bonds which formed by the carboxyl group of Met residues. The cleaved products were ultrafiltrated to remove the small fragments below 10,000 and passed an affinity column packed with Sepharose-4B linked with cobrotoxin as ligand. The molecular weight and N-terminal amino acid residues of the cleaved peptide fragments bounded to the affinity column were then assayed. RESULTS: The cleaved peptide fragments reserved toxin-binding activity were proved to be Lys2-Met485, Lys2-Met275, Lys276-Met485, Pro444-Met603. CONCLUSIONS: This result revealed that all the three domains of CSAP, along the peptide chain, possess the toxin binding activity.

Animals↗
PubMed1998

Induction of apoptosis by Elk-1 and deltaElk-1 proteins.

Elk-1, an ets related gene codes for at least two splice variants Elk-1, which regulates c-fos transcription and deltaElk-1, both of which function as transcriptional activators. To investigate the role of Elk-1 and deltaElk-1 proteins in apoptosis; we have developed rat fibroblast cell lines and human breast cancer cell lines expressing Elk-1 and deltaElk-1. The expression of Elk-1 and deltaElk-1 proteins in the Elk-1/deltaElk-1 transfectants were analysed by immunofluorescence, immunohistochemistry, and Western blot analysis. The Elk-1 unlike deltaElk-1 transfectants showed a shortened and flattened morphology compared to the parental cells. We have found that calcium ionophore treatment of Rat-1 Elk-1, MCF-7 Elk-1, Rat-1 deltaElk-1 and MCF-7 deltaElk-1 transfectants resulted in programmed cell death. These results indicate that constitutive expression of Elk-1 and deltaElk-1 proteins triggers apoptosis in Rat-1 fibroblasts and breast cancer cells when treated with calcium ionophore.

Animals↗
PubMed1998

PDGF-A and PDGFR-alpha regulate tooth formation via autocrine mechanism during mandibular morphogenesis in vitro.

Platelet-derived growth factor A (PDGF-A) binding to the PDGF receptor alpha (PDGFR-alpha) mediates signal transduction processes related to DNA synthesis, cell migrations, cytodifferentiation, and wound healing. Recent studies indicate that PDGFR-alpha functions during cranial neural crest cell migrations and first branchial arch morphogenesis (Stephenson et al. [1991] Proc. Natl. Acad. Sci. USA 88:6-10; Morrison-Graham et al. [1992] Development 115:133-142; Hu et al. [1995] Int. J. Dev. Biol. 39:939-945; Soriano [1997] Development 124:2691-2700). The present studies were designed to test the hypothesis that PDGF-A, interacts with its cognate receptor PDGFR-alpha via an autocrine mechanism that regulates the timing, rates, and size of embryonic mouse tooth morphogenesis. Both PDGF-A and PDGFR-alpha transcripts were coordinately expressed in mandibular prominences prior to and during tooth formation using reverse transcriptase-polymerase chain reaction (RT-PCR). During the dental lamina stage, ligand and receptor were present in both enamel organ epithelium and adjacent mesenchymal cells. During the bud stage, ligand and receptor were localized mainly to the enamel organ epithelium. Exogenous PDGF-A at 20 ng/ml enhanced tooth development to reach the cap stage with increased tooth size (P < 0.05) using embryonic day (E)10 mandibular explants cultured in serumless, chemically defined medium. A significant increase in DNA synthesis was observed within enamel organ epithelium at E10+4 when the mandibular explants were treated with PDGF-A at 20 ng/ml. These data suggest that PDGF-A and its cognate receptor (PDGFR-alpha) regulate the size and stage of tooth development via an autocrine mechanism during odontogenesis in vitro.

Animals↗
PubMed1998

Association of gonadotropin receptor precursors with the protein folding chaperone calnexin.

The lutropin/choriogonadotropin receptor (LHR) and follitropin receptor (FSHR) are members of the superfamily of G protein-coupled receptors. The carboxyl half of each receptor is composed of the classical seven membrane spanning regions connected by intracellular and extracellular loops. In addition, each receptor contains a large extracellular domain. Despite the complexity of the structure of G protein-coupled receptors, little is known about how these receptors assume their correct conformations during biosynthesis. Although the role of chaperone proteins in the folding of other proteins has been well documented, their role in the folding of G protein-coupled receptors has been an enigma. To better understand the folding of the LH and FSH receptors, we examined their association with the general chaperone proteins calnexin, binding protein (BiP), and the 94-kDa glucose-regulated protein (GRP94). Clonal 293 cell lines expressing comparably high levels of each receptor were solubilized, and the extracts were incubated with the appropriate antibody bound to Protein A-sepharose beads. Experiments were performed using two approaches: 1) coimmunoprecipitation of receptor/chaperone complexes with one of the antireceptor antibodies, then SDS-PAGE and Western blotting using either anticalnexin or anti-KDEL (which recognizes BiP and GRP94) antibodies; or 2) coimmunoprecipitation of receptor/chaperone complexes with anticalnexin or anti-KDEL, then Western blotting with one of the antireceptor antibodies. Using these protocols, we found that the immature forms of both the rLHR and rFSHR are associated with calnexin, but little or no association was observed for either receptor with BiP or GRP94. These experiments show that the precursor forms of the wild-type LHR and FSHR can associate with calnexin, raising the possibility that this chaperone protein may facilitate in the folding of the gonadotropin receptors.

Calcium-Binding Proteins↗
PubMed1998

A homozygous microdeletion in helix 7 of the luteinizing hormone receptor associated with familial testicular and ovarian resistance is due to both decreased cell surface expression and impaired effector activation by the cell surface receptor.

In this report, the genomic DNA was examined from two siblings with gonadal LH resistance. A 46,XY pseudohermaphrodite presented with female external genitalia and his 46,XX sister exhibited menstrual irregularities (oligoamenorrhea) and infertility. Exons 1-11 of the LH receptor (LHR) gene were amplified by the PCR using different sets of intronic primers and were directly sequenced. Sequencing revealed that both individuals carried a deletion of nucleotides 1822-1827, resulting in the deletion of Leu-608 and Val-609 within the seventh transmembrane helix. This mutation was introduced into a recombinant human (h) LHR cDNA. Transfections of 293 cells with hLHR(wt) vs. hLHR(deltaL608,V609) revealed that very little of the mutant receptor was expressed at the cell surface. This was due to both a decrease in the total amount of receptor expressed as well as to an increased intracellular retention of the mutant receptor. In spite of the decreased cell surface expression of the mutant, sufficient amounts were present to allow for assessment of its functions. Equilibrium binding assays showed that the cell surface hLHR(deltaL608,V609) binds hCG with an affinity comparable to that of the wild-type receptor. However, the cells expressing the hLHR(deltaL608,V609) exhibit only a 1.5- to 2.4-fold stimulation of cAMP production in response to hCG. In contrast, cells expressing comparably low levels of hLHR(wt) responded to hCG with 11- to 30-fold increases of cAMP levels. Therefore, the testicular and ovarian unresponsiveness to LH in these patients appears to be due to a mutation of the hLHR gene in which Leu-608 and Val-609 are deleted. As a consequence, the majority of the mutant receptor is retained intracellularly. The small percentage of mutant receptor that is expressed at the cell surface binds hormone normally but is unable to activate Gs.

Adult↗
PubMed1998

A mouse mandibular culture model permits the study of neural crest cell migration and tooth development.

A major issue in developmental biology is to determine how time and position-restricted instructions are signaled and received during morphogenesis of different phenotypes, of which tooth, Meckel's cartilage and tongue formation are classical examples. It is now evident that a hierarchy of growth factors and their downstream transcription factors regulate the timing, sequence and position of cells and tissues in forming different phenotypes during embryogenesis. Here we report the development of an early mandibular organ culture model. Explants of E8 and E9 first branchial arch were cultured and produced mandibular processes with cap stage tooth formation, Meckel's cartilage and tongue development. In tandem, vital dye (Dil) labeling studies confirmed that rhombomeres 1-4 give rise to craneal neural crest (CNC) cells which emigrate from the neural fold to the forming maxillary and mandibular arches. Furthermore, we have tested the feasibility of investigating the regulation of different phenotypes within the first branchial arch by a transcription factor using this early mandibular organ culture model. Lymphoid enhancing factor 1 (Lef1), a transcription factor, has been implicated to regulate tooth formation in vivo. We have analyzed the expression of Lef1 and studied the biological effects of Lef1 on E8 embryonic mouse first branchial arch explants in organ culture. Collectively, these results demonstrate that first branchial arch explant model is suitable for studies of rhombencephalic crest cell fate during mandibular morphogenesis and can be used as a model with direct access to investigate the molecular mechanism in regulating first branchial arch morphogenesis.

Animals↗
PubMed1997

Characterization of the fate of midline epithelial cells during the fusion of mandibular prominences in vivo.

The fusion of the mandibular prominences along the midline is achieved with the absence of medial epithelial cells at the fusion site. Failure of fusion of the mandibular prominences results in median cleft of the lower lip and mandible. Cellular and molecular events controlling mandibular fusion were examined during the fusion process in mouse embryogenesis. Cell lineage analyses at the fusion site revealed that epithelial cells migrated to the surface and oral epithelia. DiI-labeled epithelial cells were not observed within the mandibular mesenchyme at any state of fusion. Examination of the midline region did not reveal cells with ultrastructural changes characteristic of apoptotic cell death. An increase in lysosomal enzymes in the midline epithelial cells, which would be correlated with programmed cell death, was not observed. Mice lacking TGF-beta 3 did not have cleft mandible, but had clefting of the secondary palate as a feature of null mutation phenotype. We interpret our comparisons between wild type and homozygous TGF-beta 3 (-/-) mice to suggest that different developmental processes control palatal vs. mandibular fusion. We hypothesize that medical epithelial cells at the fusion site of mandibular prominences migrate to the surface epithelium during the fusion process and neither transdifferentiate into mesenchyme nor express apoptosis.

Animals↗
PubMed1997

Problem-based learning at the University of Southern California School of Dentistry.

Responding to the recent Institute of Medicine report on dental education, the Center for Craniofacial Molecular Biology (CCMB) of the University of Southern California School of Dentistry has developed a parallel track program in dental education leading to the D.D.S. degree. This program was proposed in May of 1995, and the first class of twelve students was admitted in September of that year. Currently two classes are enrolled and plans to admit a further twelve students (Class of 2001) are in place. The educational strategy for this program is totally problem-based. Students work in groups of six with a faculty facilitator, not necessarily a content expert. Facilitators are largely drawn from the multidisciplinary pool of research faculty at the center. All learning is mediated through biomedical and biodental problem cases. No formal lectures or classes are scheduled. The learning of clinical dental skills is promoted through focussed dental patient simulations in which students review clinical charts, radiographs, medical reports and then explore identified, hands-on learning needs using patient simulators in a clinical context. Early patient exposure is obtained through dental office visits and other special patient clinics. Initial experience with this program suggests that the problem-based learning (PBL) students learn as well (if not better) than their traditional program peers and develop excellent group and cognitive analytical skills. The absence of a pool of dentally related biomedical cases suitable for a PBL program has necessitated the use of innovative approaches to their development and presentation. It is believed that this educational approach will produce dental clinicians equipped with the self-motivated, life-long learning skills required in the ever-changing world of bio-dental sciences in the twenty-first century.

California↗
PubMed1994

Specific transforming growth factor-beta subtypes regulate embryonic mouse Meckel's cartilage and tooth development.

Members of the transforming growth factor-beta (TGF-beta) superfamily have emerged as critical regulators for cell growth and differentiation. Whereas the different TGF-beta subtypes are equipotent in the majority of biological assays using cell lines cultured in vitro, there are indications that in more complex systems involving epithelial-mesenchymal interactions, the TGF-beta subtypes differ in their biological activities. To test the hypothesis that TGF-beta subtypes specifically regulate either Meckel's cartilage or tooth morphogenesis, we designed experiments to compare loss of function effects of TGF-beta 1, TGF-beta 2, and TGF-beta 3 subtypes using a serumless, chemically defined medium to culture embryonic mouse E10 (42-44 somite pairs) mandibular explants. The major effect of loss of function resulting from abrogation of TGF-beta 1 using antisense treatment resulted in a 20% increase (P < 0.05) in chondrocyte number, a decrease in extracellular matrix, and dysmorphology of the rostral region of Meckel's cartilage. Exogenous TGF-beta 1 provided indistinguishable recovery to the normal phenotype. TGF-beta 2 antisense treatment produced a threefold enlargement (P < 0.05) of tooth organs and advanced their development to the cap stage. TGF-beta 2 provided recovery to the normal phenotype (e.g., reduced tooth size and development to the bud stage), whereas TGF-beta 1 or TGF-beta 3 polypeptides had no effect. TGF-beta 3 antisense treatment resulted in a reduction of approximately 15% in the length of Meckel's cartilage. We interpret these results to suggest that TGF-beta 1 functions to regulate the number of chondrogenic cells, the amount of extracellular matrix, and the rate of developmental assembly of the rostral to posterior segments in forming Meckel's cartilage. TGF-beta 2 appears to regulate tooth size and stage of development without affecting cartilage. TGF-beta 3 appears to regulate Meckel's cartilage size without altering tooth size or shape. The results are discussed in terms of the regulatory functions of the TGF-beta subtypes during embryonic craniofacial morphogenesis.

Animals↗