Biomedical subjects

D M Nelson

Publications and source records attributed to D M Nelson.

At least 55 records · Page 3Linked to original sources

PubMed1997

Transcriptional regulation of prostaglandin-H synthase-2 gene in human trophoblasts.

Abnormal PG production by placental PG-H synthase (PGHS) is associated with preeclampsia. There are two PGHS isozymes, and their regulation in trophoblasts is presently unknown. We hypothesized that the PGHS isozymes are differentially regulated in human trophoblasts. To test this hypothesis, we transfected primary trophoblasts and JEG3 cells with promoter constructs of either PGHS-1 or PGHS-2 genes. We found that in both cell systems, the basal activity of PGHS-2 promoter was 10- to 30-fold higher than the activity of PGHS-1 promoter. In response to either 12-0-tetradecanoylphorbol-13-acetate (TPA) or 8-bromo-cAMP, we observed an increase in PGHS-2 promoter activity but no change in activity of PGHS-1 promoter. Similarly, both agents enhanced PGHS-2 expression, as well as prostaglandin E2 production. The activity of PGHS-2 promoter was potentiated by coexpression of protein kinase A and inhibited by coexpression of kinase A inhibitor. Aspirin attenuated the stimulatory effect of TPA on PGHS-2 promoter. We conclude that both PGHS-1 and PGHS-2 promoters are active in trophoblasts. The activity of PGHS-2 promoter is stimulated by either TPA or cAMP, and the stimulatory effect of TPA is attenuated by aspirin. These pathways may play a role in modulation of prostanoid synthesis by trophoblasts.

Aspirin↗

PubMed1997

The expression and activity of prostaglandin H synthase-2 is enhanced in trophoblast from women with preeclampsia.

Preeclampsia is associated with altered biosynthesis of vasoactive prostanoids in placental villi. The two isozymes of prostaglandin H synthase (PGHS) are essential for prostanoid synthesis. We tested the hypothesis that PGHS-2 expression is elevated in trophoblast from preeclamptic women, compared with trophoblast from healthy women. Using immunofluorescent staining, we demonstrated a higher PGHS-2 expression in villi from preeclampsia, compared with normal pregnancy. Cytotrophoblasts cultured from placentas of preeclamptic women expressed higher levels of PGHS-2 compared with cytotrophoblasts from normal placentas. This enhanced expression of PGHS-2 correlated with increased media levels of both thromboxane and prostaglandin E2, two products of PGHS activity. The increased prostanoid production by trophoblast from preeclamptic women was markedly reduced by NS-398, a specific inhibitor of PGHS-2. We conclude that both expression and activity of PGHS-2 are enhanced in trophoblasts from preeclamptic women compared with trophoblast from normal pregnancies. The increased production of prostanoids may contribute to the clinical syndrome of preeclampsia. Our data suggest that a selective inhibitor of PGHS-2 might provide a therapeutic alternative to prophylactic low-dose aspirin in modifying the prostanoid profile in preeclampsia.

Blotting, Northern↗

PubMed1996

Determination of the viremia threshold for dental cross-infection in a mouse model.

An animal model of dental virus transmission was developed using the lactate dehydrogenase-elevating virus (LDV) of mice to study cross infection. Mouse-to-mouse cross-infection was carried out by scaling the teeth of LDV-infected donor mice with dental instruments, immediately prior to using the contaminated instruments on the teeth of recipient indicator mice. The level of donor viremia was found to correlate with the rate of virus cross-infection, with a viremia threshold level of 10(7.5) ID50/ml observed for dental cross-infection. The blood volume transferred during dental cross-infection was approximately 10(-4) to 10(-5) ml, demonstrating the inefficiency of virus cross-infection, since deposition of about 1000 virions on dental instruments was associated with the threshold limit. Virus transferred during dental cross-infection rapidly entered the blood circulation, showing that dental cross-infection was not dependent on an oral infection. The results from these model studies predict the general inefficiency of dental instrument virus cross-infection, and a further reduced likelihood of dental cross-infection with appropriately cleaned instruments.

Animals↗

PubMed1996

Apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast.

The syncytial nature and surface location of the trophoblast layer of human placental villi positions the syncytiotrophoblast to regulate maternal-fetal exchange of molecules while also providing a barrier function. However, discontinuities in the syncytiotrophoblast breach the integrity of this interface, and deposition of fibrin type fibrinoid on the trophoblast basal lamina at the syncytial discontinuity provides a matrix for trophoblast re-epithelialization. Using the electron microscope, 1 tested the hypothesis that apoptosis in the syncytiotrophoblast was one process that initiated discontinuity in the trophoblast layer of term placental villi. Ultrastructural analysis of fibrin deposits on villi from six placentae from uncomplicated term pregnancies indicated the following morphological features typical of apoptosis: condensation and margination of chromatin along an intact nuclear envelop in syncytiotrophoblast nuclei associated with villous surface fibrin deposits; loss of microvilli with membrane blebbing on the surface membrane; cytoplasmic condensation; autophagocytosis of cellular debris containing nuclear fragments; absent inflammatory response. I conclude that human placental syncytiotrophoblast undergoes apoptosis, and this process is associated with breaks in the trophoblast covering of villi. The presence of trophoblastic apoptosis, and of discontinuities in the trophoblast layer of term villi, provide new insights into the pathways for maternal-fetal exchange in the human placenta.

Apoptosis↗

PubMed1996

Fixation affects morphometric characters of Psoroptes cuniculi mites (Acari:Psoroptidae).

Thirteen morphologic characters were evaluated on Psoroptes cuniculi (Delafond) mites to determine if measurements for these variables were influenced by the use of different fixatives and varying periods of fixation. Morphologic structures composed primarily of chitin (outer opisthosomal setal lengths and right 3rd tarsal pedicle length) were not affected by either fixative type or duration of fixation. In contrast, alcohol and formalin fixation both resulted in significant reductions (P < 0.05) in mean body size measurements when compared with mites mounted in Hoyer's medium without prior fixation. The largest reduction in size was seen in mites fixed for the longest period, but there was no apparent association between size reduction and increasing concentration of alcohol or formalin. Surprisingly, when mites were remeasured 4 yr later, Hoyer's mounting medium was found to have had the opposite effect of fixation and caused an increase in body size. Mites mounted in Hoyer's medium without prior fixation had significantly increased body lengths, whereas mites mounted in Hoyer's following fixation in alcohol and formalin had significant increases in both body length and posterior body width. Our results indicate that fixation and mounting can significantly influence the morphometric analysis of mites and other arthropods. We recommend that morphometric studies be conducted using consistent methodology to reduce experimental bias, and these methodologies be reported in publications dealing with morphometric analyses.

Animals↗

PubMed1995

Correction of proliferative responses in purine nucleoside phosphorylase (PNP)-deficient T lymphocytes by retroviral-mediated PNP gene transfer and expression.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is associated with a fatal T cell immunodeficiency in children, a candidate condition for gene therapy by introduction of functional PNP sequences into either T lymphocytes or more primitive progenitor cells in the bone marrow. To test the effectiveness of PNP gene transfer in T lymphocytes, a retroviral vector (LmPSN-2) was designed and constructed to express the murine PNP cDNA under transcriptional regulation of the Moloney murine leukemia virus long terminal repeat. LmPSN-2 was first used to mediate gene transfer and expression of electrophoretically distinct murine PNP in normal (PNP-positive) human PBL. Peripheral blood leukocytes were then obtained from a PNP deficient patient and characterized phenotypically. Despite their paucity and general mitogenic unresponsiveness, T lymphocytes from this patient were successfully grown in culture by using anti-CD3 with rIL-2 and then transduced with LmPSN-2. Elevated PNP enzyme activity was observed in the transduced cell population. Mitogenic and allogeneic responses, normally depressed in PNP-deficient patients' cells, were partially corrected in the transduced cell population relative to nontransduced cells. These results suggest the possibility of effecting improved immunologic function in PNP-deficient T lymphoid cells by retroviral-mediated gene transfer as therapy for PNP deficiency.

3T3 Cells↗

PubMed1995

Differentiation and growth on a fibrin matrix modulate the cyclooxygenase expression and thromboxane production by cultured human placental trophoblasts.

Preeclampsia is associated with altered placental production of several end-products of cyclooxygenase activity. Thromboxane (TX) is one of these end-products, and trophoblast is a source of villous thromboxane. We cultured term trophoblast in the presence or absence of fibrin to study how differentiation and epithelial-matrix interactions regulate cyclooxygenase expression. The cellular trophoblast present during the first 24 h of culture on uncoated plastic produced TXB2, but little or no TX was produced in cultures grown longer than 24 h when differentiation into syncytial trophoblast occurred. Growth of cells on a fibrin matrix enhanced cellular trophoblast TX production five-fold. Medium containing 10 mumol/l arachidonic acid maximized thromboxane production in cells cultured for less than 24 h, regardless of growth surface, but this medium had little or no effect on TX production by cultures grown for more than 24 h. In contrast, exogenous arachidonic acid enhanced prostaglandin E2 (PGE2) production by both cellular and syncytial trophoblast. Cytochemical staining indicated that changes in cyclooxygenase content occurred with trophoblast differentiation. Western immunoblot analysis of cells cultured in the presence or absence of a fibrin matrix showed cyclooxygenase was induced under both growth conditions. Detectable cyclooxygenase protein disappeared beyond 24 h in cells grown on uncoated plastic. In contrast, cells grown beyond 24 h on fibrin showed sustained expression of cyclooxygenase by Western immunoblotting, and this enzyme protein expression correlated with increased PGE2 production by the differentiated trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic Acid↗

PubMed1995

Mechanisms of alphafetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy.

We investigated the mechanisms of alphafetoprotein (AFP) transfer across the human placenta by correlating measurements of AFP transfer with cytochemical localization of AFP. Placental cotyledons were dually perfused in vitro with either the fetal or maternal perfusate containing umbilical cord plasma as a source of AFP. Steady state AFP clearance, corrected for release of endogenous AFP, was 0.973 +/- 0.292 microliter/min per gram in the fetal to maternal direction (n = 10), significantly higher (P < 0.02) than that in the maternal to fetal direction (n = 5; 0.022 +/- 0.013 microliter/min per gram). Clearance of a similarly sized protein, horseradish peroxidase was also asymmetric but clearance of the small tracer creatinine was not. Using a monoclonal antibody, we localized AFP to fibrinoid deposits in regions of villi with discontinuities of the syncytiotrophoblast, to cytotrophoblast cells in these deposits, to syncytiotrophoblast on some villi, and to trophoblast cells in the decidua. We conclude that AFP transfer in the placenta is asymmetric and that there are two available pathways for AFP transfer: (a) from the fetal circulation into the villous core and across fibrinoid deposits at discontinuities in the villous syncytiotrophoblast to enter the maternal circulation; and (b) AFP present in the decidua could enter vessels that traverse the basal plate.

Biological Transport↗

PubMed1995

Effectiveness of ultrasonic cleaning of dental instruments.

PURPOSE: To quantitate blood contamination present on dental instruments used for routine prophylaxis and to assess the effectiveness of ultrasonic decontamination in reducing blood and virus contamination on dental instruments. MATERIALS AND METHODS: Human blood contamination present on dental instruments obtained after routine prophylaxis was analyzed using IgG as a blood marker. RESULTS: The estimated contaminating blood volume was found to normally range between 1.4 x 10(-6) to 2.0 x 10(-4) ml. Attempts to saturate the instruments with blood contamination suggested that the maximum possible retained blood volume was about 10-fold higher than the normal levels of contamination. Hand scrubbing of contaminated instruments was both relatively ineffective and inconsistent in removing blood contamination. Decontamination in an ultrasonic cleaner was more effective than hand washing, resulting in greater than a 100-fold reduction of blood contamination. Using a mouse model virus (lactate dehydrogenase-elevating virus, LDV), high levels of virus contamination of dental instruments and dental handpieces were achieved, as determined by assay of residual virus. Ultrasonic treatment reduced the level of virus contamination present on dental instruments by one million-fold, and virus contamination present in dental handpieces was reduced by one thousand-fold. These results provide quantitative estimations of the infection threat and its reduction by ultrasonication, posed by human-exposed dental instruments.

Animals↗

PubMed1993

Atrial natriuretic hormone: a paracrine or endocrine role within the human placenta?

There is evidence to suggest that the human placenta may synthesize atrial natriuretic hormone (ANH). To test this hypothesis we examined specimens of normal term placenta obtained at elective caesarean section for evidence of either storage or synthesis of this peptide. No immunocytochemical or ultrastructural evidence of ANH storage was found. RNA extracted from cultured cytotrophoblast cells, the chorionic or decidual plates, or from placental villi contained no ANH-encoding messenger RNA by Northern blot analysis. Polymerase chain reaction amplification of these RNA extracts using primers directed to the midportion of the ANH gene failed to demonstrate any evidence of ANH-encoding RNA. We conclude that ANH is neither stored nor synthesized within the normal term placenta, and thus a purely endocrine role for this peptide within the fetoplacental compartment is proposed.

Atrial Natriuretic Factor↗

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↗