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Biomedical subjects

W Ma

Publications and source records attributed to W Ma.

At least 289 records · Page 16Linked to original sources
PubMed1994

Analysis of the anatomical distribution of GAD67 mRNA encoding truncated glutamic acid decarboxylase proteins in the embryonic rat brain.

During development of the central nervous system (CNS) the gene that encodes the 67 kDa form of glutamic acid decarboxylase (GAD) undergoes alternative splicing. The alternatively spliced variants include an exon (referred to as ES, for embryonic stop) that contains a premature stop codon. The detection of mRNA containing the ES exon in embryonic rat brain has been previously reported (Proc. Natl. Acad. Sci., 87 (1990) 8771-8775). We have used in situ hybridization to identify the anatomical distribution of ES mRNA in the embryonic rat brain during two stages of development, embryonic day 17 (E17) and E20. At E17, GAD67 mRNA was expressed in several CNS regions that were destined to contain GABAergic neurons when mature. ES transcripts were predominantly localized to ventricular zones and other regions associated with populations of proliferative cells at E17 and E20. At both ages, however, the alternatively spliced variants were also detected in regions of brain associated with migratory or post-mitotic neurons. GAD67 transcripts that did not include the ES exon were localized to anatomical areas that contained post-mitotic, and often post-migratory neurons. The temporal and spatial disappearance of mRNA containing the ES exon generally followed a caudal-to-rostral gradient which paralleled neuronal terminal mitosis and differentiation.

Animals↗
PubMed1994

New bioactive taxoids from cell cultures of Taxus baccata.

Four new taxoids were isolated from cell cultures of Taxus baccata. Their structures were elucidated by spectroscopic analyses. Two were the aglycones corresponding to previously isolated 7-O-xylosides of taxol C [1] and 10-deacetyltaxol C [2]. The third [3] had an N-methylated side-chain, while the fourth, named taxcultine [4], contained an n-propyl group on the side-chain. All four compounds actively promoted tubulin assembly. Taxol C [1] showed potent and selective cytotoxicity in the NCI human cell line screen.

Antineoplastic Agents, Phytogenic↗
PubMed1994

Yunnanxane and its homologous esters from cell cultures of Taxus chinensis var. mairei.

From cell cultures of Taxus chinensis var. mairei, yunnanxane [2 alpha, 5 alpha, 10-beta triacetoxy-14 beta-(2'-methyl-3'-hydroxyl)-butyryloxy-4(20),11-taxadiene, [1], and four new homologous esters, 2 alpha, 5 alpha, 10 beta, 14 beta- tetra-acetoxy-4(20),11-taxadiene [2], 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-propionyloxy-4(20),11-taxadiene [3], 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-isobutyryloxy-4(20),11- taxadiene [4], and 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-(2'-methyl)-butyryloxy-4(20),11- taxadiene [5] have been isolated. Their structures were determined by spectroscopic methods.

Antineoplastic Agents, Phytogenic↗
PubMed1994

[Experimental gene transfer study using anucleated reticulocytes as target cells].

We report here a novel approach to introduce reporter beta-galactosidase genes into anucleated rabbit reticulocytes. The results indicate that the beta-galactosidase gene was expressed to certain degree in the cytoplasm of the reticulocytes. Our study has provided a useful model system for gene expression studies in anucleated eukaryotic cells.

Animals↗
PubMed1994

[A practical method of whole mount TEM sample preparation and the study of nuclear matrix-intermediate filament scaffolds in K562 cells].

We applied culture plates with bored holes covered with FORMVAR and coated with carbon, to replace the gold grids used in conventional whole mount TEM study. Human erythroleukemia cell line K562 cells plated on the plate surface were extracted using a modified protocol to study scaffolds of nuclear matrix (NM) and intermediate filament (IF). The NM of the K562 cells was found to be composed of interweaving filaments of different diameters, while the cytoplasmic IF were mainly distributed in a radialized pattern. Compared with other techniques of whole mount TEM sample preparations, this one is much more practical and economical, yielding clear NM-IF structures with few artifacts. Study of NM-IF scaffolds in K562 cells might provide a basis for further elucidation of the involvement of NM-IF in the denucleation of mammalian erythroblasts.

Humans↗
PubMed1994

Characterization of the monocyte-specific esterase (MSE) gene.

Carboxylic esterases are widely distributed in hematopoietic cells. Monocytes express the esterase isoenzyme (termed 'monocyte-specific esterase', MSE) that can be inhibited by NaF in the alpha-naphthyl acetate cytochemical staining. We examined the expression of MSE in normal cells and primary and cultured leukemia-lymphoma cells. The MSE protein was demonstrated by isoelectric focusing (IEF); MSE mRNA expression was investigated by Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The following samples were positive for MSE protein and Northern mRNA expression: 20/24 monocytic, 4/32 myeloid, and 1/20 erythroid-megakaryocytic leukemia cell lines, but none of the 112 lymphoid leukemia or lymphoma cell lines; of the normal purified cell populations only the monocytes were positive whereas, T, B cells, and granulocytes were negative; of primary acute (myelo) monocytic leukemia cells (CD14-positive, FAB M4/M5 morphology) 14/20 were Northern mRNA and 11/14 IEF protein positive. RT-PCR revealed MSE expression in 29/49 Northern-negative lymphoid leukemia-lymphoma cell lines. The RT-PCR signals in monocytic cell lines were on average 50-fold stronger than the mostly weak trace expression in lymphoid specimens. On treatment with various biomodulators, only all-trans retinoic acid significantly upregulated MSE message and protein levels but could not induce new MSE expression in several leukemia cell lines; lipopolysaccharide and interferon-gamma increased MSE expression in normal monocytes. Analysis of DNA methylation with sensitive restriction enzymes showed no apparent regulation of gene expression by differential methylation; the MSE gene is evolutionarily conserved among mammalian species; the half-life of the human MSE transcripts was about 5-6 h. The extent of MSE expression varied greatly among different monocytic leukemia samples. However, the MSE overexpression in a significant number of specimens was not associated with gene amplification, gross structural rearrangements or point mutations within the cDNA region. Taken together, the results suggest that MSE expression is not absolutely specific for, but strongly associated with cells of the monocytic lineage; MSE is either not expressed at all or expressed at much lower levels in cells from other lineages. The biological significance, if any, of rare MSE messages in lymphoid cells detectable only by the hypersensitive RT-PCR remains unclear. Further studies on the regulation of this gene and on the physiological function of the enzyme will no doubt be informative with respect to its striking overexpression in some malignant cells and to a possible role in the pathobiology of monocytic leukemias.

Base Sequence↗
PubMed1994

Expression of the FLT3 gene in human leukemia-lymphoma cell lines.

The FLT3 gene encodes a protein that appears to function as a receptor for a hematopoietic growth factor; together with the KIT and FMS receptors, FLT3 belongs to the superfamily of receptors with tyrosine kinase activity. We examined the expression of FLT3 mRNA in 36 human leukemia-lymphoma cell lines using Northern blot analysis. FLT3 transcripts were found in seven of seven pre B-ALL cell lines (derived from cases with pre B-acute lymphoblastic leukemia or chronic myeloid leukemia in lymphoid blast crisis), and in one of six B-cell lines (namely in a cell line established from a hairy cell leukemia). FLT3 message was not detected in five T-cell, five myeloid, four monocytic, four erythroid and five megakaryocytic cell lines. Two major mRNA species were expressed differentially by positive cell lines. KIT mRNA expression was also investigated in the same panel of cell lines, but was found only in cell lines with erythroid and megakaryocytic features (and not in any of the FLT3-positive cell lines). The pattern of expression of FLT3 contrasts with the transcription of FMS and KIT and suggests that the FLT3 product may play a role primary in immature lymphoid cells.

Blotting, Northern↗
PubMed1994

Sensitivity of different methods for the detection of myeloperoxidase in leukemia cells.

We examined the sensitivity of different myeloperoxidase (MPO) detection methods in leukemia cell lines. To this end the MPO-positive acute promyelocytic leukemia cell line NB-4 was diluted into cell populations of the MPO-negative myeloma cell line MM-1 at different ratios. MPO protein was identified by classical cytochemical staining and by a specific anti-MPO monoclonal antibody in an immunofluorescent reaction. Cytochemical staining detected 1% positive cells among 99% negative cells. Careful, but time-consuming observation enabled the detection of positive cells in even higher dilutions. At least a 10-fold increase in sensitivity was achieved with the immunofluorescent method, as brightly fluorescent cells are more amenable for a screening of slides at lower microscopic magnification than the cytochemically visualized cells. MPO mRNA expression was examined in whole cell populations by Northern blotting (maximal sensitivity 1%), a reverse transcriptase-polymerase chain reaction (RT-PCR) amplification assay (sensitivity 0.1%), and by RT-PCR followed by Southern blotting (sensitivity 0.05%). The high sensitivity of PCR-based techniques is offset by the fact that these methods do not allow for the identification and further characterization of the individual, MPO-positive cells. Thus, methods examining bulk populations require homogeneous cell samples in order to avoid false-positivity stemming from a few residual bystander cells. The five different techniques were used to determine the status of MPO expression in 20 randomly chosen leukemia cell lines of myelomonocytic origin. In 11 cell lines (8 positive and 3 negative) all five tests provided concordant results. Three cell lines were Northern-negative, but RT-PCR-positive and MPO protein-positive suggesting that Northern blot analysis is the least sensitive tool. Six cell lines were devoid of MPO protein, at least according to the methods used here, but trace expression of MPO message was documented by PCR. All five techniques have advantages and drawbacks and must be carefully selected in order to obtain useful data. The detection of MPO is of experimental and clinical importance in the distinction of myeloid from lymphoid leukemias, and in the lineage assignment of apparently biphenotypic or unclassifiable cases.

Blotting, Northern↗
PubMed1993

Ontogeny of GABAA receptor subunit mRNAs in rat spinal cord and dorsal root ganglia.

Relatively little is known about the development of GABAA receptor subunits and their gene expression in mammalian spinal cord. The expression of mRNAs encoding 13 GABAA receptor subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) in embryonic, postnatal, and adult rat spinal cord and dorsal root ganglia (DRG) cells were studied by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Both techniques revealed the presence of all subunit mRNAs originally found in the rat brain, except for alpha 6, which was not detectable, and delta, which was weakly detected only by RT-PCR. Two anatomically distinctive sets of subunit mRNAs were found by in situ hybridization within the ventricular zone (VZ) and mantle zone (MZ). The trio of alpha 4, beta 1, and gamma 1 subunit mRNAs emerged exclusively in neuroepithelial cells at embryonic day 13 (E13) and remained detectable in the VZ until E17. In the MZ, beta 3 subunit mRNA was first detected at E12, while alpha 2, alpha 3, alpha 5, beta 2, gamma 2, and gamma 3 transcripts appeared at E13. Expressions of the subunit mRNAs in the MZ rapidly increased and expanded in a ventrodorsal sequence from motoneurons to dorsal horn neurons before reaching a peak in the late embryonic/early postnatal period. The mRNA expressions declined during postnatal development, by region-selective depletion, with alpha 4, alpha 5, beta 1, beta 2, gamma 1, and gamma 3 subunit mRNAs becoming barely detectable. In contrast, alpha 2, alpha 3, beta 3, and gamma 2 transcripts persisted into adulthood with distinct anatomical distributions. RT-PCR analysis revealed unique developmental patterns in the intensities of PCR products, most of which were in good agreement with developmental changes in the densities of hybridized mRNA signals. However, RT-PCR amplified minute amounts of mRNAs for alpha 1, alpha 4, alpha 5, beta 1, beta 2, gamma 1, gamma 3, and delta subunits in adults, which were not found in film autoradiograms, but could be detected in a few grain-positive cells in emulsion-dipped sections. DRG cells expressed alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2 subunit mRNAs during embryogenesis but only alpha 2, beta 3, and gamma 2 subunit mRNAs were reliably detected in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Myeloperoxidase: expression and modulation in a large panel of human leukemia-lymphoma cell lines.

Myeloperoxidase (MPO) is found exclusively in the azurophilic granules (primary lysosomes) of normal myelomonocytic cells. Cytochemical staining for MPO activity is used clinically to distinguish myeloid from lymphoid leukemias. We studied the expression of MPO at the RNA and protein level in 140 continuous human leukemia-lymphoma cell lines using classical cytochemistry, immunofluorescent staining with a specific monoclonal antibody, Northern blot analysis, and a reverse transcription-polymerase chain reaction (RT-PCR) amplification assay. Seventy-eight lymphoid leukemia, myeloma, and lymphoma cell lines were negative; only 3 pre-B-acute lymphoblastic leukemia (ALL) cell lines were MPO-positive. Two of these MPO-positive pre-B-ALL cell lines showed a trace expression after RT-PCR and Southern blotting corresponding to 4% to 6% of the transcripts found in other positive myeloid cell lines. The third pre-B-ALL cell line was positive in Northern blots and cytochemical/immunofluorescent staining; however, only few cells were weakly positive in the latter assay. Although 15 of 59 cell lines assigned to the myeloid, monocytic, megakaryocytic, or erythroid lineages were MPO-positive in Northern blots, those 15 and 13 additional cell lines showed bands of mRNA after RT-PCR. MPO protein was detected in all 16 Northern-positive cell lines; on the other hand, there were 4 cell lines that were protein-positive, but Northern-negative. Differentiation induced by protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1 or by all-trans retinoic acid was associated with a decrease in MPO mRNA in all 7 initially positive cell lines studied, even leading to the complete absence of transcripts, but the enzymatic activity of the differentiated cells was only slightly less than that of unstimulated cells. MPO expression could not be induced in 10 initially negative cell lines. The half-life of MPO mRNA was found to be about 6 hours and was not shortened by prior exposure of the cells to the differentiation-inducing agents. These results confirm that MPO expression is mainly associated with myelomonocytic cells, but also underline the notion that MPO cannot be used as an absolutely lineage-specific marker for the distinction of leukemic cells. MPO can be used as an excellent parameter to characterize the various stages of normal and induced differentiation.

Base Sequence↗
PubMed1993

A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus.

In eukaryotic cells, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a signaling pathway from the ER to the nucleus. Several yeast mutants defective in this pathway map to the ERN1 gene, which protects cells from lethal consequences of stress by signaling for increased expression of BiP and other ER proteins. ERN1 encodes a 1115 amino acid transmembrane protein (Ern1p) whose glycosylated N-terminal portion is located inside microsomes and whose cytoplasmic C-terminal portion carries an essential protein kinase activity. We postulate that Ern1p is the proximal sensor of events in the ER and that binding of ligand causes transduction of information across the ER membrane, leading to activation of a specific set of transcription factors.

Amino Acid Sequence↗
PubMed1993

Analysis of taxol and related diterpenoids from cell cultures by liquid chromatography-electrospray mass spectrometry.

Authentic taxanes (taxol, 10-deacetyltaxol, cephalomannine, 10-deacetylcephalomannine, baccatin III) and extracts from cell cultures derived from various yew tree species have been analyzed by microbore high-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESMS). All gave excellent positive-ion ES spectra with dominant protonated molecules at low nozzle-to-skimmer bias value (45 V). By increasing the voltage value to 85 V, fragmentation increased and structurally informative spectra were obtained. The fragments found were both of the C-13 side-chain and of the taxane ring, so their analysis gave important information about the taxane structure and any chemical modifications at different positions of the molecule. When tandem MS was used (argon gas, 25 eV collision energy), fragments similar to those obtained from collision-induced dissociation in the source were detected. The cell culture extracts were analyzed by microbore HPLC-ESMS and excellent spectra were obtained on 5-10 ng of separated compounds; even greater selectivity and sensitivity were obtained through use of selected-ion monitoring (SIM). With SIM, 100 pg of all taxanes could readily be detected. In the HPLC-ESMS mode, only 10% of the eluent was mass-analyzed, so 90% would be available for recovery through fraction collecting.

Cells, Cultured↗
PubMed1993

Taxoid side-chain structure determination by electrospray ionization tandem mass spectrometry.

A method to elucidate the side-chain structure of novel taxoids was developed through use of electrospray ionization tandem mass spectrometry. The intact side-chain fragments produced by source fragmentation in the atmosphere-to-vacuum interface were further dissociated in the collision cell of a triple quadrupole mass spectrometer. The distinctive collisionally induced dissociation mass spectra were remarkably informative and gave detailed information on the side-chain functionalities. This technique proved valuable in the characterization of new taxoids produced by yew-tree cell cultures.

Alkaloids↗
PubMed1993

External craniofacial features, body size, and renal morphology in prenatal brachyrrhine mice.

The Brachyrrhine (Br) semidominant mouse mutant provides a useful model for studying factors responsible for midfacial hypoplasia. In order to determine early morphogenetic events responsible for midfacial hypoplasia in these mice, prenatal mutants must be differentiated from nonaffected littermates. The purpose of this study was to determine whether prenatal offspring from Br matings could be separated morphologically into groups of normal or midfacially deficient embryos. Thirty embryos resulting from Br matings were collected between day 15 and birth (Theiler stages 23-27). Qualitative observations on craniofacial morphology as well as renal morphology and histology suggested that embryos segregated into two distinct groups. External craniofacial and body measurements, collected from the embryos, as well as renal volumes, determined from computerized reconstructions of the kidneys, were subjected to quantitative analyses. A discriminant function analysis segregated the sample into two groups based primarily on midfacial length and renal volume. Bivariate regression analysis showed that midfacial length and renal volume differed significantly, and cranial length marginally so, between the two groups. The results indicate that a proportion of prenatal offspring from Br matings exhibits midfacial and renal hypoplasia and that these animals segregate completely from nonaffected embryos.

Abnormalities, Radiation-Induced↗
PubMed1993

Expression and modulation of annexin VIII in human leukemia-lymphoma cell lines.

Annexin VIII is a calcium- and phospholipid-binding protein with anticoagulant activity. Annexin VIII mRNA was found to be specifically expressed in acute promyelocytic leukemia (APL) cells; it was not found in other types of acute myeloid leukemia (AML) nor in lymphoid malignancies. Using Northern blot analysis we investigated annexin VIII expression in 142 continuous human leukemia and lymphoma cell lines at the mRNA level. While the only APL cell line, NB-4, was indeed positive, other cell lines also displayed annexin VIII mRNA: 4/22 myeloid cell lines, 8/23 monocytic cell lines, 2/8 megakaryoblastic cell lines, 5/26 lymphoma-derived cell lines, 2/10 myeloma cell lines and 1/44 lymphoid leukemia cell lines. The strongest expression was seen in NB-4 and in the Hodgkin's disease derived cell line HDLM-2. Treatment of NB-4 cells with all-trans retinoic acid (ATRA) or the phorbol ester TPA induced terminal differentiation and down-regulated annexin VIII mRNA expression rapidly within a few hours; vitamin D3 was ineffective in this regard; the protein kinase C activator Bryostatin 1 up-regulated the expression. A panel of initially negative cell lines could not be induced by any of these biomodulators to transcribe annexin VIII. The half-life (T1/2) of annexin VIII mRNA was about 3-4 h using actinomycin D as transcription inhibitor. Treatment with ATRA or TPA prior to exposure to actinomycin shortened the T1/2 to 2 h while Bryostatin 1 extended it to 6h. As 21/141 non-APL cell lines were positive, annexin VIII cannot be used as a marker gene for APL cells; however, it might be associated with myelomonocytic or erythro-megakaryoblastic precursor cells. Annexin VIII gene expression might play a unique role in the proliferation and/or differentiation of leukemic cells and could be associated with the particular abnormal hemostasis of some leukemias.

Annexins↗
PubMed1993

Use of the gamma-ray perturbed angular correlation (PAC) technique for monitoring liposomal phospholipid bilayer integrity.

A membrane labeling method based on the principle of gamma-ray perturbed angular correction (PAC) was developed to monitor the structural integrity of liposomal membranes. The reporter group was 111In(III) complexed with the lipophilic diethylenetriaminepentaacetic acid (DTPA) derivative of dipalmitoylphosphatidylethanolamine (DPPE) embedded in the phospholipid bilayers of small unilamellar liposomes. Using this method, complete chemical digestion of the constituent phospholipids in these DTPA-conjugated liposomes by phospholipase A2 or phospholipase C in the presence of Ca2+ was found not to be followed by an immediate disruption of the liposomal membrane. Compared with other methods, the method developed permits the continuous noninvasive monitoring of the microenvironment of the lipid bilayer at the molecular level. It may potentially be applicable to evaluate liposomal fusion, screen for penetration enhancers under development for enhancement in mucosal drug penetration, and monitor liposomal degradation within the living animal.

Gamma Rays↗
PubMed1993

A fluorescence quenching method for estimating chelating groups in chelate-conjugated macromolecules.

A terbium-dipicolinic acid (Tb-DPA) fluorescence quenching method for estimating free chelating groups conjugated to protein molecules was developed. This method was based on competitive displacement of DPA from binding to terbium by stronger chelating groups such as diethylenetriaminepentaacetic acid (DTPA), EDTA, nitrilotriacetic acid (NTA), DTPA-conjugated bovine serum albumin (BSA-DTPA), or DTPA-conjugated immunoglobulin G (IgG-DTPA), resulting in a significant reduction in terbium fluorescence. The chelating ability of the tested reagent, from high to low, was in the following order: BSA-DTPA > DTPA > IgG-DTPA > EDTA, NTA. At low terbium concentrations, the reduction was linear for DTPA. This fluorescence quenching method was not only rapid, simple, and as accurate as conventional radiosotopic or chromatographic methods, but also sensitive and reproductible. The detection limit was 10 nM for DTPA. The interrun coefficient of variation was at most 8%. The advantage of this method over other indirect methods is that it reveals the actual chelating ability of the tested macromolecule, unencumbered by complicating factors such as trace metal contamination and dimer/polymer formation during conjugation.

Chelating Agents↗