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Biomedical subjects

M Lee

Publications and source records attributed to M Lee.

At least 487 records · Page 27Linked to original sources
PubMed1997

Prothrombin synthesis in the adult and fetal liver.

Plasma levels of blood coagulation zymogens are lower in the newborn than in the adult, with the lowest levels being in preterm infants. It is not known if the lower coagulation factor levels reflect differences in synthesis, secretion or catabolism. Using a rabbit model we have compared prothrombin synthesis in the fetus and adult. In previous studies we attempted to compare transcription in the adult and fetal liver by extraction of mRNA, immobilization on a membrane and hybridization with a labeled cDNA for rabbit prothrombin. Comparison was impaired by the markedly dissimilar composition of fetal and adult rabbit liver; fetal liver is approximately fifty percent hematopoietic tissue even at term (1). In the present study, to obtain a more meaningful comparison we have employed in situ hybridization to compare directly prothrombin expression in adult and fetal liver. We report here that fetal liver contains more prothrombin mRNA than does adult liver. We have further compared prothrombin levels in protein extracts of adult and fetal liver and found that per microgram of extract, fetal liver contains as much prothrombin as does the adult. We conclude that the lower plasma prothrombin levels in the fetus do not reflect a lower rate of synthesis.

Animals↗
PubMed1996

Genetic analysis of macromolecular transport across the nuclear envelope.

Numerous factors that promote movement of macromolecules in and out of the nucleus have now been identified. These include both soluble cytoplasmic and nucleoplasmic proteins and proteins of the nuclear pore complex (NPC). Genetic analyses of the nuclear transport process in the model organism, the budding yeast Saccharomyces cerevisiae, have revealed remarkable conservation of all of these factors. In addition, important clues as to how these factors promote the unique bidirectional movement across the NPC have emerged from studies of yeast. We summarize the characterization and genetic interactions of the soluble transport factors and present data to illustrate how genetic experiments can be used to further define the import and export pathways.

Biological Transport, Active↗
PubMed1996

Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy.

Sixteen patients with advanced hematologic malignancies were transplanted with HLA-identical allogeneic peripheral blood stem cells (PBSCs) that were selected for CD34+ cells by an avidin-biotin immunoadsorption technique. The median age of patients was 48 years (range, 37 to 67). Patients received 12.0 or 13.2 Gy of total body irradiation followed by 120 mg/kg of cyclophosphamide. Normal donors received 16 mg/kg of granulocyte-colony stimulating factor on days 1 to 6 followed by PBSC harvests on days 4 to 7. PBSC harvests were processed each day on a single avidin-blotin column containing an antibody to the CD34 antigen and processed cells were infused without cryopreservation daily for 4 consecutive days. Prophylaxis against graft-versus-host disease (GVHD) consisted of cyclosporine alone for 5 patients and CSA plus methotrexate for 11 patients. A median of 18.64 (6.74 to 34.97) x 10(8) CD34+ cells/kg patient body weight were collected from each donor. A median of 8.96 (2.62 to 17.34) x 10(8) CD34+ cells/kg patient body weight were recovered after avidin-biotin adsorption which represented a median CD34+ cell yield of 53% (18% to 77%) with a median purity of 62% (34% to 82%). There was a reduction in CD3+ cells from a median of 557.26 (227.73 to 677.77) x 106/kg to 0.73 x 10(4)/kg (0.40 to 3.65), in CD4+ cells from 351.72 (194.47 to 520.11) x 10(6)/kg to 0.40 (0.15 to 1.03) x 10(4)/kg and in CD8+ cells from 169.74 (53.34 to 325.83) x 10(6)/ kg to 0.32 (0.12 to 2.71) x 10(4)/kg representing a median 2.8 (2.19 to 3.14) log reduction in T cells. One patient died of infection on day 3 posttransplant and was unevaluable for recovery of neutrophils. The median day to recovery of 500 neutrophils/mL was 15 (8 to 26) in the remaining 15 patients. Six of 16 patients falled to achieve a platelet count of 20,000/mL before death on days 3 to 97 of transplant-related complications. The median day to achieving platelets of 20,000 mL in the remaining 10 patients was 11 (7 to 31). Eight of 16 patients (50%) died between 3 and 97 days posttransplant, 7 of transplant-related causes, and 1 of progressive disease. Grade 2-4 acute GVHD occurred in 12 out of 14 (86%) and grades 3-4 in 6 out of 14 (43%) evaluable patients. Six of 8 evaluable patients developed clinical chronic GVHD and 1 developed subclinical chronic GVHD. Bone marrow and/or peripheral blood chimerism studies in 12 evaluable patients showed 97% to 100% donor type in 11 patients with 1 patient in relapse showing 40% donor cells 60 to 90 days posttransplant. Four of 16 patients (25%) are alive and disease-free 312 to 576 days after transplant. There were no episodes of graft failure or rejection. This study shows that allogeneic transplantation using CD34+ selected PBSC results in prompt and sustained engraftment. CD34+ selection, as employed in this preliminary study, however, resulted in an apparently higher rate of acute and chronic GVHD. However, The sample size is quite small and precludes a more definitive conclusion regarding GVHD.

Adult↗
PubMed1996

Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture.

TGF-beta1 is a known inhibitor of branching morphogenesis when added exogenously to mouse embryonic lungs in culture. However, the issue of whether endogenous TGF-beta signaling has a function in the process of lung organogenesis is not completely resolved. We utilized immunoperturbation and antisense oligodeoxynucleotide inhibitory strategies to abrogate TGF-beta type II receptor function in embryonic mouse lungs undergoing branching morphogenesis in serumless explant culture. Antisera directed against a TGF-beta type II receptor N-terminal peptide that perturbs TGF-beta ligand-receptor binding increased branching by 70%. Similarly, antisense TGF-beta type II receptor oligodeoxynucleotides (40 microM) resulted in a 58% increase in branching, compared to scrambled and mismatched sequence controls, while TGF-beta, type II receptor mRNA and its protein expression levels were suppressed by 95 and 84%, respectively. Addition of exogenous TGF-beta1 did not overcome the stimulatory effects either of TGF-beta type II receptor immunoperturbation or of antisense oligodeoxynucleotide treatment on lung branching morphogenesis. Using in situ hybridization and immunohistochemistry, both TGF-beta type II receptor mRNA and protein were localized to the epithelium lining the developing airways, and to the surrounding mesenchyme, indicating that TGF-beta type II receptor is an important regulator of epithelial-mesenchymal interaction. Exogenous TGF-beta1 decreased cyclin A mRNA levels in control embryonic lung explants, while TGF-beta type II receptor antisense oligodeoxynucleotides prevented the downregulation of cyclin A mRNA expression by exogenous TGF-beta1. In addition, PCNA immunostaining of the primitive bronchial epithelium was increased in the presence of TGF-beta type II receptor antisense oligodeoxynucleotides either alone or together with exogenous TGF-beta1, whereas TGF-beta1 alone decreased PCNA staining. Thus, abrogation of TGF-beta type II receptor expression prevented TGF-beta1-induced epithelial cell G1 arrest. These results demonstrate, for the first time, that abrogation of the TGF-beta type II receptor stimulates embryonic lung organogenesis in culture and reverses the negative influence of endogenous TGF-beta signaling upon epithelial cell cycle progression.

Animals↗
PubMed1996

The microtubule-associated protein tau is extensively modified with O-linked N-acetylglucosamine.

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimer's disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk beta(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.

Acetylglucosamine↗
PubMed1996

Calbindin-D28K in the nucleus of nerve growth factor-treated PC12 cells.

Calbindin-D28K (CB) is a calcium-binding protein found within a subset of neurons, which has been proposed to buffer intracellular calcium and to protect cells from calcium-induced neurodegeneration. The rat pheochromocytoma PC12 cell line has been an invaluable tool for studying neuronal development and characterizing the molecular actions of nerve growth factor (NGF). Using Western and RNAse protection analyses and immunocytochemical and cell fractionation techniques, we now report that NGF induces the expression of CB not only in the cytoplasm and neurites, but also within the nuclei of PC12 cells. These data indicate that PC12 cells can serve as a useful model system in which to study the functions of CB and the role of nuclear CB.

Animals↗
PubMed1996

12-Lipoxygenase in A431 cells: genetic identity, modulation of expression, and intracellular localization.

Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells.

Arachidonate 12-Lipoxygenase↗
PubMed1996

Nested retrotransposons in the intergenic regions of the maize genome.

The relative organization of genes and repetitive DNAs in complex eukaryotic genomes is not well understood. Diagnostic sequencing indicated that a 280-kilobase region containing the maize Adh1-F and u22 genes is composed primarily of retrotransposons inserted within each other. Ten retroelement families were discovered, with reiteration frequencies ranging from 10 to 30,000 copies per haploid genome. These retrotransposons accounted for more than 60 percent of the Adh1-F region and at least 50 percent of the nuclear DNA of maize. These elements were largely intact and are dispersed throughout the gene-containing regions of the maize genome.

Chromosomes, Artificial, Yeast↗
PubMed1996

Effects of dietary restriction on experimental gastric mucosal injury in Fischer 344 rats.

The present study was designed to determine whether dietary restriction would render rats more resistant to experimental gastric mucosal injury induced by either aspirin or acidified ethanol. We have found that dietary restriction rendered rats more resistant to experimental gastric mucosal injury, and that dietary restriction prevented decreases in endogenous mucosal antioxidants (such as glutathione) and energy stores (such as adenosine triphosphate) in rat stomachs that had been injured by acidified ethanol. Our findings suggest that dietary restriction may reverse some of the age-related reductions in gastric mucosal protective factors and thereby may render the aged stomach more resistant to experimental injury.

Animals↗
PubMed1996

Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities.

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. To investigate the role of gene dosage in more detail, we have generated yeast artificial chromosome transgenic mice carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. We provide evidence that not only reduced, but also increased levels of transcriptional regulators can cause developmental defects.

Animals↗
PubMed1996

Iron metabolism-related genes and mitochondrial genes are induced during involution of mouse mammary gland.

To understand molecular mechanisms of mammary gland involution, several clones were isolated after the primary differential screening of a total 40,000 pfu of involution-specific cDNA library, and further characterized. The partial sequences and Northern analysis revealed that iron metabolism-related genes and mitochondrial genes were induced during mammary gland involution. The expression of the lactoferrin gene was induced at involution days 1, 2, and 3. The expression of ferritin heavy chain gene was induced at involution days 1, 2, 3 and 4. Cytochrome oxidase subunit 1 and cytochrome oxidase subunit 2 genes were induced at involution days 4 and 7. The expression of cytochrome b gene was induced at involution day 7. These results imply that iron metabolism and mitochondrial function may be altered during mammary gland involution.

Animals↗
PubMed1996

Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.

Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event.

Animals↗
PubMed1996

Tau protein is phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II within its microtubule-binding domains at Ser-262 and Ser-356.

Phosphorylation of tau protein at Ser-262 has been shown to diminish its ability to bind to taxol-stabilized microtubules. The paired helical filaments (PHFs) found in Alzheimer's disease brain are composed of PHF-tau, which is hyperphosphorylated at multiple sites including Ser-262. However, protein kinase(s) able to phosphorylate this site are still under investigation. In this study, the ability of cyclic AMP-dependent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-356, is demonstrated by use of a monoclonal antibody (12E8) which has been shown to recognize tau when these sites are phosphorylated. Cleavage of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thiocyanobenzoic acid, which cuts the protein into essentially two fragments and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphorylates both Ser-262 and Ser-356. In addition, phosphorylation with cAMP-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both had been mutated to alanines, clearly demonstrated that cAMP-PK and CaMKII were able to phosphorylate both sites. Mitogen-activated protein kinase or protein kinase C did not phosphorylate tau at Ser-262 and/or Ser-356. Finally, evidence is presented that phosphorylation of both these sites occurs in cultured nerve cells under certain conditions, indicating their potential physiological relevance.

Amino Acid Sequence↗