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Biomedical subjects

B Knowles

Publications and source records attributed to B Knowles.

18 recordsLinked to original sources
PubMed1999

A new family of mouse genes homologous to the human MAGE genes.

The human MAGE genes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. The MAGE genes are located on the X chromosome, in three clusters denoted MAGE-A, B, and C, mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 human MAGE-A genes. Using a MAGE-A probe, we isolated 8 new murine genes that are homologous to the MAGE genes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with the MAGE-A ORFs than with the MAGE-B or MAGE-C ORFs. Furthermore, like MAGE-A genes, they encode acidic proteins, whereas the MAGE-B genes encode basic proteins. Accordingly, these 8 murine genes were named Mage-a1 to 8 (approved symbols Magea1 to 8). Mage-a genes were mapped in two different loci on the mouse X chromosome. Mage-a4 and Mage-a7 are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts, Mage-a genes were found to be transcribed in adult testis, but not in other tissues. Expression of some Mage-a genes was also detected in tumor cell lines. Two Mage-a genes were found to be expressed in blastocysts.

Animals↗
PubMed1992

Gene expression during preimplantation mouse development.

To develop a resource for the identification and isolation of genes expressed in the early mammalian embryo, large and representative cDNA libraries were constructed from unfertilized eggs, and two-cell, eight-cell, and blastocyst-stage mouse embryos. Using these libraries, we now report the first stages at which the cytokines interleukin (IL)-6, IL-1 beta, and interferon (IFN)-gamma are transcribed in the developing embryo and the presence of IL-7 transcripts in the unfertilized egg. Transcripts for IL-1 alpha, -2, -3, -4, or -5 were not detected at these stages. To identify novel genes expressed on activation of the embryonic genome, the egg and eight-cell stage-specific cDNA libraries were subtracted from the two-cell library, yielding a specialized cDNA library enriched for transcripts expressed at the two-cell stage. Sequence and Southern blot analysis of several of these cDNAs expressed predominantly at the two-cell stage of embryogenesis revealed them to be from novel genes, thereby providing the first molecular tools with which to approach the study of gene expression in the early mammalian embryo.

Animals↗
PubMed1990

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).

Amino Acid Sequence↗
PubMed1985

Expression of histocompatibility antigens H-2K, -D, and -L is reduced in adenovirus-12-transformed mouse cells and is restored by interferon gamma.

Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of class I antigens H-2K, -D, and -L on the cell surface and were capable of forming tumors in syngeneic animals, whereas cells transformed by Ad5 continued to express class I antigens and were nontumorigenic. Cells from a tumor, generated by injection of Ad12-transformed mouse cells into a syngeneic mouse, also expressed low levels of H-2 antigens, indicating that this phenotype is maintained in vivo. In all Ad12-transformed cells, synthesis of the H-2 heavy chain was not detected whereas the beta 2-microglobulin light chain was synthesized. Furthermore, the level of cytoplasmic H-2 mRNA in the Ad12 lines was greatly reduced. Reduction of H-2 expression is instructed solely by the transforming region of the viral genome, since this repression occurred in cells transformed by a DNA fragment containing only Ad12 E1A and E1B genes. Addition of recombinant murine interferon gamma strongly stimulated expression of class I antigens in the Ad12 transformants as well as in cells from the Ad12 tumor. This result indicates that Ad12 does not preferentially transform cells that are deficient for class I genes and that Ad12 does not mutate the class I genes in cells it transforms. The correlation between tumorigenicity and loss of H-2 expression in Ad12-transformed cells is discussed.

Adenoviridae↗
PubMed1985

Tumor location using F(ab')2 mu from a monoclonal IgM antibody: pharmacokinetics.

A monoclonal IgM antibody (anti-SSEA-1) and its divalent antigen-binding peptic fragment [F(ab')2 mu] were compared as in vivo tumor localization reagents in mouse teratocarcinomas. F(ab')2 mu is cleared more rapidly than whole antibody from the whole body, blood, and all tested organs (t1/2 for whole antibody approximately 18 hr; t1/2 for F(ab')2 mu, 12 hr). A corresponding average improvement in tumor-to-tissue ratio is observed 48 hr after injection and earlier. However, the affinity of the F(ab')2 mu for antigen is much lower, and a smaller fraction of the antibody fragment is retained in the tumor than with whole antibody. The fragment was not retained by animals bearing nonantigenic tumors.

Animals↗
PubMed1985

Detection of lecithin: cholesterol acyltransferase (LCAT) in a human hepatoma cell line.

A human hepatoma cell line (HepG-2) was probed for the presence of lecithin: cholesterol acyltransferase (LCAT) using an antiserum to human plasma LCAT. Double immunodiffusion analysis using antiserum to human plasma LCAT revealed a single precipitin line in the sonicated cell homogenate. This precipitin line showed a reaction of identity with highly purified plasma LCAT. The presence of LCAT within the hepatoma cells was also confirmed by an immunofluorescence test. In contrast, the cell culture supernate showed a weak and inconsistent precipitin line. These data suggest that HepG-2 cells synthesize LCAT but secretion of the enzyme by these cells into the culture medium may be partially or totally impaired.

Carcinoma, Hepatocellular↗
PubMed1984

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2.

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.

Binding, Competitive↗
PubMed1984

Receptor modulating properties of an antibody directed against the epidermal growth factor receptor.

A murine antiserum with specificity for the human epidermal growth factor (EGF) receptor was used to investigate EGF receptor function. The IgG fraction of this antiserum displayed no EGF-like mitogenic activity, even when cross-linking was ensured by sequential treatment with rabbit anti-(mouse IgG). The interaction of antibody with solubilized purified EGF receptor was characterized in detail. The binding of 125I-antibody to the receptor was not blocked by EGF, but the binding of 125I-EGF to the receptor was blocked by the immune IgG. Scatchard analysis of this reaction revealed a reduction in maximal EGF binding but an enhanced EGF binding affinity. In addition, at low concentrations, the immune IgG was found to enhance receptor kinase activity in the absence of EGF. The enhancement of kinase activity, as measured by receptor phosphorylation, was due to a decreased Km for ATP, and an increased V. These results suggest that the antibody is capable of altering conformations at receptor active sites by binding to non-active species-specific epitopes.

Animals↗
PubMed1969

Response of simian virus 40-transformed cell lines and cell hybrids to superinfection with simian virus 40 and its deoxyribonucleic acid.

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.

Adsorption↗