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PubMed1998

Quantitative analysis of carcinoembryonic antigen messenger RNA in peripheral venous blood and portal blood of patients with pancreatic ductal adenocarcinoma.

One major therapeutic failure of pancreatic adenocarcinoma treatment is metastasis to the liver. To screen patients with high risk for such hematogenous dissemination, we previously developed a very sensitive system to detect carcinoembryonic antigen (CEA) in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing both preoperative peripheral blood and intraoperative portal blood for the presence of CEA mRNA. CEA mRNA was not detected in the peripheral venous blood of any of the three patients examined, but it was identified in the portal blood without fail. In addition, the quantities of CEA mRNA identified in the portal blood before and after pancreatectomy were different. This study suggests that analysis of the portal blood seems to be important for the precise evaluation of hematogenous dissemination and of the pathophysiology of pancreatic ductal adenocarcinoma.

Adenocarcinoma↗
PubMed1991

Prolactin receptor gene expression in rat mammary gland and liver during pregnancy and lactation.

The expression of two forms of PRL receptor messenger RNA was measured at different stages of pregnancy and lactation in mammary gland and liver from Sprague-Dawley rats, using 32P-labeled complementary DNA probes encoding the extracellular part of the receptor (E probe), common to the two forms and a probe encoding the intracellular part of the long form of the receptor (I probe), that only recognizes sequences specific to the long form of the receptor. Hybridizations were performed in Northern blots obtained from electrophoreses of poly (A+) enriched RNA preparations from mammary glands and livers of rats on days 0, 6, 12, 19, and 21 of pregnancy and 5, 10, 15, and 20 of lactation. The Northern blots were also hybridized with a chicken beta-actin probe, to correct for the amount of mRNA added and the different metabolic states of the tissues. Both tissues expressed the same forms of PRL receptor mRNAs, namely bands at 2.5, 3, and 5.5 kilobases encoding the long form of the receptor and a major band at 1.8 kilobases encoding the short form. The liver expressed all the receptor mRNA forms in much higher quantity than the mammary gland, independent of the reproductive state. In liver there was an increase of all the transcripts on day 19 of pregnancy, followed by an abrupt decline at the onset of lactation, to levels lower than those of virgin rats. In contrast, mammary gland PRL receptor mRNAs were low in virgin and pregnant animals, increased significantly at day 21 of pregnancy, and continued to increase throughout lactation. Treatment of day 19 pregnant rats with the antiprogesterone RU 486 induced, 24 h later, PRL receptor mRNAs in mammary gland but not in liver. There were no significant differences in the relative proportions of long to short forms of PRL receptor mRNAs at the different reproductive states, but the proportion of the long form was slightly greater in mammary gland than in liver. Membrane PRL receptor concentrations were also measured in the same tissues used for the mRNA study by binding to a 125I-labeled monoclonal antibody (U5), which specifically recognizes the PRL receptor at a site different from the hormone binding site. The quantity of receptor measured by U5 binding was approximately 3 times higher than that measured with 125I-labeled ovine PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Premature termination codon mutations in the type VII collagen gene in recessive dystrophic epidermolysis bullosa result in nonsense-mediated mRNA decay and absence of functional protein.

The severe mutilating Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is characterized by the absence of anchoring fibrils that consist of type VII collagen. We have previously identified premature termination codon (PTC) mutations in both alleles of the type VII collagen gene (COL7A1) in HS-RDEB patients. In this study we have defined the mechanism by which these mutations elicit their phenotypic consequences in a family. The extent of nonsense-mediated mRNA decay induced by these mutations was assessed by quantitation of the level of expression of the corresponding mRNA from each of the mutant alleles by RT-PCR of parental RNA. The level of expression of the paternal mutant allele with a PTC in exon 2 was approximately 30% of that of the wild-type allele whereas that of the maternal mutant allele with a PTC in exon 104 was reduced to about 80% of the normal allele. Immunoprecipitation of newly synthesized type VII collagen with a monoclonal antibody revealed reduced quantities of alpha1(VII) polypeptides in both parents' cells, whereas their synthesis was entirely absent in the proband's keratinocytes. Thus, a consequence of these premature termination codon mutations in COL7A1 is nonsense-mediated mRNA decay, with a dramatic reduction in type VII collagen synthesis, and the absence of anchoring fibrils in the proband. These results establish a mechanistic link between the presence of premature termination codon mutations in both alleles of COL7A1 and the clinical phenotype of HS-RDEB.

Alleles↗
PubMed2005

Dissection of the sets of genes that control the behavior of biglycan-deficient pre-osteoblasts using oligonucleotide microarrays.

Biglycan (bgn) is a small leucine-rich proteoglycan (SLPR) that is enriched in the extracellular matrix of skeletal tissues. Bgn-deficient mice develop age-related osteopenia with a phenotype that resembles osteoporosis. In order to identify sets of genes that play a key role in the skeletal abnormality, we determined the global gene expression patterns in bgn-deficient (bgn-KO) pre-osteoblasts using oligonucleotide microarray technology. Calvarial cells were harvested from newborn mice and cultured in the presence or absence of BMP-4 for 7 days. The total RNA was purified, labeled and hybridized to Affymetrix chips (U74A), and analyzed with a software program called GeneSpring. Our data suggested that biglycan regulates the activity of osteoblastic progenitors through sets of genes associated with cell cycle, cell growth, and differentiation. The biological outcome from the altered expression of these genes could cause a defect in the quantity and quality of osteoblastic progenitors, which could contribute to the development of age-related osteopenia in bgn-KO mice. Moreover, the data from this approach also revealed that biglycan deficiency affected the genes that control inflammation, immune response, and growth of tumor cells. These are new and unexpected findings that lead to the formation of new paradigms for biglycan function. Based on these findings, we propose that the reduction of this small proteoglycan with aging may increase the risk of infection and autoimmune diseases, impair wound healing, and cause higher incidences of malignancy. This study provides a broad and deep foundation for understanding SLRP function at a more complex level.

Animals↗
PubMed1989

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle.

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.

Animals↗
PubMed2006

The expression of phosphatidic acid phosphatase 2a, which hydrolyzes lipids to generate diacylglycerol, is regulated by p73, a member of the p53 family.

p73, a p53-related gene, is essential for a development of animals, while p53 is important for tumor formation. And little is known about the target genes specifically regulated by p73. Identifying the specific targets of p73 is important to understand the physiological roles of p73. To identify the genes specifically regulated by p73, we conducted serial analysis of gene expression to quantitatively evaluate messenger RNA populations. We found that the gene for phosphatidic acid phosphatase 2a (PAP2a), an enzyme that hydrolyzes lipids to generate diacylglycerol, was specifically upregulated by ectopic production of p73beta. The promoter region of this gene contains an element that is functionally responsive to p73beta. And the quantity of PAP2a protein was upregulated by ectopic production of p73beta. These results suggest that the expression of PAP2a is directly regulated by p73.

Cell Line↗
PubMed1967

Functional chloroplast polyribosomes from tobacco leaves.

Incubation of isolated to bacco chloroplasts with ingredients re quired for protein synthesis resulted in liberation of 70S ribosomnes and poly ribosomes that no longer sedimented with the chloroplasts. With increasing time of incubation, polyribosomes broke down to 70S monosomes. Similarly, microgram quantities of ribonuclease caused chloroplast polyribosomes to break down into monosomes. Both polyribosomes and 70S ribosomes that were isolated on sucrose density gra dients and tested separately in cell-free systems were capable of protein syn thesis; however, polyribosomes formed more protein per unit of RNA than monosomes did.

Carbon Isotopes↗
PubMed2003

KDBI: Kinetic Data of Bio-molecular Interactions database.

Understanding of cellular processes and underlying molecular events requires knowledge about different aspects of molecular interactions, networks of molecules and pathways in addition to the sequence, structure and function of individual molecules involved. Databases of interacting molecules, pathways and related chemical reaction equations have been developed. The kinetic data for these interactions, which is important for mechanistic investigation, quantitative study and simulation of cellular processes and events, is not provided in the existing databases. We introduce a new database of Kinetic Data of Bio-molecular Interactions (KDBI) aimed at providing experimentally determined kinetic data of protein-protein, protein-RNA, protein-DNA, protein-ligand, RNA-ligand, DNA-ligand binding or reaction events described in the literature. KDBI contains information about binding or reaction event, participating molecules (name, synonyms, molecular formula, classification, SWISS-PROT AC or CAS number), binding or reaction equation, kinetic data and related references. The kinetic data is in terms of one or a combination of the following quantities as given in the literature of a particular event: association/dissociation or on/off rate constant, first/second/third/. order rate constant, equilibrium rate constant, catalytic rate constant, equilibrium association/dissociation constant, inhibition constant and binding affinity constant. Each entry can be retrieved through protein or nucleic acid or ligand name, SWISS-PROT AC number, ligand CAS number and full-text search of a binding or reaction event. KDBI currently contains 8273 entries of biomolecular binding or reaction events involving 1380 proteins, 143 nucleic acids and 1395 small molecules. Hyperlinks are provided for accessing references in Medline and available 3D structures in PDB and NDB. This database can be accessed at http://xin.cz3.nus.edu.sg/group/kdbi/kdbi.asp.

DNA↗
PubMed1990

Growth hormone regulation of hepatic glutamine synthetase mRNA levels in rats.

The expression of glutamine synthetase (GS) in the rat liver is dependent on pituitary growth hormone (GH). RNA blot hybridizations revealed that in hypophysectomized rats the level of glutamine synthetase mRNA was dramatically reduced in liver but not brain. This drop of GS mRNA in the liver results in a reduction of GS enzyme activity as well. Two other messages, phosphoenolpyruvate carboxykinase and glycerol phosphate dehydrogenase were not diminished in the liver, indicating that the effects of hypophysectomy on hepatic GS expression are specific and not part of a general reduction in transcription due to lack of pituitary factors. Daily administration of rat pituitary growth hormone caused an increase in the levels of hepatic GS mRNA as well as enzyme activity. In situ hybridization of normal liver sections with the GS antisense message showed an abundant amount of message confined to the region around each central vein of the hepatic acini, while in the hypophysectomized animal the message for GS is greatly reduced but still only located in hepatocytes surrounding the central vein. Hypophysectomized animals given GH replacement showed a substantial increase in the amount of exposed silver grains only around the central veins. This indicates that GH does not influence the cellular position of GS expression nor the viability of those hepatocytes that express the enzyme, but it does regulate the quantity of GS in the liver through changes in the levels of GS mRNA.

Animals↗
PubMed1995

Isolation and characterization of heparin-binding growth factors in human leiomyomas and normal myometrium.

Uterine leiomyomas (fibroids) are benign, smooth muscle cell (SMC) tumors of the myometrium containing abundant extracellular matrix (ECM). Heparin-binding growth factors present in leiomyoma and normal myometrial fresh tissue were isolated using heparin-affinity fast protein liquid chromatography. Purification of these growth factors was monitored by the stimulation of [3H]thymidine incorporation into BALBc-3T3 cells and myometrial SMC. Western blot analysis confirmed that two consistent peaks of growth factor activity (eluting at 0.5 M NaCl and 1.7 M NaCl) were platelet-derived growth factor (PDGF), 31 kDa, and basic fibroblast growth factor (bFGF), 18 kDa, respectively. Northern blot analysis of leiomyoma and myometrial tissue revealed three RNA transcripts (2.8, 2.3, and 1.9 kb) for PDGF-A chain, one RNA transcript (4.0 kb) for PDGF-B chain, and two RNA transcripts (3.7 and 3.5 kb) for bFGF. RNase protection assay showed elevated expression of the bFGF mRNA transcript in leiomyomas in 3 out of 5 patients. Immunoperoxidase staining of paraffin-embedded tissue showed that PDGF was predominantly intracellular in both vascular and myometrial SMC. Basic FGF, by contrast, was found primarily bound to the ECM of myometrium and fibroids. Leiomyomas showed much stronger staining for bFGF due to the large areas of ECM in these tumors. A third mitogenic peak eluting at 1.1 M NaCl was also seen in both myometrial and leiomyoma tissue. This peak was not definitively identified by Western blotting. However, Northern analysis for heparin binding-epidermal growth factor (HBEGF), which also elutes at 1.1 M NaCl, detected one RNA transcript for HBEGF (2.5 kb) in normal myometrium but little or no expression in the corresponding leiomyoma tissue. Immunoperoxidase staining showed that HBEGF was a cell-membrane-associated protein in both normal myometrial and leiomyoma SMC with more intense staining in normal myometrium. These results show that both leiomyomas and myometrium synthesize a number of heparin-binding growth factors. The enhanced growth of leiomyomas may be due, in part, to the presence of large quantities of bFGF that are stored in the ECM of these tumors. In addition, the level of HBEGF mRNA declines during the transformation of myometrial SMC into leiomyomas.

Animals↗
PubMed1993

cis-acting elements in the 5'-untranslated region of rat testis proenkephalin mRNA regulate translation of the precursor protein.

We found that a rat testis cDNA library contained two types of proenkephalin cDNA, corresponding to somatic and germ cell-specific transcripts. The somatic form is identical in primary sequence to previously characterized transcripts from rat brain and heart. The longer germ cell-specific cDNA contains a novel 5' terminus derived from the somatic intron A. We found that germ cell-specific transcripts translate in vitro with significantly lower efficiency than do somatic-type transcripts. Deletion analysis indicated that translational inefficiency is mediated primarily by the presence of four upstream AUG codons followed by short open reading frames within the leader sequence. Secondary structure in the form of a stem-and-loop upstream of the initiator codon for proenkephalin was found to have a slightly inhibitory effect on translation. These results, coupled with the very low abundance of the somatic transcript, provide a possible explanation for the low levels of opioid peptides found in adult rat testis in spite of the abundant quantity of germ cell-specific proenkephalin transcript.

Animals↗
PubMed1992

Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1.

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

Amino Acid Sequence↗
PubMed1990

Variation within and between nucleolar organizer regions in Australian hylid frogs (Anura) shown by 18S + 28S in-situ hybridization.

Five distinct classes of secondary constriction are found in the hylid frogs from the genera Litoria and Cyclorana, each of which is defined by its C-banding pattern and morphology (King, 1980, 1987). In-situ hybridization experiments utilizing 18S + 28S copy RNA probes derived from Xenopus and Drosophila rDNA templates, were made on nine species of frogs possessing the major constriction types. Types 1, 2, 4, and 5 are confirmed as being NORs. These results also indicate that type 1 and 2 constriction types are not differentially despiralized as previously suggested, but show absolute differences in the quantity of ribosomal DNA present. This variation took two forms, deletion polymorphism and amplification polymorphism. These differences were observed between homologues within cells and between cells within individuals. Animals possessing these 'despiralized' constrictions are therefore mosaics for both deletion and amplification polymorphisms. Polymorphism frequencies vary greatly between constriction types. Some specimens have a higher level of presence/absence heterozygosity, (L. moorei, type 2, L. nannotis type 5, L. raniformis (animal A, pair 8 type 2), than do others (L. peronii, L. rothii, L. caerulea). The above species also vary markedly in the degree and frequency of amplification of the NORs. The type 4 constrictions analysed (L. coplandi, L. lesueuri and C. novaehollandiae) have a particularly low frequency of presence/absence heterozygosity, and they have fewer size heteromorphisms between homologues. The type 3 ephemeral constrictions did not hybridize to cRNA probes at any stage. In all but one of the species studied, a single pair of chromosomes possessed an NOR. However, in L. raniformis these occurred on two pairs of chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Quantitative polymerase chain reaction of lysyl oxidase mRNA in malignantly transformed human cell lines demonstrates that their low lysyl oxidase activity is due to low quantities of its mRNA and low levels of transcription of the respective gene.

Lysyl oxidase (EC 1.4.3.13), an extracellular copper amino oxidase, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues. We developed here a polymerase chain reaction (PCR) method for the quantification of lysyl oxidase mRNA in which a synthetic RNA is used as an internal standard for coamplification with the targeted mRNA. The amount of lysyl oxidase mRNA when studied by Northern blot analysis and the number of lysyl oxidase mRNA molecules when determined by the quantitative PCR method were found to be markedly low in various malignantly transformed cell lines relative to control cell lines, quantitative PCR indicating values of about 2-10% of those in the controls. No difference was found in the number of beta-actin mRNA molecules between the transformed cells and the controls. Nuclear runoff experiments indicated that most if not all of the decrease in the number of lysyl oxidase mRNA molecules can be explained by diminished transcription of the respective gene.

Adult↗
PubMed1990

Anti-RNA polymerase I antibodies in the urine of patients with systemic lupus erythematosus.

Urine samples from patients with systemic lupus erythematosus (SLE) (n = 80), patients with rheumatoid arthritis (RA) (n = 21), and healthy controls (n = 36) were analyzed by radio-immunoassay (RIA) for anti-RNA polymerase I (RPI) antibodies. Significant levels of anti-RPI antibodies were detected in the urine of 46% of the patients with SLE but in only 19% of the patients with RA and in no sample from healthy individuals. The presence of anti-RPI antibodies in the urine was confirmed by demonstrating that IgG purified from the urine of patients with SLE was capable of inhibiting the transcriptional activity of RPI in vitro. If the quantity of anti-RPI antibodies excreted is related to disease activity, analysis of urine for these antibodies may be a useful alternative for the purpose of monitoring the progression of disease in individuals with SLE because of the ease by which the sample can be collected.

Adolescent↗
PubMed2005

Dependence of RNA secondary structure on the energy model.

We analyze a microscopic RNA model, which includes two widely used models as limiting cases; namely, it contains terms for bond as well as for stacking energies. We numerically investigate possible changes in the qualitative and quantitative behavior while going from one model to the other; in particular, we test whether a transition occurs when continuously moving from one model to the other. For this we calculate various thermodynamic quantities, at both zero temperature and finite temperatures. All calculations can be done efficiently in polynomial time by a dynamic programming algorithm. We do not find a sign for the transition between the models, but the critical exponent nu of the correlation length, describing the phase transition in all models to an ordered low-temperature phase, seems to depend continuously on the model. Finally, we apply the epsilon -coupling method to study low-energy excitations. The exponent theta describing the energy scaling of the excitations seems to depend not much on the energy model.

Algorithms↗
PubMed1986

Identification of the C-terminal portion of a protein by comparative peptide mapping.

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.

14-3-3 Proteins↗
PubMed1993

Reverse transcription-polymerase chain reaction: an overview of the technique and its applications.

The polymerase chain reaction (PCR) has galvanized molecular biologists by virtue of its ability to provide them with large quantities of any desired fragment (up to 11kb) of DNA. This power combined with its flexibility has also inspired many useful applications, including new methods of DNA sequencing, cloning and mutagenesis. One logical variation of PCR has been its application to the detection and analysis of messenger RNA by the addition of a reverse transcription step prior to performing PCR. Due to the exquisite sensitivity of PCR, reverse transcription-PCR (RT-PCR) has been used to characterize mRNAs previously undetectable by established methods of RNA analysis such as Northern hybridization and RNase protection assays. Furthermore, its capacity as a method of quantitative analysis is currently being developed. RT-PCR has also been used to diagnose the presence of certain diseases. Recently, RT-PCR has been employed to identify and isolate genes that are differentially expressed in different cells or environmental conditions.

Journal Article↗