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D C Eichler

Publications and source records attributed to D C Eichler.

At least 19 recordsLinked to original sources
PubMed2000

Stimulation of receptor-mediated nitric oxide production by vanadate.

Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore- (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to approximately 125 kDa and 2 minor bands corresponding to approximately 105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.

Animals↗
PubMed1999

A spun-column assay for determination of leptin binding in serum.

Serum collected from 27 patients was assayed simultaneously using a spun-column assay (SPC) and a traditional exclusion gel-filtration assay (GFC) to determine specific leptin binding. The levels of serum leptin binding determined by either assay correlated inversely with serum leptin levels (SPC, r = 0.63, P < 0.001; GFC, r = 0.79, P < 0.0001). Although specific leptin binding as determined by the traditional exclusion gel-filtration assay was generally higher than that obtained by the spun-column assay (mean = 18.3% vs 14.0%, P < 0. 02, respectively); the values obtained between the two assay methods were highly correlative (r = 0.89, P < 0.0001). By varying either the amount of 125I-leptin or the amount of competitor, analysis was carried out using the spun-column assay to determine the intrinsic properties of serum leptin binding. Results yielded a Kd = 0.3 nM, where each variable amount of leptin or competitor was carried out in duplicate. The complete analysis was carried out in the time that it typically takes for a single sample determination by the traditional exclusion gel-filtration assay. We conclude that the "spun-column" assay is a useful method for rapid and accurate quantification of leptin binding in serum.

Adult↗
PubMed1999

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCbetaII mRNA in vascular smooth muscle cells.

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCbeta gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCbetaII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCbetaI levels were unaltered. PKCbeta mRNA levels were depleted by 60-75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCbeta expression via the common promoter for PKCbetaI and PKCbetaII, deletion constructs of the PKCbeta promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (-411 to +179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5' promoter region of the PKCbeta gene. In actinomycin D-treated A10 cells, a 60% decrease in PKCbeta mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose-induced posttranscriptional destabilization of PKCbetaII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to posttranscriptionally destabilize PKCbetaII mRNA, but not the PKCbetaI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.

Animals↗
PubMed1998

Insulin regulates protein kinase CbetaII expression through enhanced exon inclusion in L6 skeletal muscle cells. A novel mechanism of insulin- and insulin-like growth factor-i-induced 5' splice site selection.

The protein kinase Cbeta (PKCbeta) gene encodes two isoforms, PKCbetaI and PKCbetaII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCbeta pre-mRNA was examined in L6 myotubes. Mature PKCbetaII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCbetaI mRNA and protein levels remained unchanged. Mature PKCbetaII mRNA resulted from inclusion of the PKCbetaII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCbetaII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCbetaII mRNA, and suggest that PKCbetaII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5'-splice site variant of PKCbetaII was also observed. After 30 min, the longer 5'-splice site variant became the predominate species through activation of a downstream 5' splice site. Similar results were obtained using IGF-I. Although the role of this new PKCbetaII mRNA species is presently unknown, inclusion of either PKCbetaII-specific exon results in the same PKCbetaII protein.

Alternative Splicing↗
PubMed1997

Demonstration of a leptin binding factor in human serum.

Serum leptin levels are elevated in subjects with exogenous obesity, indicating that obesity is associated with leptin resistance. Since in man no abnormalities have yet been found in either the genes for leptin or its receptor, the mechanism of leptin resistance in obesity remains unknown. To determine if resistance might be related to leptin binding by a serum component, we assessed the carrier status of leptin in serum. The presence of a specific leptin binding factor in human serum has been established by (1) demonstrating [125I]-leptin binding to a serum component that is saturable and specifically displaceable only by unlabeled leptin and not by human growth hormone, pork insulin, insulin-like growth factors I and II, luteinizing or follicle stimulating hormones, transforming growth factor-beta 1, interleukin-6, or leukemia inhibiting factor; (2) fractionating the leptin bound serum complex and the serum leptin binding component on a molecular sieving column revealing a mass of approximately 450 kDa; and (3) identifying an inverse correlation between the concentration of serum leptin and the quantity of the leptin binding component. It is suggested that binding of leptin by this serum component may influence the physiologic response to leptin.

Animals↗
PubMed1994

Characterization of a nucleolar 2'-O-methyltransferase and its involvement in the methylation of mouse precursor ribosomal RNA.

A nucleolar 2'-O-methyltransferase, partially purified from isolated mouse nucleoli, catalyzes the methylation of each of the four nucleosides, although to different levels depending on the RNA substrate. Similar to most methyltransferases which use S-adenosyl-L-methionine (SAM) as the methyl donor, the nucleolar 2'-O-methyltransferase was shown to bind S-adenosyl-L-homocysteine (SAH) (Kd = 0.17 microM), a product of the transfer reaction, as tightly as SAM (Kd = 0.24 microM). Binding assays also demonstrated stereospecificity about the sulfonium center of SAM. The naturally occurring S-chiral form of SAM had a 10-fold higher binding affinity than the R-chiral form. In addition, the alpha-amino group of the methionine moiety and the 6-amino group of the adenine moiety were shown to be required for maximal binding. The relative high affinity for both SAM and SAH may reflect a mechanism by which ribosome biogenesis is, in part, coordinated with cell growth, since a decrease in SAM:SAH ratio correlates with decreasing levels of 2'-O-methylation. The availability of unmethylated, in vitro-derived rRNA transcripts has made it possible to explore questions relating to the specificity for the RNA substrate. Using an in vitro-derived 28S rRNA transcript, the enzyme selectively methylated the sequence AmGmCm that occurs in a single-stranded bridge spanning two highly conserved structural domains of 28S rRNA. These results demonstrated that the purified nucleolar 2'-O-methyltransferase was sufficient to accurately methylate this region of 28S rRNA, and were taken to support the involvement of this nucleolar enzyme in the posttranscriptional methylation of the 47S precursor ribosomal RNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1994

Processing of eukaryotic ribosomal RNA.

In summary, it can be argued that the understanding of eukaryotic rRNA processing is no less important than the understanding of mRNA maturation, since the capacity of a cell to carry out protein synthesis is controlled, in part, by the abundance of ribosomes. Processing of pre-rRNA is highly regulated, involving many cellular components acting either alone or as part of a complex. Some of these components are directly involved in the modification and cleavage of the precursor rRNA, while others direct the packaging of the rRNA into ribosome subunits. As is the case for pre-mRNA processing, snoRNPs are clearly involved in eukaryotic rRNA processing, and have been proposed to assemble with other proteins into at least one complex called a "processosome" (17), which carries out the ordered processing of the pre-rRNA and its assembly into ribosomes. The formation of a processing complex clearly makes possible the regulation required to coordinate the abundance of ribosomes with the physiological and developmental changes of a cell. It may be that eukaryotic rRNA processing is even more complex than pre-mRNA maturation, since pre-rRNA undergoes extensive nucleotide modification and is assembled into a complex structure called the ribosome. Undoubtedly, features of the eukaryotic rRNA-processing pathway have been conserved evolutionarily, and the genetic approach available in yeast research (6) should provide considerable knowledge that will be useful for other investigators working with higher eukaryotic systems. Interestingly, it was originally hoped that the extensive work and understanding of bacterial ribosome formation would provide a useful paradigm for the process in eukaryotes. However, although general features of ribosome structure and function are highly conserved between bacterial and eukaryotic systems, the basic strategy in ribosome biogenesis seems to be, for the most part, distinctly different. Thus, the detailed molecular mechanisms for rRNA processing in each kingdom will have to be independently deciphered in order to elucidate the features and regulation of this important process for cell survival.

Animals↗
PubMed1993

Selection of a preribosomal RNA processing site by a nucleolar endoribonuclease involves formation of a stable complex.

A nucleolar endoribonuclease from mouse Ehrlich ascites tumor cells, that has been implicated in the endonucleolytic cleavage of mouse precursor ribosomal RNA, specifically and stably binds an in vitro-derived rRNA transcript containing the +650 early processing site. The specificity of binding was demonstrated by mobility shift analysis, glycerol gradient velocity sedimentation analysis, and UV-crosslinking studies. Binding did not require Mg2+ and therefore was not dependent on cleavage; however, binding was dependent on the presence of the early +650 processing site since a pre-rRNA transcript with the +650 processing site deleted failed to compete in binding. A small nucleolar RNA component was not required for the formation of this stable complex or for the specific cleavage of a processing competent pre-rRNA transcript. UV crosslinking studies using 32P-labeled 5-azidouridine-substituted pre-rRNA with bound nucleolar endoribonuclease identified three closely sized polypeptides of approximately 50, approximately 48, and approximately 45 kDa, respectively, that specifically crosslinked to the processing competent rRNA transcript. These three polypeptides species were identified following ribonuclease digestion and electrophoresis on a SDS-polyacrylamide gel. An identical pattern of labeled polypeptides was also identified from gel mobility shift analysis where the specifically shifted material was U.V. crosslinked. The largest of these polypeptides corresponded to the estimated size of the nucleolar endoribonuclease, while the lower molecular weight species may represent partially proteolyzed enzyme. Overall, these results suggest that the unique specificity of the nucleolar endoribonuclease may, in part, be attributed to the formation of a stable complex at the +650 processing site for mouse preribosomal RNA, and that formation of this unique stable complex affords a means to specifically label the limited amount of available partially purified enzyme for sequence analysis.

Animals↗
PubMed1993

Information processing and gene expression.

The process by which information is transferred from DNA to RNA to protein accommodates the further editing and altering of information, particularly in messenger RNA. From one gene, a cell may derive different protein products by altering the information content of mRNA to accommodate the very specialized functions of the cell. Information processing in the form of posttranscriptional RNA processing allows for incredible complexity and differentiation of tissue and tissue function. Most importantly, the posttranscriptional processing of mRNA does not alter the indelible information stored in the DNA which must be maintained and passed on from one generation to the next. Rather, the alteration of information occurs during the transient flow of information from DNA to RNA at the level of messenger RNA. Developmental and differentiation processes are dependent on this information processing which endows the cell with another level of control in gene expression.

Animals↗
PubMed1991

A nucleolar 2'-O-methyltransferase. Specificity and evidence for its role in the methylation of mouse 28 S precursor ribosomal RNA.

Methylation of ribose moieties appears to be an essential post-transcriptional event in ribosomal RNA maturation. Although the sites of ribose methylation have been identified, the components involved in the 2'-O-methylation of precursor ribosomal RNA in mammalian cells have not yet been elucidated. To investigate the involvement of a recently isolated nucleolar 2'-O-methyltransferase in this process, an in vitro synthesized 28 S rRNA transcript containing a unique tandem triple 2'-O-methylated ribose site was used as a substrate. Activity assays demonstrated that this transcript served as a substrate for the nucleolar 2'-O-methyltransferase. The distribution of incorporated methyl groups was determined by hydrolyzing the 2'-O-methylated transcript with RNase followed by chromatography of the digested products on an anion-exchange high performance liquid chromatography column. Results showed one unique RNase-resistant 2'-O-methylated product, a tetramer. The position of the tetrameric sequence in the 28 S rRNA transcript was determined using RNase protection analysis which mapped the methylations to a 20-nucleotide region spanning the unique tandem triple 2'-O-methylated ribose site in 28 S rRNA. To confirm the absolute specificity of methylation, direct sequence analysis was carried out on the tandem triple 2'-O-methylated tetramer. The sequence determined for the tetramer, AmGmCmA, corresponded exactly with that reported from in vivo studies. These findings demonstrate that the purified nucleolar 2'-O-methyltransferase can accurately methylate at a specific site of an in vitro derived preribosomal RNA transcript and support the proposed involvement of this nucleolar enzyme in ribosomal RNA maturation.

Animals↗
PubMed1990

In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

Animals↗
PubMed1989

The specificity of interaction between S-adenosyl-L-methionine and a nucleolar 2'-O-methyltransferase.

The structural features of S-adenosyl-L-methionine (SAM)3 required for optimal binding to a nucleolar 2'-O-methyltransferase were elucidated using various analogs of SAM with modifications of the amino acid, sugar, sulfonium center, and base portions of the molecule. Equilibrium binding constants for SAM and each analog were determined by a nitrocellulose filter binding assay. To ensure the chiral and chemical purity of the 3H-labeled SAM used in the binding experiments, a cation-exchange HPLC procedure was developed to separate degradation products of SAM such as adenine and 5'-deoxy-5'-methylthioadenosine, as well as to separate the (S,S)-SAM from the biologically inactive (R,S)-SAM stereoisomer. Results from these studies demonstrated that S-adenosyl-L-homocysteine, a product of the methyltransferase reaction, bound equally as well as (S,S)-SAM, indicating that neither the charge nor the methyl group at the sulfonium center of (S,S)-SAM is essential for maximal binding. Other modifications of the sulfonium center demonstrated that a sulfur to carbon atom replacement had little effect on binding affinity, whereas substituting an ethyl group for the methyl group greatly reduced the binding affinity. In addition, the chirality at the sulfonium center was important. The naturally occurring S-chiral form had a 10-fold higher binding affinity than the R-chiral form. No significant stereospecificity was observed relative to the chiral alpha-carbon of the methionine moiety in SAM. The alpha-amino group of methionine and the 6-amino group of adenine were both required for maximal binding, while the loss of the 2'-hydroxyl group on the ribose moiety was not. Taken together, these results defined some of the specific geometric and functional group requirements which affect the specificity of interaction between S-adenosyl-L-methionine and the nucleolar 2'-O-methyltransferase.

Animals↗
PubMed1988

Ribosomal RNA processing. Limited cleavages of mouse preribosomal RNA by a nucleolar endoribonuclease include the early +650 processing site.

A highly purified nucleolar associated endoribonuclease was tested for possible involvement in the processing of precursor ribosomal RNA at a primary cleavage site approximately 650 nucleotides downstream from the transcription initiation site. Preribosomal RNA sequences containing the +650 region were synthesized in vitro and subsequently digested over a range of concentrations of the nucleolar endoribonuclease. Cleavages generated by the nucleolar endoribonuclease were localized both by S1 nuclease protection analysis and primer extension analysis. A more precise determination of the specificity of cleavage was achieved by chemical cleavage DNA sequence analysis. These data demonstrated that the purified nucleolar endoribonuclease specifically cleaved the precursor ribosomal RNA transcript at the +650 site. Additional enzyme-dependent cleavages were observed upstream to the +650 site in a region which is rapidly degraded following processing at the +650 site in vivo. No major cleavages were observed for a distance of approximately 250 nucleotides downstream from the +650 site in a conserved region of sequence previously shown to be important in specifying processing at the +650 site. As a control, pancreatic ribonuclease, a single strand-specific endoribonuclease, was shown not to produce similar cleavages in the +650 region, indicating that cleavage by the nucleolar RNase was not simply due to accessibility of the RNA at the +650 site. Taken together, these results suggest that the nucleolar endoribonuclease may be necessary and sufficient to catalyze one of the initial endonucleolytic cleavages in preribosomal RNA processing.

Animals↗
PubMed1988

Specificity of a nucleolar 2'-O-methyltransferase for RNA substrates.

In this study we examined the specificity of a nucleolar 2'-O-methyltransferase isolated from nucleoli of Ehrlich ascites tumor cells. The nucleolar methyltransferase was capable of methylating each of the four nucleosides of RNA, however, the level of methylation at a particular nucleoside varied with the type of RNA. Both kinetic analysis and the stimulation of methylation by polyamines suggested that the structure of RNA was critical in influencing the discrimination and apparent specificity of nucleolar 2'-O-methyltransferase.

Adenosine↗
PubMed1987

Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes.

In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.

Animals↗
PubMed1987

Isolation and characterization of a nucleolar 2'-O-methyltransferase from Ehrlich ascites tumor cells.

A 2'-O-methyltransferase that transfers the methyl group from S-adenosylmethionine to the 2'-hydroxyl group of ribose moieties of RNA has been purified from Ehrlich ascites tumor cell nucleoli. The partially purified enzyme is devoid of other RNA methylase activities and is free of ribonucleases. The enzyme has optimal activity in tris(hydroxymethyl)aminomethane buffer, pH 8.0, in the presence of 0.4 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, and 50 mM KCl, and has an apparent Km for S-adenosylmethionine of 0.44 microM. Gel filtration studies of this enzyme gave a Stokes radius of 43 A. Sedimentation velocity measurements in glycerol gradients yield an S20,w of 8.0 S. From these values, a native molecular weight of 145,000 was calculated. The enzyme catalyzes the methylation of synthetic homoribopolymers as well as 18S and 28S rRNA; however, poly(C) is the preferred synthetic substrate, and preference for unmethylated sequences of rRNA was observed. For each RNA substrate examined, only methylation of the 2'-hydroxyl group of the ribose moieties was detected.

Animals↗
PubMed1987

Anti-flavin antibodies.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.

Animals↗