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Biomedical subjects

W Leung

Publications and source records attributed to W Leung.

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

Human hematopoietic stem/progenitor cells generate CD5+ B lymphoid cells in NOD/SCID mice.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation model is increasingly utilized to study both human lymphohematopoietic stem/progenitor cells and committed cell types. Human B lymphoid cells develop and proliferate in this model. We found high numbers of CD19+CD5+ B lymphoid cells in the bone marrows and spleens of NOD/SCID mice transplanted with human CD34+ stem/progenitor cells. The CD5+ cells accounted for a particularly large percentage of the B lymphoid cells in the spleens of chimeras analyzed three months after transplantation. CD19+CD5+ cells from all the analyzed chimeras coexpressed HLA-DR, surface IgM, CD20, CD38, CD43, and CD45. However, CD19+CD5+ cells were negative for kappa light chain, CD10, CD11a, CD11b, CD15, CD21, CD22, CD23, CD25, CD34, CD35, CD44, CD62L, CD69, and CD71. Cell surface expression of the lambda light chain, surface IgD, CD9, and CD40 antigens was detected in some but not all chimeras. Thus, the CD19+CD5+ cell population detected in our study has the phenotype of previously described CD5+ B lymphoid cells in humans and other species. The origin and role of the B lymphoid cells which express CD5 cell surface glycoprotein are poorly understood. The malignant cells in B lymphoid chronic lymphocytic leukemia express CD5, and the numbers of CD5+ B lymphoid cells are elevated in several autoimmune conditions. The human-NOD/SCID chimera system may provide an in vivo model to investigate the maturation and development of this cryptic human CD5+ B lymphoid cell subpopulation.

Animals↗
PubMed1999

A human-murine chimera model for in utero human hematopoietic stem cell transplantation.

To date, 16 in utero hematopoietic stem cell (HSC) transplants for diseases other than immunodeficiency disorders have been reported. No therapeutic level of engraftment was detected in 15 of these transplants. To overcome engraftment failure, we transplanted a very large number (5 billion paternal CD34+ cells/kg) of HSCs to a fetus with leukodystrophy during the first trimester of gestation. As reported previously, the fetus died in utero 7 weeks after the procedure and the cause of death appeared to be overwhelming donor engraftment. In the present investigation, we developed a human-murine chimera model to test for the optimal donor cell dose for human in utero transplantation. We found a strong correlation between the level of donor engraftment in three human fetuses transplanted for leukodystrophy during the first trimester of gestation and the results of parallel xenotransplants of the same human donor cells using the NOD/SCID mouse model. This small animal model appears to predict both extremes of hyperengraftment (seen in the first human fetus transplanted) and engraftment failure (seen in the second and third human fetuses transplanted in utero). These and future correlated clinical and laboratory assay results may be useful for the development of in utero transplants for a variety of congenital disorders.

Animals↗
PubMed1999

Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood.

Cord blood (CB) and autologous mobilized peripheral blood stem/progenitor cells (PBSC) are now used widely for clinical transplantation. We characterized the short-term (<8 weeks) and long-term (>8 weeks) engraftment in NOD/SCID mice resulting from transplanted CD34+ cells from these two sources. We also quantified the frequency of long-term engrafting cells, and the average proliferative capacity of individual engrafting cells by a competitive repopulation assay with binomial variance-covariance modeling. We found that 0.5 million CD34+ CB cells were able to generate sustained, high-level, multilineage human hematopoiesis, whereas a sixfold higher number of CD34+ PBSC (3 million) from cancer patients undergoing chemotherapy generated comparable short-term, but much lower sustained multilineage human hematopoiesis after transplantation. In comparison to CD34+ cells from PBSC from cancer patients, long-term engrafting cells were approximately eightfold enriched in CB CD34+ cells, and each CB long-term engrafting cell had an approximately 15-fold higher multilineage proliferative capacity. Thus, the number and function of transplantable hematopoietic cells were remarkably different between these two sources of stem/progenitor cells.

Animals↗
PubMed1999

Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros.

Development of the embryonic kidney results from reciprocal signaling between the ureteric bud and the metanephric mesenchyme. To identify the signaling molecules, we developed an assay in which metanephric mesenchymes are rescued from apoptosis by factors secreted from ureteric bud cells (UB cells). Purification and sequencing of one such factor identified the tissue inhibitor of metalloproteinase-2 (TIMP-2) as a metanephric mesenchymal growth factor. Growth activity was unlikely due to TIMP-2 inhibition of matrix metalloproteinases because ilomastat, a synthetic inhibitor of these enzymes, had no mesenchymal growth action. TIMP-2 was also involved in morphogenesis of the ureteric bud, inhibiting its branching and changing the deposition of its basement membrane; these effects were due to TIMP-2 inhibition of matrix metalloproteinases, as they were reproduced by ilomastat. Thus, TIMP-2 regulates kidney development by at least 2 distinct mechanisms. In addition, TIMP-2 was secreted from UB cells by mesenchymal factors that are essential for ureteric bud development. Hence, the mesenchyme synchronizes its own growth with ureteric morphogenesis by stimulating the secretion of TIMP-2 from the ureteric bud.

Animals↗
PubMed1998

Temperature and pH-dependent supramolecular self-assembly of amelogenin molecules: a dynamic light-scattering analysis.

Evidence for the molecular self-assembly of amelogenin proteins to form quasi-spherical particles ("nanospheres") in solution, both in vitro and in vivo, has recently been documented. A particle-size distribution analysis of dynamic light-scattering data was undertaken to investigate the influence of temperature on this molecular self-assembly process at three different pH's. The long-term objective was to correlate these observations to the unusual physiochemical characteristics of the protein, to improve understanding of the molecular mechanisms involved in the generation of amelogenin "nanospheres" and understanding of their putative relation to amelogenin function in vivo. We analyzed data using two different algorithms: Dynamics and DynaLS. It was found that at pH 8, in a temperature range between 5 and 25 degrees C, the size of the recombinant amelogenin nanospheres is monodisperse, giving rise to particles of 15-18 nm in hydrodynamic radius. However, heterogeneous distribution of particle size was observed at temperature ranges between 27 and 35 degrees C, becoming monodisperse again with larger particles (60-70 nm) after the temperature was elevated to 37-40 degrees C. We interpret these results to suggest that amelogenin molecular self-association possesses a second stage assembly process at temperatures of 30-35 degrees C, creating larger entities which apparently are structured and stable at 37-40 degreesC. The effect of pH on the size of amelogenin "aggregates" was much more noticeable at 37 degrees C compared to that at 25 degrees C. This observation suggests that at physiological temperature (i.e., 37 degrees C) amelogenin molecular self-assembly is extremely sensitive to pH changes. This finding supports the notion that local pH changes in the microenvironment of the enamel extracellular matrix may play critical roles in controlling the structural organization of the organic matrix framework.

Amelogenin↗
PubMed1998

Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing's sarcoma.

Many poor-risk neuroblastomas and tumours of the Ewing's sarcoma family (ET) recur despite autologous transplants. Recurrence may be due to tumor cells contained in the BM harvests or PBSC harvests. The objectives of this prospective study were to: (1) determine the incidence and degree of tumor cell contamination in paired BM and PBSC harvests; and (2) determine the efficacy of tumor cell purging by immunomagnetic CD34+ cell selection. 198 samples from 11 consecutive patients with neuroblastoma or Ewing's sarcoma were analyzed. We assayed tumor contamination by RT-PCR assay for PGP 9.5, plus immunohistochemistry for neuroblastoma-specific antigens (the latter in neuroblastoma only). None of these patients had tumor cells detected in their BM by clinical histology immediately before BM or PBSC harvests. However, 82% of PBSC and 89% of backup BM harvests were contaminated with tumor by RT-PCR and/or immunocytochemistry assays. Unselected PBSC and BM harvests contained similar quantities of tumor cells (median, approximately 200000 cells). Cyclophosphamide plus G-CSF mobilization did not affect the incidence or level of contamination in PBSC harvests, as compared to blood obtained before mobilization. Immunomagnetic CD34+ cell selection depleted tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for BM harvests.

Adolescent↗
PubMed1998

Classification and genetic characterization of pattern-forming Bacilli.

One of the more natural but less commonly studied forms of colonial bacterial growth is pattern formation. This type of growth is characterized by bacterial populations behaving in an organized manner to generate readily identifiable geometric and predictable morphologies on solid and semi-solid surfaces. In our first attempt to study the molecular basis of pattern formation in Bacillus subtilis, we stumbled upon an enigma: some strains used to describe pattern formation in B. subtilis did not have the phenotypic or genotypic characteristics of B. subtilis. In this report, we show that these strains are actually not B. subtilis, but belong to a different class of Bacilli, group I. We show further that commonly used laboratory strains of B. subtilis can co-exist as mixed cultures with group I Bacilli, and that the latter go unnoticed when grown on frequently used laboratory substrates. However, when B. subtilis is grown under more stringent semiarid conditions, members of group I emerge in the form of complex patterns. When B. subtilis is grown under less stringent and more motile conditions, B. subtilis forms its own pattern, and members of group I remain unnoticed. These findings have led us to revise some of the mechanistic and evolutionary hypotheses that have been proposed to explain pattern growth in Bacilli.

Bacillus↗
PubMed1998

Quantitative analysis of amelogenin solubility.

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habits of forming enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins at various pH (from 4.0 to 9.0) at constant ionic strength (IS), and to examine the influence of buffer composition, IS, and divalent metal ions (including Ca2+, Mg2+, and Zn2+) on amelogenin solubility. The solubility of the recombinant murine amelogenin ("rM179") was minimum near its isoelectric point and increased rapidly below and above, regardless of buffer composition. A similar trend was observed for the native porcine ("25K") amelogenin. Porcine "23K" amelogenin was only sparingly soluble from pH of 4.0 to 9.0, in contrast to the analogous recombinant "rM166", which was more soluble in acidic solutions. The synthetic amelogenin polypeptide "TRAP" was extremely insoluble, while synthetic LRAP was readily soluble. Porcine "20K" amelogenin solubility increased strikingly as the solution pH was lowered from 7.0 to 6.0. Increasing IS decreased the solubility of rM179. While Zn2+ reduced rM179 solubility, Ca2+ and Mg2+ showed no significant effects. We conclude that the solubility of amelogenin was dependent on the primary structure, solution pH, and IS, and the low solubility of amelogenins under physiological conditions may result from their tendency to form quaternary (aggregate) structures in vivo.

Amelogenin↗
PubMed1998

The pH dependent amelogenin solubility and its biological significance.

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habit of enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins in various pH (4.0-9.0) solutions with an ionic strength (IS) of 0.15 M using the Micro BCA protein assay at 25 degrees C or 37 degrees C. The solubility of the recombinant amelogenin rM179 was lowest (0.7 mg/ml) close to its isoelectric point and it increased below and above this point. The solubility of the recombinant amelogenin rM166 remained almost the same (1-2 mg/ml) as the pH rose from 6.0 to 9.0 and it increased as the solution became more acidic. Synthetic "tyrosine-rich amelogenin polypeptide" (TRAP) was extremely insoluble (<0.2 mg/ml) in the pH range studied while synthetic "leucine-rich amelogenin polypeptide" (LRAP) was readily soluble (>3.3 mg/ml). The native porcine amelogenin with apparent molecular weight 25 kDa shared similar solubility behavior to rM179. The porcine 23 kDa amelogenin was only sparingly soluble (0.3-0.8 mg/ml) over a wide range of pH. Interestingly, the porcine 20 kDa amelogenin was remarkably soluble in the pH range of 4.0 to 6.0 (approximately 12 mg/ml), but the solubility dropped strikingly to only approximately 0.2 mg/ml at pH larger than approximately 7.0. The strong dependence of amelogenin solubility on solution pH may be involved in the regulation of aggregation, enzymatic degradation and the binding properties of amelogenins, thus playing an important role in enamel biomineralization.

Amelogenin↗
PubMed1998

Does amelogenin nanosphere assembly proceed through intermediary-sized structures?

We have proposed that these nanosphere structures are functionally involved in the organization and control of initial enamel biomineralization at the ultrastructural level. Based on the observed nanosphere hydrodynamic radii (18-20 nm diameter) computation suggests these structures to be compounded of some 100 amelogenin monomers, raising the question as to the possible molecular mechanism for the assembly of such structures? Based on recent dynamic light scattering experiments using the recombinant murine amelogenin M179, and employing a newer size distribution algorithm we now report that the size distribution data for M179 are better described by a bimodal distribution model, than the monomodal distribution as previously described. We suggest that amelogenin nanosphere assembly proceeds through intermediate structures (perhaps represented in vivo by "stippled material") of some 4-5 nm hydrodynamic radius, and computed to comprise 4-6 amelogenin monomers. We suggest that such intermediary, sub-unit structures, assemble through inter-molecular hydrophobic interactions to generate the 20 nm diameter nanospheres observed by TEM in the secretory stage enamel matrix.

Amelogenin↗
PubMed1998

Effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro.

The objective of the present study was to determine the effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro. Current experimental data, together with previous reports support the view that among the different proteinases present in the enamel extracellular matrix, serine proteinase(s) are responsible for the massive degradation of amelogenins during the maturation stage. For our in-vitro experiments we used the recombinant amelogenin M179 as substrate and a "65%-satd. (NH4)2SO4" fraction of enamel proteins as well as chymotrypsin as sources for serine-proteinase activity. We report preliminary experiments of amelogenin proteolysis in the presence of apatite crystals resulting in a different proteolysis pattern when compared to amelogenin proteolysis without apatite crystals. Quantitative analysis of the HPLC peaks corresponding to the proteolysis products indicates that the presence of apatite crystals in the proteolysis solution inhibits the ability of the serine-proteinases to degrade amelogenin. The present observations support the hypothesis that amelogenin degradation correlates with apatite crystal growth during enamel maturation.

Amelogenin↗
PubMed1998

In vivo engraftment potential of clinical hematopoietic grafts.

BACKGROUND: Quantitative assays are needed to characterize the multilineage engraftment potential of clinical hematopoietic grafts. After we observed a dose-response relationship between the number of human hematopoietic cells transplanted into nonobese diabetic-scid/scid (NOD/SCID) mice and the number of human CD45+ cells recovered in the chimeras' marrows and spleens, we sought to develop a multiple linear regression model that allows quantitative comparisons of human cell engraftment in vivo. METHODS: We used this NOD/SCID xenotransplant model to compare the engraftment potential of cord blood vs. adult marrow or mobilized blood, after either of 2 commonly used clinical graft engineering procedures: CD34+ cell selection or T cell depletion. RESULTS: The engraftment per transplanted cell was > 20 fold higher for cord blood cells, as compared to hematopoietic cells from adults. However, there was no difference in engraftment per CD34+ cell transplanted between marrow and mobilized blood. Levels of human cell engraftment from all sources could be increased by administration of human hematopoietic growth factors to human/mouse chimeras after transplantation. Correlation analysis of the number of human CD13+ myeloid cells and CD19+ B lymphoid cells in the chimeras' marrows 8 weeks after transplantation provided evidence that almost all the human myeloid and B lymphoid cells were derived from the same primitive precursor cells. CONCLUSIONS: These findings and assay may be useful in the development of clinical hematopoietic cell therapies.

Adult↗
PubMed1997

Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction.

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Saccharomyces cerevisiae, by expression of the site-specific HO endonuclease. Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment. This occurred at a frequency of only 2 x 10(-4), despite the fact that nearly all cells successfully repaired, by single-strand annealing, the chromosome break created by liberating the fragment. The frequency of Leu+ recombinants was 6- to 25-fold higher in pms1 strains lacking mismatch repair. In 70% of these cases, the colony was sectored for Leu+/Leu-. Similar results were obtained when a 4. 1-kb fragment containing adjacent LEU2 and ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutations on the chromosome. These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each close to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire region, by the assimilation of one of the two strands of the linear duplex DNA. Moreover, the recovery of Leu+ transformants is frequently defeated by the cell's mismatch repair machinery; more than 85% of mismatches in heteroduplex DNA are corrected in favor of the resident, unbroken (mutant) strand.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Mammographic density in women on postmenopausal hormone replacement therapy.

BACKGROUND: Studies have suggested that mammographic density and pattern are affected by hormone replacement therapy (HRT) and may influence breast diagnosis. Because 40% of breast cancers diagnosed at our center are mammographically detected while still clinically occult, mammographic sensitivity is crucial. For this reason we studied the effect of HRT on mammographic density. METHODS: During a period of 18 months we studied consecutive women older than 54 years attending for breast screening. We recorded HRT use and dosing regimes. A breast density score (BDS) was developed and applied to all mammograms. RESULTS: Mammograms of 148 HRT users were compared with those of 158 nonusers. HRT users had a significantly higher mean density score (4.7 versus 3.4; p < 0.001). Only 11% of non-HRT users had high scores compared with 37% among HRT users (p < 0.001). The significant difference remained when women were stratified by age. Duration of HRT (longer or shorter than 5 years) did not affect density scores. CONCLUSIONS: HRT is associated with a significant increase in breast density. In turn, density and mammographic sensitivity are related. The possibility that increased breast density will hamper mammographic diagnosis of clinically occult cancers is worrisome.

Age Factors↗
PubMed1997

An uncommon urologic presentation of a supernumerary breast.

Polythelia and polymastia are common developmental abnormalities of the breast and nipple which usually present as small lesions along the mammary line, an embryologic line that extends bilaterally from the axillary regions to the inguinal ligaments. These lesions have been reported in various locations outside the mammary line. We report an unusual location of polymastia in the perineum of a newborn male. A brief discussion of the clinical relevance of such lesions is also included.

Breast↗
PubMed1996

Identification of a novel proteinase (ameloprotease-I) responsible for the complete degradation of amelogenin during enamel maturation.

During enamel formation the proteins of the extracellular matrix, particularly amelogenins, are removed prior to maturation. In order to investigate this process and to improve our understanding of the function of proteinases during enamel maturation, proteinase fractions were isolated from developing pig enamel and assayed for proteolytic activity in vitro. A recombinant murine amelogenin, M179, was used as a substrate. Two major groups of enamel proteinases were defined as high-molecular-mass ['high-molecular-weight' in Moradian-Oldak, Simmer, Sarte, Zeichner-David and Fincham (1994) Arch. Oral Biol.39, 647-656] and low-molecular-mass proteinases. Here we report the characterization of one of the proteinases present in the low-molecular-mass group. We demonstrate that this proteinase is a serine proteinase capable of degradation of M179 following cleavage of the tyrosine-rich amelogenin polypeptide from the N-terminal region. A partial N-terminal sequence of the proteinase was obtained (LPHVPHRIPPGYGRPXTXNEEGXNPYFXFFXXHG). An anti-peptide antibody directed against a synthetic peptide corresponding to the first 14 amino acids of the above sequence was produced. The presence of the proteinase in the acetic acid extract was confirmed by Western blotting. Searching using the amino acid sequence determined in this study showed it to be also present in the 32 kDa and 89 kDa enamelin proteins reported by Fukae, Tanabe, Murakami and Tohi [(1996) Adv. Dent. Res., in the press]. We therefore identify the 32 kDa enamelin as an enamel proteinase ('ameloprotease-I') which is responsible for amelogenin degradation in maturing enamel. We propose that the 89 kDa enamelin is a precursor of ameloprotease-I, the first enamel protein for which a function has been defined.

Amelogenin↗
PubMed1994

Magnetic resonance imaging of radiation necrosis after radiotherapy for acromegaly: report of a case.

A total 5,600 cGy in 28 fractions in 44 days was delivered to a 53-year-old woman with acromegaly after transsphenoid removal of a pituitary adenoma. Sixteen months later, she suffered a progressive loss of vision. Radiation optic neuropathy was diagnosed. A T1-weighted imaging showed gadolinium-enhanced lesions in the optic chiasm, pituitary stalk and hypothalamus. Magnetic resonance imaging is uniquely suited to detect radiation injury to the sellar region. The complications of radiation therapy for benign tumor can be severe even by current standard procedures.

Acromegaly↗