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Biomedical subjects

J R Emanuel

Publications and source records attributed to J R Emanuel.

At least 19 recordsLinked to original sources
PubMed1997

Viral RNA in duodenal bile of cirrhotic patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) has been detected in blood, saliva, urine, semen, breast milk, and tears. To our knowledge, bile has not yet been investigated. We observed histologic immunoreactivity in bile with an antibody to c100 protein in four of five HCV-positive cirrhotic livers, but also in two HCV-negative controls owing to a focally present cross-reacting antigen. METHODS: We collected duodenal bile from 13 cirrhotic patients during endoscopic evaluation of varices (10 HCV, three controls) and assayed for HCV by reverse transcriptase polymerase chain reaction. RESULTS: Viral RNA was detected in the bile of 8 of 10 seropositive patients and in 0 of 3 seronegative controls. CONCLUSION: Hepatitis C virus RNA and an antigen immunoreactive with anti-c100 protein are present in bile in a proportion of cirrhotic patients with chronic HCV. It remains to be determined whether the virus is intact or degenerate, and whether it is shed into bile from hepatocytes or is a contaminant from blood or other secretions.

Adult↗
PubMed1996

Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations.

Mutation detection by single-strand conformational polymorphism (SSCP) analysis is more difficult when the variant is limited to a small proportion of target sequences in a sample. Use of SYBR-Green II, a sensitive, nonradioactive, minimally hazardous nucleic acid stain, permits detection of Ki-ras mutants present as less than 0.5% of the target sequences. The polymerase chain reaction (PCR) primers we have selected produce an amplicon that distinguishes all clinically observed variants in Ki-ras codons 12 and 13 from the wild type. We compared mutant discrimination and SYBR-Green II detection sensitivity in three formats: (a) standard MDE gel SSCP, (b) rapid minigel MDE using an internal gel temperature controller, and (c) rapid resolution in chilled 15% (37.5:1) acrylamide minigels. All these gels are easily evaluated by standard ultraviolet transillumination and digital image analysis. This ssDNA staining method is rapid, highly reproducible, and minimally hazardous, and minigels use 25% the reagents of most other systems. Our improvements are relevant for the detection of mutations in pathologic samples with minimal targets, such as fine-needle aspirates, and body fluids in which mutated alleles of a gene may be present at low levels but carry a high level of diagnostic or prognostic importance.

DNA↗
PubMed1996

Identification of mutations in the Ki-ras gene in human retinoblastoma.

PURPOSE: To investigate the mutational status of the Ki-ras gene in retinoblastoma and to evaluate a correlation of the genotype with clinical and histopathologic variables. METHODS: Tumor samples were microdissected from sectioned archival paraffin-embedded tissue. Ki-ras genomic sequences (exons 1 and 2) were amplified by polymerase chain reaction then analyzed by single-strand conformation polymorphism and direct sequencing. Tissue sections, flanking the analyzed samples, were stained with hematoxylin and eosin for examination by light microscopy. RESULTS: Four of 12 tumors had mutation in exon 1, codon 12 of the Ki-ras gene; none had mutation in exon 2. Signal intensity of the mutated alleles indicates clonal mutation in the tumor cell populations. One of four bilateral tumors was mutated, and all three samples with undifferentiated histologic appearance harbored a clonal Ki-ras mutation. However, only one of nine moderately to poorly differentiated tumors harbored a mutation. CONCLUSIONS: Ki-ras, an oncogene seldom altered in neuroectodermal neoplasms, is mutated in one third of the retinoblastomas studied. The Ki-ras mutations are clonal, suggesting that affected cells have a selective growth advantage. The mutations are present and are likely to play a pathogenetic role in heritable and sporadic retinoblastomas. These results suggest that mutations in Ki-ras are preferentially associated with undifferentiated tumors.

Adolescent↗
PubMed1991

Cell-specific expression of mRNAs encoding Na+,K(+)-ATPase alpha- and beta-subunit isoforms within the rat central nervous system.

We have used in situ hybridization histochemistry to analyze the subcellular distribution of mRNAs encoding Na,K-ATPase alpha- and beta-subunit isoforms in the rat central nervous system. Substantial differences in the cell-specific pattern of expression were found for the genes encoding three isoforms of the alpha subunit. Transcripts of alpha 1-subunit gene were detected in virtually all cell types and structures examined. Expression of alpha 2-subunit mRNA was characteristic of glia, whereas alpha 3-subunit transcripts were predominant in neurons. Transcripts encoding the beta 1 subunit were detected in neurons, whereas beta 2-subunit mRNA expression was characteristic of glia. mRNA encoding both beta-subunit isoforms was present in choroidal epithelial cells. The distribution pattern of alpha- and beta-subunit mRNAs in structures throughout the central nervous system is consistent with the possibility of six structurally distinct Na+,K(+)-ATPase isoenzymes.

Animals↗
PubMed1990

Expression of Na,K-ATPase alpha and beta subunit genes during preimplantation development of the mouse.

Na,K-ATPase is a plasma membrane enzyme that plays a critical role in eutherian blastocoel formation (cavitation) by pumping Na+ into the extracellular space enclosed by the trophectoderm. Previous experiments with the mouse had shown that the alpha (catalytic) subunit of the enzyme becomes detectable by immunocytochemistry in the late morula, just prior to the onset of cavitation. In the present study we have used cDNAs corresponding to three mRNA isoforms of the alpha subunit and a beta subunit to determine which genes are expressed during preimplantation development and to explore the timing of their expression. Of the three alpha subunit cDNAs tested by Northern blot hybridization with blastocyst RNA, only alpha 1 produced a hybridization signal, recognizing a single mRNA about 4 kb in length. This mRNA is relatively abundant in zygotes but barely detectable by the 2-cell stage and then accumulates steadily thereafter to reach its preimplantation maximum in blastocysts. The beta 1 cDNA detected mRNA of about 2.6-2.8 kb. This mRNA is present in zygotes but could not be detected in 2-, 4-, or 8-cell stages; it is present at a low level in late morulae and is abundant in blastocysts. The temporal profile of accumulation of beta 1 mRNA thus matches more closely than does alpha 1 the timing of appearance of the catalytic subunit. This suggests that the beta subunit may regulate production of the holoenzyme and hence the timing of cavitation.

Animals↗
PubMed1990

1 alpha,25-dihydroxyvitamin D3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells.

1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH)2D3 could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, we analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH)2D3 in a dose- and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH)2D3 was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH)2D3 regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.

Animals↗
PubMed1990

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.

Amino Acid Sequence↗
PubMed1989

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression.

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.

Amino Acid Sequence↗
PubMed1989

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.

Animals↗
PubMed1988

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit.

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.

Algorithms↗
PubMed1987

Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+-ATPase alpha subunit.

The molecular basis for the marked difference between primate and rodent cells in sensitivity to the cardiac glycoside ouabain has been established by genetic techniques. A complementary DNA encoding the entire alpha 1 subunit of the mouse Na+- and K+-dependent adenosine triphosphatase (ATPase) was inserted into the expression vector pSV2. This engineered DNA molecule confers resistance against 10(-4) M ouabain to monkey CV-1 cells. Deletion of sequences encoding the carboxyl terminus of the alpha 1 subunit abolish the activity of the complementary DNA. The ability to assay the biological activity of this ATPase in a transfection protocol permits the application of molecular genetic techniques to the analysis of structure-function relationships for the enzyme that establishes the internal Na+/K+ environment of most animal cells. The full-length alpha 1 subunit complementary DNA will also be useful as a dominant selectable marker for somatic cell genetic studies utilizing ouabain-sensitive cells.

Animals↗
PubMed1987

Genes encoding alpha and beta subunits of Na,K-ATPase are located on three different chromosomes in the mouse.

We have made use of a panel of mouse-hamster somatic cell hybrids and restriction fragment length polymorphisms between two mouse species (Mus musculus and Mus spretus) to determine the chromosomal localization of genes encoding the alpha and beta subunits of the Na,K-ATPase (Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3). DNA probes for three distinct isoforms of the Na,K-ATPase alpha subunit mapped to three different mouse chromosomes: the alpha 1 gene (Atpa-1) cosegregated with the Egf gene on chromosome 3; alpha 2 (Atpa-2) with the cytochrome P-450PB gene family/coumarin hydroxylase locus on chromosome 7; alpha 3 (Atpa-3) with the alpha-spectrin gene on chromosome 1. The Na,K-ATPase beta-subunit gene (Atpb) mapped to the same region of chromosome 1, but it was not tightly linked to the Atpa-3 gene. These results indicate that three isoforms of the Na,K-ATPase alpha subunit are encoded by three distinct genes. The dispersion of Na,K-ATPase genes suggests that their expression is not likely to be controlled by a common cis-acting regulatory element.

Animals↗
PubMed1987

Differential expression of Na+,K+-ATPase alpha- and beta-subunit mRNAs in rat tissues and cell lines.

We have analyzed Na+,K+-ATPase (EC 3.6.1.3) alpha- and beta-subunit mRNA expression in rat tissues and cell lines derived from the rat central nervous system. Substantial differences in the tissue and developmental specificity of expression were found for the genes encoding three isoforms of the alpha subunit. Transcripts of the alpha 1-subunit gene were detected in all tissues tested, whereas alpha 2- and alpha 3-subunit mRNA species were expressed predominantly in brain. The pattern of expression of beta-subunit mRNA also was complex and tissue specific but was distinct from that of any of the alpha-subunit mRNAs. Cell lines derived from the rat central nervous system and the pheochromocytoma PC12 expressed the mRNAs for all three alpha-subunit isoforms, whereas beta-subunit mRNA was detected only in PC12 cells. The distinct expression patterns of rat Na+,K+-ATPase mRNAs suggest that different members of the ATPase family may have specialized functions.

Actins↗
PubMed1987

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications.

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

Amino Acid Sequence↗