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Biomedical subjects

D A Young

Publications and source records attributed to D A Young.

At least 19 recordsLinked to original sources
PubMed1992

An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus.

A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.

Bacteriochlorophylls↗
PubMed1992

cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase.

The antiinflammatory glucocorticoids are potent inhibitors of cyclooxygenase, a key regulator of prostaglandin synthesis; yet, the mechanism(s) by which this occurs is not fully understood. We have cloned a 4.1-kilobase (kb) cDNA, distinct from the previously cloned cyclooxygenase (2.8 kb), that confers cyclooxygenase activity to transfected cells. The mRNA for this newly discovered cyclooxygenase is unique for its long 3' untranslated region containing many AUUUA repeats. Levels of the 4.1-kb cyclooxygenase mRNA are rapidly increased by serum or interleukin 1 beta in mouse fibroblasts and human monocytes, respectively, and decreased by glucocorticoids, whereas levels of the 2.8-kb cyclooxygenase mRNA do not change. Similar effects are seen in the presence of cycloheximide where the 4.1-kb, but not the 2.8-kb, mRNA is greatly superinduced. Thus, there are both constitutive (2.8 kb) and regulated (4.1 kb) cyclooxygenase species, the latter most likely being a major mediator of inflammation.

Amino Acid Sequence↗
PubMed1992

Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation.

The mouse 3T3-L1 fibroblastic cell line rapidly differentiates to an adipocyte phenotype when post-confluent cells are treated for 48 h in fetal calf serum-containing medium supplemented with 1 microM dexamethasone (D), 0.5 mM methylisobutylxanthine (M) and 10 micrograms/ml insulin (I). D and I act synergistically to commit the cells to differentiate 24-48 h after initiating treatment, and this is blocked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. In order to identify cellular proteins involved in the differentiation process we analyzed differentiating 3T3-L1 cells using two-dimensional electrophoresis on large format gels. We observed changes in over 300 proteins during differentiation (over 100 within 5 h of initiating differentiation) and many of these are also changed at the level of mRNA (by analysis of in vitro translation products). About 75% of the initial changes were maximally induced by treatment with a combination of M and I, while no more than 10 proteins and their corresponding mRNAs were maximally induced by D within 3.5 h. Another 10 proteins were synergistically regulated by the combination of all three agents (DMI) within 3.5 h. Additional species were induced at later times. Five of these were synergistically induced by treatments that lead to differentiation, were first expressed at elevated levels during commitment and remained elevated in fully differentiated adipocytes. One or more of these proteins could well have a functional role in the commitment to and/or expression of the adipocyte differentiation program.

3T3 Cells↗
PubMed1992

Glucocorticoid modulation of transformed cell proliferation is oncogene specific and correlates with effects on c-myc levels.

In the presence of the glucocorticoid hormone dexamethasone, bovine papillomavirus-1 (BPV-1)-transformed C127 mouse fibroblasts assume a flattened morphology and reach a saturation density of only 50% of that attained without hormone. This phenotypic reversion of transformation is dependent on the continued presence of dexamethasone and occurs with concentrations as low as 1 nM. Dexamethasone also suppresses the growth of the parental C127 cells as well as that of cells transformed by polyoma middle-T. In contrast, the growth of C127 cells transformed by the oncogenes v-H-ras, v-mos, or v-fes is inhibited by low concentrations of dexamethasone (1 nM) and stimulated by higher concentrations (0.1-1 microM), possibly due to dexamethasone-induced transcription from the viral long terminal repeat promoters as is shown for v-H-ras. On the other hand, inhibition of BPV-transformed cell line growth by dexamethasone does not appear to be related to hormone effects on BPV-1 oncogene transcription. Indeed, in several cases, dexamethasone increases the steady state transcript levels of the BPV-1 oncogenes, E5 and E6-E7, while suppressing cellular proliferation. Dexamethasone also rapidly reduces the steady state levels of c-myc in the BPV-transformed cells but has less effect on c-myc expression in the ras-transformed cells. These results demonstrate that the growth-promoting actions of the papillomavirus transforming genes, but not those of several retroviral oncogenes, may be overcome by dexamethasone, which appears to act by down-regulation of c-myc expression.

Animals↗
PubMed1992

Imaging techniques in the evaluation of pulmonary parenchymal neoplasms.

Conventional PA and lateral chest radiographs continue to be the initial examination of choice to evaluate patients who are suspected of having a pulmonary parenchymal neoplasm. A lung lesion can be characterized as probably benign or malignant based on its radiographic appearance (size, shape, margins, presence of calcification, cavitation or air bronchograms, growth rate). A spiculated or lobulated lesion greater than 3 cm in size that is noncalcified is highly suspicious for malignancy. A lung lesion less than 3 cm in size with smooth borders that appears noncalcified on conventional radiographs should be examined by CT, including densitometry to detect calcification or fat, which indicates benignity. In patients with known lung cancer, CT can help to stage the tumor by indicating hilar or mediastinal involvement, or distant metastases. Currently, MR imaging has a limited role, but can be used as a "problem solving" modality for selected cases in evaluating pulmonary parenchymal neoplasms.

Diagnosis, Differential↗
PubMed1991

A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein.

The profound influence of glucocorticoid hormones on the inflammatory response includes a rapid and significant reduction in the synthesis of cyclooxygenase (prostaglandin G/H synthase, PGHS), the key enzyme for prostaglandin biosynthesis. In analyzing the glucocorticoid effects on PGHS synthesis in C127 mouse fibroblasts, we detected a novel 4-kilobase (kb) mRNA that is related to a PGHS cDNA cloned from an ovine seminal vesicle library. This RNA is much more prevalent in cycloheximide-treated cells and, based on stringency analysis and preliminary sequence data, arises from a gene distinct from that transcribed into the previously cloned 2.8-kb PGHS cDNA. Furthermore, the 4-kb mRNA encodes a 70-kDa protein that is specifically immunoprecipitated by anti-PGHS serum. The abundance of the 4-kb mRNA is strongly decreased by dexamethasone and increased by serum within 2 h whereas the 2.8-kb PGHS mRNA, which is also seen in these cells, does not consistently change. These changes in the level of the 4-kb mRNA with serum and dexamethasone treatment parallel changes in the level of synthesized PGHS protein detected in both metabolically labeled cells and in in vitro translated mRNAs. This discovery of a cyclooxygenase-related gene that is transcriptionally regulated by serum and glucocorticoid hormones in a manner identical to that reported for cyclooxygenase activity may help clarify issues regarding cyclooxygenase regulation and suggests that two distinct and differentially regulated cyclooxygenase species exist.

Amino Acid Sequence↗
PubMed1991

Amylin or CGRP (8-37) fragments reverse amylin-induced inhibition of 14C-glycogen accumulation.

The peptides amylin and calcitonin-gene related peptide (CGRP) have been shown to have similar effects on glycogen metabolism in vivo and in vitro. However, it is not clear whether they act via separate receptors. Peptide fragments based on the amino acid sequence of amylin or CGRP were evaluated for their ability to inhibit the action of the peptides in vitro. Insulin-stimulated glycogen turnover, as measured by 14C-glycogen accumulation, was inhibited about 70% by amylin (10nM) and 85% by CGRP (10nM). In the absence of exogenous peptide, peptide fragments based on the 8-37 and 10-37 amino acid sequences of rat amylin (10 uM) had no affect on 14C-glycogen accumulation. In the presence of amylin (10nM), the 8-37 and 10-37 fragments blocked amylin-induced inhibition of 14C-glycogen accumulation 100% and 11.4%, respectively. The 8-37 and 10-37 amylin fragments blocked CGRP inhibition of 14C-glycogen accumulation by 23.2% or 28.6%, respectively. The CGRP 8-37 fragment was equally effective as the amylin 8-37 reversing the effects of amylin than at reversing the effects of CGRP. These results demonstrate that amylin (8-37) completely antagonizes the effects of amylin with limited ability to block CGRP. Removing the eighth and ninth amino acids reduced the effectiveness of the inhibitor by about 90%.

Amyloid↗
PubMed1991

Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus.

Rhodobacter capsulatus has been known to possess two ferredoxins (I and II) with distinct physicochemical and structural properties: ferredoxin I is a 2[4Fe-4S] type and the other is a [3Fe-4S] [4Fe-4S] type. To analyze their possible functional differences, their genes (fdxN and fdxA) were cloned, sequenced, and subjected to interposon mutagenesis experiments. The former gene was adjacent to a gene encoding a chloroplast-type [2Fe-2S] ferredoxin (fdxC). Mutants with inactivated fdxN and/or fdxC were obtained, and they showed virtually no growth under nitrogen-fixing conditions. Complementation experiments confirmed that both fdxN and fdxC were required for nitrogen fixation. On the other hand, we have not been able to disrupt fdxA under the screening conditions surveyed, including conditions that do not require nitrogenase activity for growth, suggesting that ferredoxin II could have an unknown essential role(s). These indicate functional differences among multiple ferredoxins in one bacterium other than in cyanobacterial heterocysts and indispensability of certain ferredoxins in nitrogen fixation other than Rhizobium meliloti FdxN.

Amino Acid Sequence↗
PubMed1991

The truncated glucocorticoid receptor in the P1798 mouse lymphosarcoma is associated with resistance to glucocorticoid lysis but not to other glucocorticoid-induced functions.

Glucocorticoid receptors in lines of the P1798 mouse lymphosarcoma either sensitive or resistant to glucocorticoid-induced lysis have been characterized and their functional significance determined. The glucocorticoid receptor from the cortisol-sensitive tumor is an Mr approximately 98,000 protein with a Stokes radius of 7.4 nm in the oligomeric, non-DNA-binding state and 5.6 nm in the transformed, DNA-binding state. This receptor binds glucocorticoid and reacts with the BUGR-2 monoclonal antibody. In contrast, two abnormal receptor species were identified in the cortisol-resistant tumor. One is an Mr approximately 98,000 non-steroid-binding but immunologically reactive protein. The other is an Mr approximately 45,000 species which contains both steroid- and DNA-binding sites but exhibits little or no reactivity with BUGR-2, suggesting that its NH2 terminus is truncated in a region within or adjacent to the BUGR epitope. This species had Stokes radii of 5.8 and 3.5 nm in nontransformed and transformed states, respectively. In both tumor lines, glucocorticoids stimulated the activities of glutamine synthetase and 5'-nucleotidase and the synthesis of glucocortin. However, glucocorticoid-induced tumor regression occurred only in the cortisol-sensitive tumor. Additionally, the glucocorticoid inducibility of a specific protein in the sensitive, but not in the resistant, tumor was demonstrated, as well as the presence of a protein specific to the resistant line. Taken together, these results suggest that the truncated glucocorticoid receptor in the P1798 lymphosarcoma is functional, although possibly in a more restricted gene-specific manner, and that the lysis defect, while possibly resulting from a truncated receptor, may also result from the inability of glucocorticoids to induce a critical protein in the pathway of programmed cell death and/or from the presence of a protein which inhibits the lytic response.

5'-Nucleotidase↗
PubMed1991

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.

Amyloid↗
PubMed1991

Interactions of dimethyl sulfoxide and granulocyte-macrophage colony-stimulating factor on the cell cycle kinetics and phosphoproteins of G1-enriched HL-60 cells: evidence of early effects on lamin B phosphorylation.

We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.

Cell Cycle↗
PubMed1991

Bovine papillomavirus type 1 alters the processing of host glucose- and calcium-modulated endoplasmic reticulum proteins.

We have previously characterized five proteins induced by the presence of the E2 open reading frame (ORF) region of bovine papillomavirus type 1 (BPV-1) in C127 mouse fibroblasts (R. M. Levenson, U. G. Brinckmann, M. K. O'Banion, E. J. Androphy, J. T. Schiller, F. Tabatabai, L. P. Turek, K. Neary, M. T. Chin, T. R. Broker, L. T. Chow, and D. A. Young, Virology 172:170-179, 1989). By specific immunoprecipitation, we now find that one of the papillomavirus-associated proteins (pvp1) is a highly glycosylated form of glucose-regulated protein 100 (grp100), a major constituent of the endoplasmic reticulum. A second set of pvps (2, 3, and 4) are shown to be related precursors of another protein already present in C127 cells (protein B). Based on their induction by the calcium ionophore A23187 and their positions on giant two-dimensional gels, we have tentatively identified pvp2, -3, and -4 and B as forms of calcium-regulated protein 55, another constituent of the endoplasmic reticulum (D. R. J. Macer and G. L. E. Koch, J. Cell Sci. 91:61-70, 1988). The mechanism by which BPV-1 brings about these changes is not yet defined; however, it is unlikely to involve calcium level perturbations or transformation per se, since ionophore treatment changes other proteins in C127 cells not seen with BPV and the papillomavirus-associated proteins are found in nontransformed cells harboring the E2 ORF region. Furthermore, the BPV changes are not associated with increased grp mRNA levels, as occurs in ionophore-treated cells. Rather, it appears that BPV-1 somehow retards the normal processing of these resident endoplasmic reticulum proteins that are believed to serve as critical regulators of host protein processing and assembly.

Animals↗
PubMed1991

Interactions of sanguinarine and zinc on oral streptococci and Actinomyces species.

Sanguinaria extract, which contains benzophenanthridine alkaloids, has been used as a folk medicine for many years. Minimum inhibitory and minimum bactericidal concentrations (MIC and MBC values) for sanguinarine were determined for common and etiologically important plaque bacteria. Because the efficacy of sanguinarine is believed to be enhanced by zinc, isobolograms were assessed to determine their mode(s) of interaction. Hydrogen ion concentration influenced the inhibitory activity of both sanguinarine and zinc. For sanguinarine, at the optimum pH (6.5), MIC values were 4 or 8 micrograms/ml for Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Actinomyces viscosus and Actinomyces naeslundii. MIC values were 0.125-0.50 mmol Zn/ml. MBC values ranged from 1 to 8 mmol Zn/ml at pH 5.5. Isobologram data revealed that sanguinarine and zinc interacted synergistically. Viadent oral rinse, which contained 300 micrograms sanguinaria extract/ml and 0.2% zinc chloride (14.9 mmol Zn/l), was inhibitory to all strains tested. MIC values were 1 or 2% (ml Viadent oral rinse/100 ml aqueous solution) for all strains except A. viscosus for which the MIC value was 12% (vol/vol).

Actinomyces↗
PubMed1991

Suppression of tumorigenicity of a Wilms' tumor cell line is associated with a decrease in synthesis of two proteins.

Wilms' tumor has been associated with deletions in two loci on chromosome 11, and the introduction of a translocated human chromosome [t(X;11)] into a Wilms' tumor cell line (G401.6TG.6) by microcell hybridization suppresses tumor formation in nude mice. The tumorigenic phenotype is restored in segregants of these microcell hybrids, in which the introduced chromosome is lost. We have used ultrahigh-resolution 'giant' two-dimensional gel electrophoresis of metabolically labeled cellular proteins and in vitro translation products of isolated mRNA to identify changes in cellular gene expression that occur in these cell lines. The changes in gene expression associated with these chromosomal manipulations per se are quite minimal. However, we have identified two proteins (p16 and p28) whose synthesis is consistently decreased in three non-tumorigenic (suppressed) microcell hybrid clones relative to parental and segregant tumorigenic lines. They are also decreased at the level of mRNA in at least two of the non-tumorigenic clones. The decrease of these proteins represents markers of the suppressed phenotype, and their down-regulation may conceivably mediate the suppression of tumorigenicity.

Child, Preschool↗
PubMed1990

Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity.

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.

Antibody-Dependent Cell Cytotoxicity↗