PubMed Health⌕ Search

Biomedical subjects

P Nugent

Publications and source records attributed to P Nugent.

At least 37 records · Page 2Linked to original sources
PubMed1995

Patterns of cyclic AMP-dependent protein kinase gene expression during ontogeny of the murine palate.

Normal growth and differentiation of embryonic palatal tissue depends on regulated levels of intracellular cAMP. Cyclic AMP-dependent protein kinases (PKA) act to mediate the biological activities of cAMP. PKA isozyme protein profiles demonstrate a clear pattern of temporal alterations in embryonic palatal tissue during its development. In order to ascertain the molecular basis for changing PKA isozyme profiles during palatal ontogeny, the spatial and temporal expression of mRNAs for regulatory (RI alpha, RII alpha, and RII beta) and catalytic (C alpha) subunits of PKA was examined. RNA extracted from murine embryonic palatal tissue (days 12-14 of gestation) was examined by Northern blot analysis. Significant levels of constitutively expressed RI alpha and C alpha mRNA were seen on all days of gestation examined. RI alpha transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of RII alpha and RII beta mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of RII beta, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of RII beta was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, RII beta was predominantly localized to palatal epithelial cells. Direct activation of adenylate cyclase with forskolin in murine embryonic palate mesenchymal (MEPM) cells resulted in an increase in RII alpha mRNA levels but had no effect on steady state levels of RII beta or C alpha mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of RI alpha mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP-dependent protein kinase regulatory and C alpha subunits in murine embryonic palatal tissue. In addition, we have demonstrated adaptational changes of this kinase in MEPM cells in response to conditions of increased intracellular levels of cAMP.

Animals↗
PubMed1995

TGF-beta modulates the expression of retinoic acid-induced RAR-beta in primary cultures of embryonic palate cells.

We have previously shown that both transforming growth factor-beta (TGF-beta) and retinoic acid (RA) regulate the expression of cellular retinoic acid binding proteins (CRABP) I and II and TGF-beta 3 mRNAs in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. We now describe additional cross-talk between the RA and TGF-beta signal transduction pathways--the ability of TGF-beta, including the endogenous form(s), to modulate the expression of the nuclear retinoic acid receptor-beta (RAR-beta). Northern blot hybridization revealed that RA induced the expression of RAR-beta mRNA, there being little or no detectable expression in untreated MEPM cells. Induction by 3.3 microM RA was abrogated by simultaneous treatment with TGF-beta 1 (5 ng/ml). TGF-beta 1 alone had no effect on RAR-beta mRNA expression. Determination of RAR-beta mRNA half-life by treatment with actinomycin D indicated that TGF-beta 1 did not alter the stability of RAR-beta mRNA. Conditioned medium (CM) from MEPM cells contained little active TGF-beta protein; heat treatment of the CM dramatically increased the amount of active TGF-beta as assessed by the mink lung epithelial cell bioassay. Furthermore, heat- or acid-activated CM also inhibited CRABP-I and RA-induced RAR-beta expression. The effect of heat-activated conditioned medium could be abrogated with panspecific neutralizing antibodies to TGF-beta, confirming that endogenous TGF-beta is the biologically active factor in heat-activated CM. These results provide evidence for complex interactions between TGF-beta and RA in the regulation of gene expression in embryonic palatal cells and suggest a role for endogenous TGF-beta in the regulation of expression of genes encoding elements of the RA signal transduction pathway.

Animals↗
PubMed1994

Regulation of TGF beta 3 gene expression in embryonic palatal tissue.

The TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Interactions between the transforming growth factor beta (TGF beta) and retinoic acid signal transduction pathways in murine embryonic palatal cells.

Regulation of expression of transforming growth factor-beta 3 (TGF-beta 3) and the cellular retinoic acid-binding proteins-I and II (CRABP-I, -II) by retinoic acid (RA) and TGF-beta was examined in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. Northern blot hybridization revealed that RA and TGF-beta 1, beta 2 and beta 3 stimulated the expression of TGF-beta 3 mRNA within 24 hours of treatment. RA down-regulated the expression of CRABP-I mRNA and up-regulated the expression of CRABP-II mRNA in a time- and dose-dependent fashion. TGF-beta 1, beta 2 and beta 3 also down-regulated the expression of CRABP-I mRNA, while epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) were without effect. TGF-beta 1 also stimulated a dose-dependent increase in the expression of CRABP-II mRNA. Again EGF and TGF-alpha were without effect. Basic fibroblast growth factor (bFGF) elicited a slight inhibitory effect on CRABP-II and a slight stimulatory effect on CRABP-I mRNA expression. Thus, cells derived from the mammalian developing palate express CRABP-I and CRABP-II mRNAs, both of which may be regulated by RA and TGF-beta. These data constitute the first demonstration of an effect of TGF-beta on the expression of CRABP-I and CRABP-II and provide further evidence for cross-talk between RA and TGF-beta signal transduction pathways.

Animals↗
PubMed1993

Milk-alkali syndrome. A consequence of chronic antacid abuse.

The case described here illustrates the need to consider milk-alkali syndrome in the differential diagnosis of hypercalcemia, even in this age of histamine2 blockers and mucosal protectors. Thorough history taking and gastrointestinal workup help rule out alternative causes of hypercalcemia, such as cancer, hyperparathyroidism, and other metabolic and endocrine diseases. Milk-alkali syndrome is managed by administration of saline solution and loop diuretics and avoidance of thiazide diuretics. Patient education regarding the hazards of abusing calcium-containing antacids is essential.

Antacids↗
PubMed1993

Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells.

A series of antisense oligonucleotides were developed to inhibit specifically expression of a mutated exogenous gene for collagen without inhibiting expression of an endogenous gene for the same protein. The test system consisted of mouse NIH 3T3 cells that were stably transfected with an internally deleted construct of the human gene for the pro alpha 1(I) chain of type I procollagen [Olsen et al. (1991) J. Biol. Chem. 266, 1117]. The target site was a region at the 3' end of exon 1 and the first few nucleotides of intron 1 of the exogenous human gene that differed in sequence by nine nucleotides from the sequence of the endogenous mouse gene. Expression of the two genes was assayed by Western blot with cross-reacting antibodies and by steady-state levels of mRNAs. None of the oligonucleotides were effective in concentrations up to 25 microM when administered without any carrier. However, when administered with 5 or 10 micrograms/mL lipofectin, one of the oligonucleotides in concentrations of 0.1-0.2 microM inhibited expression of the exogenous gene from 50% to 80% without significant inhibition of expression of the endogenous gene. Also, a missense version of the same oligonucleotide had no significant effect, and the inhibition observed with the most effective oligonucleotide was abolished by a single base change. Time course experiments indicated that, after a 4-h treatment, inhibition appeared at 8 h and persisted for at least 22 h.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1992

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts.

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.

3T3 Cells↗
PubMed1992

Multidisciplinary cycles for protein engineering: site-directed mutagenesis and X-ray structural studies of aspartic proteinases.

The specificity and pH profile of aspartic proteinases have evolved to include not only pepsin with a broad specificity and an optimal activity in acid media, but also renin, with high specificity for angiotensinogen and activity close to neutral pH. Comparisons of the structures and catalytic activities of aspartic proteinases provide helpful clues for engineering new activity profiles. We illustrate an approach that involves recombinant DNA techniques, biochemistry, structure determination and biocomputing. We use the 3-D structures of inhibitor complexes of several aspartic proteinases to define likely intermediates and specificity sub-sites. The multidisciplinary research is organised as cycles, in which each cycle tests a design hypothesis proposed in the previous cycle. We use one member of the aspartic proteinase family, chymosin, to illustrate these ideas in engineering enzymes with altered pH optima and specificities.

Amino Acid Sequence↗
PubMed1991

Effect of the SV40 T antigen on the posttranscriptional regulation of the proliferating cell nuclear antigen and DNA polymerase-alpha genes.

tk-ts13 cells are G1-specific temperature-sensitive mutants of the cell cycle that arrest in G1 at the restrictive temperature. In these cells the mRNAs for early growth-regulated genes (for instance, c-myc) are inducible by serum at both permissive and restrictive temperatures. In contrast, the mRNAs for late growth-regulated genes [such as histones, proliferating cell nuclear antigen (PCNA), and DNA polymerase-alpha] are not detectable at the restrictive temperature, although they are normally induced by serum at the permissive temperature. Despite the absence of their mRNAs at the restrictive temperature, transcription rates for DNA polymerase-alpha, PCNA, and histone H3 are the same in serum-deprived cells and in cells that are serum stimulated at either the permissive or the restrictive temperature. Since the half-lives of the mRNAs are not substantially different at the two temperatures, the conclusion is that in tk-ts13 cells the mRNA levels of these late growth-regulated genes are regulated at a posttranscriptional level, presumably during hnRNA processing. When serum-deprived tk-ts13 cells carrying a stably integrated SV40 T antigen-coding gene (T-neo cells) are stimulated with serum, they are capable of one additional round of DNA replication at the restrictive temperature. At 20 h after stimulation of T-neo cells, the mRNAs for the late growth-regulated genes are detectable at the restrictive temperature in amounts not substantially different than those at the permissive temperature. Transcription rates in T-neo cells are increased for histone H3 (in comparison to tk-ts13 cells) but not for PCNA and DNA polymerase-alpha. The presence of the T antigen does not seem to seriously affect the half-lives of the mature mRNAs. The conclusion is that the presence of the SV40 T antigen in tk-ts13 cells promotes the appearance of mature mRNAs for DNA polymerase-alpha and PCNA. These experiments suggest that T antigen, in this instance, may intervene either directly or indirectly at a posttranscriptional level in the regulation of the steady state mRNA levels of certain cellular genes.

Animals↗
PubMed1991

Venous thrombosis in patients who have undergone major hip or knee surgery: detection with compression US and impedance plethysmography.

To compare the sensitivity, specificity, and predictive values of compression ultrasonography (US) in postoperative orthopedic patients with those of (a) impedance plethysmography in postoperative patients and (b) compression US in symptomatic outpatients, the authors performed an investigator-blinded cohort study. One hundred thirty-four consecutive inpatients who had undergone elective knee-replacement surgery or surgery for a fractured hip and 65 consecutive outpatients with clinically suspected venous thrombosis who had undergone venography were evaluated. Compression US allowed detection of 11 of 21 (52.4%) proximal-vein thrombi but was insensitive to calf-vein thrombi in the orthopedic patients. Compression US had a significantly greater specificity and positive predictive value than impedance plethysmography for all thrombi in orthopedic patients; compression US also had greater sensitivity. The sensitivity of compression US for proximal-vein thrombi was significantly higher (92.1%) in symptomatic outpatients than in orthopedic patients. The authors conclude that compression US has significant advantages over impedance plethysmography in the detection of proximal-vein thrombi in patients who have undergone hip- or knee-replacement surgery.

Hip Joint↗
PubMed1988

Fibrin glue sealing of polytetrafluoroethylene vascular graft anastomoses: comparison with oxidized cellulose.

To evaluate potential clinical applications of nonautologous fibrin glue (FG) as a hemostatic agent in vascular surgery, we compared its efficacy to oxidized regenerated cellulose (OC) in hemostatically sealing polytetrafluoroethylene (PTFE) vascular graft anastomoses. PTFE grafts (4 mm wide and 4 to 6 cm in length) were placed to each femoral artery in a heparinized canine model, in end-to-end fashion in half of the dogs and in end-to-side fashion in the remaining dogs. Each set of graft-arterial anastomoses was then sealed with either FG or OC, determined randomly, followed by simultaneous measurement of blood loss through the graft anastomoses and needle holes. There was significantly less bleeding from anastomoses sealed with FG compared with those sealed with OC, regardless of whether the anastomoses sealed with FG compared with those sealed with OC, regardless of whether the anastomosis was constructed in end-to-end (p less than 0.03) or end-to-side (p less than 0.004) fashion; overall, the operative blood loss for grafts sealed with FG was 14 +/- 6 (mean +/- standard error of the mean) vs 99 +/- 27 ml/min for those sealed with OC (p less than 0.001). In the early postoperative period, significant groin hematomas occurred more frequently in grafts sealed with OC compared with those sealed with FG. Microscopic examination of graft-arterial specimens harvested at postoperative intervals ranging from 1 day to 3 months revealed no significant inflammatory reaction with either hemostatic agent; after 2 to 3 weeks, paired specimens appeared histologically similar despite previous treatment with either FG or OC.(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗
PubMed1986

The surgeon's role in treating acquired immunodeficiency syndrome.

The ever-increasing number of patients with acquired immunodeficiency syndrome (AIDS) will involve more surgeons in their diagnosis and treatment. The surgeon should be aware of the cause of AIDS, mode of transmission, method of diagnosis, usual cutaneous and abdominal manifestations, complications needing operative procedures, and precautions needed during surgery and the postoperative period. The gravity of AIDS requires the surgeon to be aware of the potential risks to other surgical patients by contaminated blood transfusions. From 110 cases of AIDS, we analyzed the indications, types of surgical procedures, and effect on final outcome in patients with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Breast cancer and pregnancy.

Previous studies have found a poor prognosis for breast cancer occurring during pregnancy due to the intense hormonal stimulation produced by the pregnancy. In our study of 176 patients, pregnancy did not seem directly to affect the prognosis of breast cancer. Rather, poor survival was related to the patients' youth (less than 40 years old) and to the large number of estrogen receptor-negative tumors. Of the pregnant patients, 71% had estrogen receptor-negative tumors, implying hormonal insensitivity. Terminating the pregnancy on this basis does not seem warranted. Subsequent pregnancies in young patients did not seem to affect survival adversely. Future pregnancy in patients with stage I tumors can be considered after two years. Survival is so poor in patients with stage II or III tumors that subsequent pregnancies should be discouraged for socioethical reasons.

Abortion, Therapeutic↗
PubMed1983

Heterogeneity of very-low-density lipoprotein metabolism in hyperlipidemic subjects.

Very-low-density lipoproteins (VLDL) are triglyceride-rich lipoproteins that have been shown, by physicochemical means, to comprise more than one group of particles. Because of the potential atherogenicity of catabolized VLDL, we used the technique of heparin-affinity chromatography to separate VLDL into two classes of particles, one of which appears to contain partly catabolized VLDL. This observation is based on the higher cholesterol/triglyceride and higher apoprotein E/apoprotein C ratios in VLDL particles that are bound to heparin, resembling in this way intermediate-density lipoproteins (IDL), which are certainly derived in the main through VLDL catabolism. Further studies showed separate metabolic characteristics for the unbound and heparin-bound VLDL particles. Radiolabeled whole VLDL or unbound particles were reinjected into seven hypertriglyceridemic subjects and the kinetics studied in serial samples of plasma over the next 18-48 hours. The specific radioactivity-time curves of apoprotein B in the unbound and bound particles showed that the bound particles were derived wholly or partly from the unbound particles and in turn, were the precursors of IDL. This confirmed that heparin-bound VLDL particles represented VLDL undergoing catabolism, although in one subject about one-half of the bound particles appeared to have an origin other than through VLDL catabolism. These studies show that VLDL metabolism is heterogeneous, that the kinetics of total VLDL must be interpreted accordingly, and that the technique of heparin-affinity chromatography can be used for more detailed studies of VLDL.

Adult↗
PubMedDate not supplied

Antisense oligonucleotides to CRABP I and II alter the expression of TGF-beta 3, RAR-beta, and tenascin in primary cultures of embryonic palate cells.

The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor beta (TGF-beta). These cells also express high levels of TGF-beta 3, which is also regulated by RA and TGF-beta. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 microM or 0.33 microM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-beta 3, retinoic acid receptor beta (RAR-beta), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-beta 3, RAR-beta, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-beta 3, RAR-beta, and tenascin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗