Biomedical subjects

D M Nelson

Publications and source records attributed to D M Nelson.

At least 19 recordsLinked to original sources

PubMed1992

Isolation and expression of a murine purine nucleoside phosphorylase-encoding cDNA and sequence similarity with the human message.

To isolate murine purine nucleoside phosphorylase-encoding cDNA sequences (PNP), a murine BALB/c liver cDNA library in lambda gt10 was screened for recombinants hybridizing to a human PNP cDNA probe. Two of three clones recovered included inserts large enough to contain the full-length coding sequence. Sequence analysis of the largest clone revealed an 867-nucleotide open reading frame encoding 289 amino acids with 84% residue identity to that encoded by human PNP and 351 bp of 3'-untranslated region. The 5' end of the murine PNP message was specifically amplified by PCR using the RACE (rapid amplification of cDNA ends) protocol, revealing a 5'-untranslated region of 78 bp. Northern hybridization using the murine PNP cDNA sequence as a probe identified a message of approx. 1.6 kb in mouse NIH3T3 cells which was slightly smaller than the human message observed in HeLa cells. The cloned murine PNP cDNA coding sequence was inserted into a mammalian expression vector under transcriptional regulation of the Moloney murine leukemia virus long terminal repeat. Transfection of this plasmid into human 293 cells resulted in the expression of PNP activity which co-focused with murine PNP activity extracted from NIH3T3 cells, verifying that the isolated murine PNP cDNA clone encoded catalytically active PNP protein.

3T3 Cells↗

PubMed1992

Induction of manganese superoxide dismutase in cultured human trophoblast during in vitro differentiation.

The antioxidant responses of human cell differentiation and membrane fusion are not known and may be important in understanding cellular response to injury in the human placenta. We studied the regulation of antioxidant enzymes in human trophoblasts which differentiate from mononucleated cellular trophoblasts to synctium in vivo and in culture. We characterized morphological and biochemical differentiation of cultured trophoblasts from term placenta in the presence or absence of serum, on different growth surfaces, and with a range of plating densities. Culture of cellular trophoblasts consistently and transiently induced the mRNAs of the mitochondrial antioxidant manganese superoxide dismutase (Mn SOD) but not the mRNAs for the antioxidant enzymes copper zinc SOD or catalase. Fibrin and type I collagen substrates modulated only the expression of the placental specific proteins, human chorionic gonadotropin, and human placental lactogen. Both Mn SOD induction and terminal differentiation, as reflected by human chorionic gonadotropin expression, were dependent on trophoblastic plating density. Increased levels of a smaller Mn SOD mRNA species correlated temporally with an increase in Mn SOD enzyme activity in cultured trophoblasts. These results demonstrate that Mn SOD gene expression and enzyme activity precede or are coordinately regulated with morphological and biochemical trophoblastic differentiation.

Antioxidants↗

PubMed1992

Correction of purine nucleoside phosphorylase deficiency by retroviral-mediated gene transfer in mouse S49 T cell lymphoma: a model for gene therapy of T cell immunodeficiency.

To determine the effectiveness of retroviral-mediated purine nucleoside phosphorylase (PNP) gene transfer and expression for metabolic correction of PNP deficiency, we used as a gene transfer target the NSU-1 subline of murine S49 T lymphoma cells, an in vitro genetic model of PNP deficiency. NSU-1 cells were transduced with recombinant retroviruses that express either the murine or human PNP coding sequences under transcriptional regulation of the Moloney murine leukemia virus (Mo-MLV) long terminal repeat (LTR), resulting in expression of substantial levels of PNP activity. Untransduced or control virus-transduced NSU-1 cells were extremely sensitive to deoxyguanosine, a PNP substrate that is toxic for lymphoid cells. However, PNP-virus transduction of NSU-1 cells metabolically corrected the sensitivity of these cells to deoxyguanosine, resulting in near wild-type levels of growth inhibition. These results demonstrate that retroviral-mediated PNP gene transfer and expression corrects the metabolic defect observed in PNP-deficient murine lymphoid cells, suggesting that PNP gene transfer and expression in human lymphoid cells might similarly correct substrate-mediated toxicity and provide an effective genetic therapy.

Animals↗

PubMed1992

Promoter elements required for positive control of transcription of the Escherichia coli uhpT gene.

The uhpABCT locus of Escherichia coli encodes the transport system which allows the cell to accumulate a variety of sugar phosphates in unaltered form. The expression of uhpT, the gene encoding the transport protein, is regulated by the uhpABC gene products. The UhpA protein is required for expression; its deduced amino acid sequence shows that it belongs to a subfamily of bacterial transcription regulators including NarL, DegU, and FixJ. Members of this subfamily have an amino-terminal phosphorylation domain characteristic of so-called two-component regulators, such as OmpR, CheY, PhoB, and NtrC, and a carboxyl-terminal domain conserved among many transcriptional activators, including LuxR and MalT. The major sequence elements in the uhpT promoter that are needed for uhpT expression were investigated. Northern (RNA) hybridization analysis showed that the uhpT transcript was only present in cells induced for UhpT transport activity. The start site of transcription was identified by primer extension. Comparison of the regions upstream of the uhpT transcription start site in E. coli and Salmonella typhimurium suggested the presence of four sequence elements that might be involved in promoter function: a typical -10 region, a short inverted repeat centered at -32, a long inverted repeat centered at -64, and a cyclic AMP receptor protein-binding sequence centered at -103. Deletion and linker substitution mutations in the promoter demonstrated that the presence of the cyclic AMP receptor protein-binding site resulted in about an eightfold increase in promoter activity and that the -64, -32, and -10 elements were essential for promoter function. In vivo titration of transcriptional activator UhpA by the intact or mutant promoters on multicopy plasmids identified the -64 element as the UhpA-binding site. The two halves of the -64 inverted repeat did not contribute equally to promoter function and did not have to be intact for UhpA titration. The sequence recognized by UhpA is predicted to be 5' -GGCAAAACNNNGAAA.

Bacterial Proteins↗

PubMed1992

Interventions related to respiratory care.

Nursing interventions provide the link between nursing diagnosis and patient outcomes. The validation of nursing interventions is the starting point for a standardized nursing language. This study adapted a method for the validation process for nursing interventions and identified interventions that nursing experts believe are vital to the care of respiratory patients. The study served as the pilot for further surveys by the Iowa classification project. Based on the results of this study, the group made modifications in the survey process. Specifically, national samples of nurses were used, definitions were included in the surveys, and standard deviations were calculated for each activity. The subsequent articles in this issue demonstrate these changes.

Critical Care↗

PubMed1991

Axillary granular parakeratosis.

The term axillary granular parakeratosis is proposed for a unique axillary eruption with distinct histopathologic features. Four middle-aged to elderly patients (three women, one man) had unilateral or bilateral, usually pruritic, hyperpigmented to bright red patches in the axillae. Biopsy specimens revealed severe compact parakeratosis with the stratum corneum measuring 80 to 250 microns in maximal thickness, maintenance of the stratum granulosum, remarkable retention of keratohyaline granules throughout the stratum corneum, and vascular proliferation and ectasia. A contact reaction to an antiperspirant/deodorant is suspected as the cause. We speculate that the offending agent alters the maturation sequence of the stratum granulosum and stratum corneum, possibly by interfering with the degradation of filaggrin precursor to filaggrin units.

Aged↗

PubMed1991

An improved method for determining 89Sr and 90Sr in urine.

A method has been developed for the rapid isolation and quantitation of 89Sr and 90Sr in urine samples. The radiostrontium is concentrated from the bulk urine sample by coprecipitation with calcium phosphate. The precipitate is then wet ashed with nitric acid, and a solution of the resulting residue in 2 M HNO3-0.5 M Al(NO3)3 is passed through an extraction chromatographic column containing a supported crown ether that preferentially retains strontium. Sorbed strontium may then be eluted from the column with either dilute HNO3 or water and counted via liquid scintillation. A new counting scheme that permits quantitation of both 89Sr and 90Sr on the same day the separation is performed is described.

Humans↗

PubMed1991

Pentose phosphate pathway in cellular trophoblasts from full-term human placentas.

Glucose metabolism was investigated in cellular trophoblasts isolated from full-term human placentas. The specific yields of 14CO2 from D-[1-14C]glucose and D-[6-14C]glucose were used to determine glucose metabolism via the pentose cycle for cells freshly isolated or cells grown in culture for 1 and 3 days. Cells were mononucleated on day 1 but fused to form multinucleated syncytiotrophoblasts by day 3. The principal product of glucose metabolism under all conditions was lactate, accounting for approximately three-fourths of recovered 14C in products. Pentose cycle activity contributed 0.57 +/- 0.01, 0.39 +/- 0.06, and 0.21 +/- 0.05% of the glucose metabolized by cells freshly isolated, cultured for 1 day, and cultured for 3 days, respectively. In the presence of the electron acceptor methylene blue, pentose cycle activity increased to 16.5 +/- 2.1, 13.8 +/- 1.5, and 18.2 +/- 1.7% for cells freshly isolated, cultured for 1 day, and cultured for 3 days, respectively. Trace amounts of 14C were recovered in other products including amino acids and glycogen. These data suggest that pentose cycle activity in cellular trophoblasts from full-term placenta, like those in full-term villous tissue, is a minor component of glucose metabolism. However, these cultured cells maintain a capacity to oxidize glucose via the pentose cycle at relatively high rates.

Carbon Radioisotopes↗

PubMed1990

Human placental insulin binding in normal and well-controlled diabetic patients.

Previous studies of insulin binding to placentas of both insulin-dependent and untreated gestational diabetic patients have described placentas from diabetics to contain fewer insulin receptors than placentas from nondiabetic gravidas. However, these studies were done using membrane fractions prepared from the placentas and at a time when adequacy of antepartum glycemic control in the diabetic patients was not routinely evaluated by self blood sugar measurement or hemoglobin A1 assay. The current study compares specific 125I-insulin binding in vitro to intact placental villi from 15 normal patients with insulin binding to intact villi obtained from 15 insulin-dependent diabetic mothers whose fasting and postprandial blood sugars and hemoglobin A1 levels were maintained in a range normal for term pregnancy. We demonstrate that insulin binding to intact placental villi is the same in this group of diabetic patients as in the nondiabetic patients.

Adult↗

PubMed1990

Trophoblast interaction with fibrin matrix. Epithelialization of perivillous fibrin deposits as a mechanism for villous repair in the human placenta.

The authors have used morphometric, immunocytochemical, and electron optical techniques to study fibrin deposits associated with villi from 14 normal term placentas, and have examined the response of cultured cellular trophoblast to fibrin matrix in vitro. Morphometric analysis of 3477 villous profiles showed that 5.5% of villi examined had fibrin deposition at sites of syncytial denudation and that fibrin deposition was highly associated with villous epithelial denudation, as evidenced by loss of cytokeratin staining. The perivillous fibrin deposits were strongly immunoreactive for the B beta chain of fibrin II, consistent with local thrombolytic cleavage of fibrinogen to fibrin. Deposits were frequently surfaced by a discontinuous layer of cytokeratin-positive trophoblastic cells that showed type IV basement membrane collagen immunoreactivity at the interface between trophoblast and fibrin. Ultrastructurally, damage to the syncytial trophoblast was apparent at the edge of some deposits, where syncytial denudation was accompanied by a fibrin coating of residual cellular trophoblast and the trophoblastic basal lamina. Other deposits were surfaced by syncytial trophoblast with underlying cellular trophoblast and a new basal lamina external to the basal lamina of the villous core. Cultured cellular trophoblast grown on a fibrin matrix, but not on uncoated plastic, showed morphologic differentiation into a trophoblast layer like that on term villi. The authors suggest that epithelialization of perivillous fibrin deposits is a form of villous repair and that trophoblast-fibrin interactions can modulate trophoblastic differentiation.

Chorionic Villi↗

PubMed1989

High glucose levels decrease proliferation of cultured human fetal cells from placenta.

We used the placenta as a source of undifferentiated cells to study the effect high glucose levels can have on human fetal cell proliferation in vitro. Cells were subcultured in a modified minimum essential medium with 10% fetal bovine serum containing either 5.5 mmol/L (100 mg/dl) D-glucose (control), 11 mmol/L (200 mg/dl) D-glucose, or 22 mmol/L (400 mg/dl) D-glucose. Cells grown in mannitol-containing media were used as controls for osmolality. After 3 and 7 days' growth in different media, the labeling index was determined by autoradiographic analysis, and cell numbers were determined with a Coulter counter. The labeling indices for cells grown 3 days in 11 or 22 mmol/L D-glucose were 89% (p less than 0.002) and 84% (p less than 0.001), respectively, of control cells grown in 5.5 mmol/L D-glucose. After 7 days' growth, the labeling indices of cells grown in 11 or 22 mmol/L D-glucose were 84% (p less than 0.002) and 70% (p less than 0.001), respectively, of cells grown in 5.5 mmol/L D-glucose media. There was a significant decrease in the number of cells present at both 3 and 7 days in cultures grown in 22 mmol/L D-glucose compared with control. We conclude that a few day's exposure to high glucose levels can have an effect on proliferation of human placental cells in vitro. We suggest that a glucose effect on proliferation of other cells derived from the products of conception might be one mechanism contributing to abnormal development in some pregnancies of diabetic women.

Autoradiography↗

PubMed1989

Aspirin differentially affects thromboxane and prostacyclin production by trophoblast and villous core compartments of human placental villi.

Low-dose aspirin has been used as prophylactic treatment for preeclampsia and fetal growth retardation, but the physiologic mechanisms for the beneficial effect of this therapy are unknown. We studied the effects of aspirin on eicosanoid production by different compartments of normal term human placental villi. Duplicate parallel incubations (n = 7) of whole villi, villous core tissues, and trophoblast were established on Millicell platform dishes in minimum essential medium in the presence or absence of 1 x 10(-4) or 1 x 10(-5) mol/L aspirin. Production rates of thromboxane A2 and prostacyclin were estimated by radioimmunoassay of their stable hydrolysis products, thromboxane B2 and 6-keto-prostaglandin F1 alpha, respectively. Our results indicate that 1 x 10(-4) mol/L aspirin inhibits thromboxane production in whole villi and villous core tissues denuded of their trophoblast layer but not in isolated trophoblast cells. The same concentration of aspirin also inhibits prostacyclin production in the isolated villous core but not in whole villi and not in isolated trophoblast. We conclude that aspirin can selectively inhibit thromboxane production in whole placental villi and differentially affects thromboxane and prostacyclin production by the trophoblast and villous core compartments.

Aspirin↗

PubMed1989

Thromboxane and prostacyclin production by different compartments of the human placental villus.

We separated the trophoblast and villous core of human placental villi to compare thromboxane (Tx) and prostacyclin production in these two compartments with eicosanoid production by intact villi. TxB2 and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), the stable metabolites of TxA2 and prostacyclin, respectively, were measured in serum-free media from 48-h incubations of intact villi, villous core tissue denuded of its trophoblast layer, and trophoblast cells. In villi, the medium TxB2 concentrations increased rapidly to a peak level of 20 +/- 9 (+/- SE) (n = 11) pg/microgram protein at 48 h; 6-keto-PGF1 alpha was first detected in medium at 20 h, and it increased to 19.6 +/- 4.0 pg/micrograms protein (n = 11) by 48 h. Compared to villi, villous core tissue denuded of its surface trophoblast layer had a 7-fold greater TxB2 level (136 +/- 17 pg/micrograms protein; n = 11) by 48 h, but a comparable level of 6-keto-PGF1 alpha (22.5 +/- 3.7 pg/micrograms protein). Trophoblast cultures produced predominantly TxB2 (109 +/- 18 pg/micrograms protein; n = 11) and had the lowest 6-keto-PGF1 alpha production among the three groups (11.4 +/- 2.6 pg/micrograms protein). At 48 h, the mean TxB2/6-keto-PGF1 alpha ratio was 1.0 in medium from intact villi, 6.2 in medium from villous core tissue, and 13.3 in medium from trophoblast cells. Indomethacin inhibited production of both eicosanoids in all cultures. Our studies indicate that intact placental villi produce equal amounts of Tx and prostacyclin, the trophoblast compartment produces predominantly Tx, and the villous core compartment produces an increased amount of Tx when denuded of its trophoblast layer. These data also suggest that the trophoblast produces an inhibitor or provides a catabolic function that limits villous core Tx production.

6-Ketoprostaglandin F1 alpha↗

PubMed1989

Pertussis toxin-sensitive G protein mediation of PGE2 inhibition of cAMP metabolism and phasic glucose-induced insulin secretion in HIT cells.

Although prostaglandin E2 (PGE2) is known to inhibit glucose-induced insulin secretion, it is uncertain whether PGE2 actions on the beta-cell are direct, whether they are equipotent for both phases of hormone secretion, and whether the same mechanism of action prevails throughout. Study of the HIT cell, a clonal line of pancreatic beta-cells, provides answers to these questions because perifusion with glucose and 3-isobutyl-1-methylxanthine stimulates biphasic insulin secretion. Perifusion with PGE2 decreased both the first and second phases of glucose-induced insulin release to 47 +/- 4% of controls. Pretreatment with pertussis toxin partly prevented PGE2 inhibition to 80 +/- 4% of controls for first phase and 79 +/- 4% of controls for second phase. To evaluate whether the partial prevention of PGE2 inhibition seen with pertussis toxin pretreatment was caused by Gi heterotrimer association between the preincubation period and the end of perifusion, PGE2 actions were also examined during continuous treatment with pertussis toxin. Under these conditions, PGE2 inhibition of both phases was totally prevented. However, no difference was observed in membrane protein ADP ribosylation when cells were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pretreatment or continuous treatment with pertussis toxin. Cyclic AMP (cAMP) accumulation was inhibited by PGE2 (from 3263 +/- 153 to 1549 +/- 158 fmol/10(6) cells) but less so after pretreatment with pertussis toxin (correlation between insulin release and cAMP accumulation during perifusion; n = 18, r = .85, P less than .001). Thus, PGE2 equally inhibits both phases of glucose-induced insulin secretion and cAMP generation through a pertussis toxin-sensitive G protein-mediated direct effect on the pancreatic beta-cell.

1-Methyl-3-isobutylxanthine↗

PubMed1988

Cord blood acid-base status in neonates delivered by Silastic vacuum cup extraction: comparison with forceps and spontaneous deliveries.

Umbilical cord blood gases were measured as objective parameters of neonatal outcome in patients delivered by Silastic vacuum extractor and were compared with those values from patients delivered spontaneously, by forceps, or by sequential use of vacuum and forceps. The results demonstrated no clinically significant differences among the groups in cord blood pH, pCO2, pO2, base excess, or bicarbonate values. These observations provide reassurance that the Silastic vacuum extractor is a safe alternative for vaginal delivery of the appropriately selected term fetus.

Acid-Base Equilibrium↗

PubMed1979

Instrumental analysis of trace elements in thumbnails of human subjects.

Human thumbnails were analyzed for trace elements by instrumental analysis using thermal neutron activation technique. The average concentration of metals studied in clinically symptom-free adult female and male subjects were: zinc, 184 vs. 153 ppm; chromium, 6.8 vs. 4.2; selenium, 0.9 vs. 0.6; gold, 2.6 vs. 0.4; mercury, 1.9 vs. 0.4; silver, 0.7 vs. 0.3; cobalt, 0.07 vs. 0.04. A summary of literature reported concentration of metals in human nail is also presented.

Adult↗

PubMed1978

Nonuniform distribution and grouping of insulin receptors on the surface of human placental syncytial trophoblast.

These studies were designed to investigate the cytologic localization and topographic distribution of insulin receptors in human placental villi. Biochemical studies showed placental villi to specifically bind 125I-insulin. Radioautographic studies showed the specific binding to be localized to the surface of the syncytial trophoblast. Topographic distribution of insulin binding was determined with ferritin-insulin. Initial studies using ferritin-insulin containing some oligomers of ferritin revealed the insulin receptors to be specifically associated with the glycocalyx region of the surface membranes of microvilli. No insulin receptors were detectable in association with the intermicrovillous plasma membrane even though its glycocalyx is in direct continuity with the glycocalyx of microvilli. Monomeric ferritin-insulin showed the same nonuniform distribution of the insulin receptor, which suggests that there is not complete freedom of lateral mobility of the insulin receptors in the surface membrane of this tissue. The insulin receptors were found to occur as singletons or in groups of two or more. Incubations with monomeric ferritin-insulin at 4 degrees or with tissue prefixed with formaldehyde showed that the groups of insulin receptors were naturally occurring, i.e., they are present prior to and independent of insulin binding and thus not secondary to ligand-induced aggregation. The physiologic meaning of the nonuniform distribution and the groups of insulin receptors is unclear at present.

Cell Membrane↗

PubMed1977

Characterization of a microvillous membrane preparation from human placental syncytiotrophoblast: a morphologic, biochemical, and physiologic, study.

The syncytiotrophoblast microvillous membrane must play a vital role in many essential functions of the placenta. In order to better understand the functional characteristics of this membrane, we have investigated an isolated membrane preparation by a variety of techniques. Electron microscopic observations showed membranous structures similar to microvilli of intact placental villi in size, shape, and microfilamentous content. Similarities in colloidal iron staining and transferrin localization were also shown. The preparation was enriched in enzymes characteristic of surface membranes and diminished in enzymes characteristic of intracellular organelles. Sialic acid content was also increased. SDS gel electrophoresis revealed the presence of a 45,000 molecular weight band, which may be actin. The preparation transported serine, glycine, and alpha-aminoisobutyric acid by a temperature-dependent, saturable process.

Alkaline Phosphatase↗