Biomedical subjects

C A Brenner

Publications and source records attributed to C A Brenner.

33 records · Page 2Linked to original sources

PubMed1999

Search for a human homologue of the mouse Ped gene.

The Ped gene influences the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. The mouse Ped gene product is a major histocompatibility complex (MHC) class Ib protein called Qa-2. Studies from many human in-vitro fertilization (IVF) clinics suggest that the mouse Ped gene has a human homologue because embryos fertilized at the same time have different cleavage rates, and those embryos that cleave at a faster rate are more likely to result in a viable pregnancy. Candidates for the human homologue of the mouse Ped gene include the MHC class Ib genes HLA-E, HLA-F, and HLA-G. The presence of mRNA for these three genes was tested in 108 spare day 3 human preimplantation embryos from 25 couples by using reverse transcription-polymerase chain reaction (RT-PCR). Of the 86 embryos tested for HLA-E mRNA, 72 were positive (84%), and of the 88 embryos tested for HLA-G mRNA, 39 were positive (44%). None of the 17 embryos tested for HLA-F mRNA were positive (0%). Studies of expression of HLA-G protein were undertaken to ascertain whether HLA-G was attached to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage similar to that found in Qa-2 protein. Treatment of JEG-3 cells, an HLA-G expressing cell line, with phospholipase C did not result in removal of HLA-G showing that HLA-G, unlike Qa-2, is not GPI linked to the cell surface. The pros and cons of HLA-E, HLA-F, and HLA-G as candidates for the human Ped gene are discussed.

Animals↗

PubMed1999

Alternative splicing of the telomerase catalytic subunit in human oocytes and embryos.

The human telomerase catalytic subunit (hTCS) is a ribonucleoprotein which synthesizes telomere repeats on the ends of chromosomes. Telomerase activity is thought to be essential in maintaining normal telomere length in immortal (cancer) and germ cells. The objective of this study was to determine the gene expression of telomerase mRNA in human oocytes at different meiotic stages and in embryos. Normal and abnormal human oocytes, preimplantation embryos, and blastocysts were analysed for the presence and expression of the hTCS transcripts. Multiple telomerase mRNA products were identified by reverse transcription-polymerase chain reaction (RT-PCR) using primers within the reverse transcriptase domain. DNA sequencing of these amplicons suggest that there are alternative splicing variants which align to other telomerase reverse transcriptase (RT) consensus domains. Surprisingly, in unfertilized and immature gametes, as well as preimplantation embryos, hTCS expression revealed three different PCR product sizes, 457, 421 and 275 bp. The frequency of the 275 bp DNA product was 6.6% in oocytes (two out of 30) compared with 56.6% (17 out of 30) in poorly developing human preimplantation embryos (P < 0.005). The presence of alternately spliced mRNA variants in human preimplantation embryos may suggest a lack of telomerase activity and thus chromosomes associated with shortened telomeres.

Alternative Splicing↗

PubMed1998

Genetic regulation of egg and embryo survival.

In both mice and humans, 15-50% of embryos die during the preimplantation period from mechanisms that are largely unknown. Two major criteria predict preimplantation embryo quality, the rate of development and the degree of fragmentation. We review evidence that both of these criteria have a genetic basis. Rate of development and subsequent embryo survival are controlled by a gene, Ped, we discovered in the mouse. Although progress is being made in the search for the human homologue of the mouse Ped gene, it has not yet been identified. Fragmentation, observed in both mouse and human embryos, is probably the result of apoptosis. We analysed transcription of two genes that regulate apoptosis, bcl-2 and bax, and found that both are transcribed in mouse and human preimplantation embryos. Overall, the literature reviewed and new data presented in this paper support the concept that there is a genetic basis for preimplantation egg and embryo survival.

Animals↗

PubMed1998

Mitochondrial DNA deletion in human oocytes and embryos.

Mitochondrial DNA (mtDNA) deletions are present in both human oocytes and embryos. It has been found that these tissues contain a mtDNA mutation which is present in high amounts in patients with Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia. In the present study, the frequency of this KSS deletion was investigated in human oocytes and embryos. Using a nested primer polymerase chain chian reaction (PCR) strategy, the frequency of the KSS deletion in 74 human oocytes and 137 embryos was found to be 32.8 and 8.0% respectively. Using a 'long PCR-short PCR' nested primer strategy, the frequency of the KSS deletion in 181 human oocytes and 104 embryos was found to be 47.0 and 20.2% respectively. There was no statistical correlation between the age of the patients at the time of oocyte retrieval and the presence of the deleted molecules. There was a statistical difference between the presence of the deleted molecules in oocytes versus embryos using either technique (P < 0.0001). The relevance of these findings to the accumulation of low levels of deleted mtDNA in both oocytes and embryos is discussed in this study.

Adult↗

PubMed1996

Matrix metalloproteinase expression during mouse peri-implantation development.

PROBLEM: The purpose of this study was to define the temporal expression and to quantitate the mRNA levels of collagenase, 72 kDa, 92 kDa, and membrane-type matrix metalloproteinase during the peri-implantation period of pregnancy in the mouse uterus. Embryonic expression of 72 kDa and 92 kDa matrix metalloproteinases, as well as interleukin 1 alpha, was also investigated. METHODS: Uterine matrix metalloproteinases were detected using gelatin substrate gel electrophoresis (zymography) and reverse-transcription polymerase chain reaction methodology was used to detect and quantitate different mRNA species in the mouse uterus and blastocyst. RESULTS: Collagenase, 72 kDa, and 92 kDa matrix metalloproteinases are developmentally regulated during the peri-implantation period of pregnancy, but membrane-type matrix metalloproteinase appears to be expressed constitutively. Matrix metalloproteinase mRNA levels have been quantitated and confirm the observed developmental expression patterns. Prominent expression of bot 92 kDa matrix metalloproteinase and interleukin 1 alpha was observed in blastocysts during outgrowth while weak expression of the 72 kDa matrix metalloproteinase was detected. CONCLUSIONS: The date provide evidence of matrix metalloproteinase expression in vivo and substantiate their potential role in tissue remodeling prior to and during blastocyst implantation. Expression of interleukin 1 alpha, 92 kDa, and 72 kDa matrix metalloproteinases suggests that these proteins are important for trophoblast invasion associated with implantation of the early embryo.

Animals↗

PubMed1990

Natural killer lines and clones with apparent antigen specificity.

Fresh CD3-, CD16+ lymphocytes that adhered to selected allogeneic lymphoblastoid cell lines (LCL) were cultured with LCL in the presence of IL-2-containing medium. The resulting lines as well as clones derived from these lines expressed CD16 and/or CD56, but lacked detectable CD3 or TCR-alpha/beta or TCR-gamma/delta complexes on the cell surface. Northern blot analysis failed to detect CD3 epsilon or TCR-beta transcripts, but revealed the presence of a TCR-gamma chain transcript in one of these lines. In addition to displaying potent cytolytic activity against K562 erythroleukemia cells (a classical NK target), the vast majority of these lines and clones lysed their specific stimulator LCL to a significantly greater extent than irrelevant LCL. This selective killing was inhibited by the addition of cold stimulator LCL or K562 cells, or anti-LFA 1 mAbs, but not by irrelevant LCL or mAbs to CD3, class I or class II MHC antigens. These results indicate that some CD3- lymphocytes, phenotypically indistinguishable from NK cells, can recognize and lyse allogeneic targets in a specific manner.

Antibodies, Monoclonal↗

PubMed1990

Soluble suppressive molecule released by human T lymphocytes induced by heat-inactivated allostimulator cells in the presence of cyclosporine.

E-rosette-positive peripheral blood lymphocytes (E+PBL) stimulated initially with heat-inactivated allogeneic lymphoblastoid cells in the presence of cyclosporine (CsA/HI) produce a soluble molecule that suppresses fresh lymphocytes in a primary mixed lymphocyte reaction. Cell lines were derived from the E+PBL cells after one and two weeks of culture. These lines were CD4+ by both FACS and mRNA analysis. The cells produce a potent soluble molecule (supernates often containing greater than 1000 units of suppressive activity per milliliter). The factor has an apparent molecular weight of 90 k and is sensitive to both pH and boiling. The molecule is not the suppressive cytokine TGF beta, based on neutralization with anti-TGF beta antibody and mRNA expression. None of the available cytokines expressed by these cells was suppressive when titrated into an MLR, alone or in combination. These results support the conclusion that CsA/HI-activated T cell lines produce a novel cytokine that is not antigen-specific or MHC-restricted.

Antigens, CD↗

PubMed1990

Expression of extracellular matrix-degrading metalloproteinases and metalloproteinase inhibitors is developmentally regulated during endoderm differentiation of embryonal carcinoma cells.

The differentiation of F9 and PSA-1 embryonal carcinoma cells to embryoid bodies composed of a mixture of parietal and visceral endoderm was accompanied by changes in their secretion of metalloproteinases. Differentiation was induced by retinoic acid and dibutyryl cyclic AMP (for F9 cells) or by removing cells from a substrate of feeder cells to alter cell-cell interaction and adhesion (for PSA-1 cells). The embryoid bodies attached to gelatin-coated dishes, and the parietal endoderm cells spread out over the matrix. The differentiated cells secreted specific gelatin- and casein-degrading proteinases, including enzymes that comigrated with proenzyme forms of collagenase and stromelysin. Total proteinase activity as well as specific collagenase activity increased with the time of differentiation. All of the gelatin- and casein-degrading proteinases detectable by substrate gel zymography were inhibited by inhibitors of metalloproteinases but not by inhibitors of serine or cysteine proteinases, indicating that they were metalloproteinases. Both cell lines showed increased collagenolytic activity, which was activated by treatment with plasmin. In addition, both cell lines showed increased secretion of specific metalloproteinase inhibitors, including tissue inhibitor of metalloproteinases, with differentiation. Analysis of mRNA from undifferentiated and differentiated F9 cells by RNA blot analysis or reverse transcription coupled with the polymerase chain reaction showed that increased expression of genes for collagenase, stromelysin and tissue inhibitor of metalloproteinases is associated with differentiation of these cells. These results suggest that the expression of extracellular matrix-degrading metalloproteinases and their inhibitors is developmentally regulated during the differentiation and spreading of the parietal endoderm.

Animals↗

PubMed1989

Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction.

A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide and a conserved 3' constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.

Amino Acid Sequence↗

PubMed1989

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation.

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.

Amino Acid Sequence↗

PubMed1989

Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development.

Extracellular matrix (ECM) remodeling accompanies cell migration, cell-cell interactions, embryo expansion, uterine implantation, and tissue invasion during mammalian embryogenesis. We have found that mouse embryos secrete functional ECM-degrading metalloproteinases, including collagenase and stromelysin, that are inhibitable by the tissue inhibitor of metalloproteinases (TIMP) and that are regulated during peri-implantation development and endoderm differentiation. mRNA transcripts for collagenase, stromelysin, and TIMP were detected as maternal transcripts in the unfertilized egg, were present at the zygote and cleavage stages, and increased at the blastocyst stage and with endoderm differentiation. These data suggest that metalloproteinases function in cell-ECM interactions during growth, development, and implantation of mammalian embryos.

Amino Acid Sequence↗

PubMed1988

Developmental expression of PDGF, TGF-alpha, and TGF-beta genes in preimplantation mouse embryos.

Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors from embryonic or maternal sources. With the use of single-cell messenger RNA phenotyping, the simultaneous expression of growth factor transcripts in single or small numbers of preimplantation mouse embryos was examined. Transcripts for platelet-derived growth factor A chain (PDGF-A), transforming growth factor (TGF)-alpha, and TGF-beta 1, but not for four other growth factors, were found in whole blastocysts. TGF-alpha, TGF-beta 1, and PDGF antigens were detected in blastocysts by immunocytochemistry. Both PDGF-A and TGF-alpha were detected as maternal transcripts in the unfertilized ovulated oocyte, and again in blastocysts. TGF-beta 1 transcripts appeared only after fertilization. The expression of a subset of growth factors in mouse blastocysts suggests a role for these factors in the growth and differentiation of early mammalian embryos.

Animals↗

PubMed1987

Characterization of a cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.

Age Factors↗

PubMedDate not supplied

Quantification of human ooplasmic mitochondria.

It is likely that there is an association between the fitness of mitochondria and their ability to support normal cellular function. Oocytes are greatly enriched in the number of mitochondria as they support essential developmental processes such as oocyte maturation and embryonic development, while their replication is deferred until gastrulation. The mitochondrial DNA (mtDNA) copy number in 87 human oocytes from 29 patients was evaluated after DNA extraction and real-time quantitative polymerase chain reaction (PCR). The average mtDNA copy number was 795,000 (+/- 243,000) in metaphase II oocytes. mtDNA content varied considerably between oocytes, even within the same patient. No relationship was found between mtDNA copy number and maternal age. The findings suggest that mtDNA replication is fully accomplished by the germinal vesicle stage in the fully developed oocyte.

DNA, Mitochondrial↗

PubMedDate not supplied

Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells.

A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and enzymatic amplification of cytokine-specific DNA fragments using the polymerase chain reaction. In this study, the technique has been applied to the analysis of cytokines produced by lymphoid cells ranging in number from a single cell to 10(6) cells. The technique should be applicable to virtually any tissue or cell type.

Base Sequence↗